pHLIP® peptide-mediated epitope tethering at cell surfaces
20200246420 · 2020-08-06
Assignee
Inventors
- Yana K. Reshetnyak (Saunderstown, RI, US)
- Oleg A. Andreev (Saunderstown, RI, US)
- Anna Moshnikova (Warwick, RI, US)
- Donald M. Engelman (New Haven, CT)
Cpc classification
A61K47/00
HUMAN NECESSITIES
A61K45/05
HUMAN NECESSITIES
A61K38/16
HUMAN NECESSITIES
C07K2319/33
CHEMISTRY; METALLURGY
A61K47/10
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
A61K38/16
HUMAN NECESSITIES
Abstract
The invention features methods and compositions for eliciting an anti-tumor response in a subject comprising administering to the subject a pHLIP construct comprising an antibody recruiting molecule linked to one or more pHLIP peptides by a non-cleavable linker compound. The construct increases the amount of the antibody recruiting molecule on the surface of a diseased cell.
Claims
1. A method of eliciting an immune response in a subject comprising administering to said subject a pHLIP construct comprising an antibody recruiting molecule or an immune cell recruiting molecule linked to one or more pHLIP peptides, wherein said construct increases the amount of said antibody or immune cell recruiting molecule on the surface of a diseased cell.
2. The method of claim 1, wherein said antibody recruiting molecule or said immune cell recruiting molecule comprises an epitope.
3. The composition of claim 1, wherein said construct comprises the formula of
Epitope-Linker-Pept wherein Epitope is an antibody or immune cell recruiting molecule; wherein Linker is a non-cleavable linker compound or a membrane non-inserting end of the pHLIP peptide further comprises an amino acid extension; wherein Pept is a pHLIP peptide comprising the sequence AXDDQNPWRAYLDLLFPTDTLLLDLLW (SEQ ID NO: ______) or AXDQDNPWRAYLDLLFPTDTLLLDLLW (SEQ ID NO: ______), where X is a functional group, selected from a lysine, a cysteine, or an Azido-containing amino acid; wherein each is a covalent bond.
4. The method of claim 1, wherein said construct comprises an antibody recruiting molecule
5. The method of claim 4, wherein 2 antibody recruiting molecules are linked to pHLIP peptide.
6. The method of claim 1, wherein said construct comprises the formula of
Epitope 1-Linker-Pept-Linker-Epitope1 wherein Epitope1 is an antibody recruiting molecule; wherein Linker is a polyethylene glycol linker; wherein Pept is a pHLIP peptide comprising the sequence TABLE-US-00043 (SEQIDNO:_) Ac-AKQNDDQNKPWRAYLDLLFPTDTLLLDLLWA or (SEQIDNO:_) Ac-AKQNDNDNKPWRAYLDLLFPTDTLLLDLLWA or (SEQIDNO:_) ACQNDDQNCPWRAYLDLLFPTDTLLLDLLWA or (SEQIDNO:_) ACQNDNDNCPWRAYLDLLFPTDTLLLDLLWA wherein each is a covalent bond.
7. The method of claim 2, wherein said epitope comprises a peptide with a length less than 50 amino acids.
8. The method of claim 1, wherein said diseased cell comprises a tumor cell.
9. The method of claim 1, wherein said diseased cell comprises a cell in inflamed tissue.
10. The method of claim 1, wherein said antibody recruiting molecule or immune cell recruiting molecule comprises an epitope.
11. The method of claim 2, wherein said epitope comprises a peptide with a length less than 50 amino acids.
12. The method of claim 2, wherein said epitope comprises a length of between 5 to 20 amino acids.
13. The method of claim 2, wherein said epitope is a HA peptide.
14. The method of claim 12, wherein said peptide comprises the amino acid sequence of YPYDVPDYA (SEQ ID NO: ______).
15. The method of claim 2, wherein said epitope is selected from the group consisting of QVSHWVSGLAEGSFG (SEQ ID NO: ______), LSHTSGRVEGSVSLL (SEQ ID NO: ______), QMWAPQWGPD (SEQ ID NO: ______); MASMTGGQQMG (SEQ ID NO: 4); EQKLISEEDL (SEQ ID NO: 5); YTDIEMNRLGK (SEQ ID NO: 7); KETAAAKFERQHMDS (SEQ ID NO: 8); GKPIPNPLLGLDST (SEQ ID NO: 9); DYKDDDDK (SEQ ID NO: 10); GAPVPYPDPLEPR (SEQ ID NO: 11); HHHHHH (SEQ ID NO: 12); TKENPRSNQEESYDDNES (SEQ ID NO: 13); WSHPQFEK (SEQ ID NO: 14); and PDRVRAVSHWSS (SEQ ID NO: 15).
16. The method of claim 2, wherein said epitope comprises a protein epitope with a length of 200 or less amino acids.
17. The method of claim 16, wherein said protein epitope comprises a cytokine.
18. The method of claim 17, wherein said cytokine comprises an interleukin (IL).
19. The method of claim 18, wherein said interleukin comprises IL-1, IL-2, IL-6, IL-7, IL-12, and IL-17.
20. The method of claim 17, wherein said cytokine comprises tumor necrosis factor (TNF).
21. The method of claim 17, wherein said cytokine comprises a chemokine (CXC).
22. The method of claim 21, wherein said chemokine is CXCL9, CXL10, or CXL11.
23. The method of claim 21, wherein said chemokine comprises CXCL10 comprising the amino acid sequence: TABLE-US-00044 (SEQIDNO:) MNQTAILICCLIFLTLSGIQGVPLSRTVRCTCISISNQPVNPRSLEKLEII PASQFCPRVEIIATMKKKGEKRCLNPESKAIKNLLKAVSKERSKRSP.
24. The method of claim 2, wherein said epitope comprises a small molecule.
25. The method of claim 24, wherein said small molecule comprises a dinitrophenyl (DNP) or a derivative thereof.
26. A composition comprising an antibody or immune cell recruiting molecule linked to one or more pHLIP peptides by a non-cleavable linker compound.
27. A composition comprising an epitope linked to one or more pHLIP peptides, wherein the epitope is a protein epitope and is an extension of the non-inserting end of the pHLIP peptide.
28. The composition of claim 27, wherein said extension comprises a peptide epitope.
29. A composition comprising an epitope linked to one or more pHLIP peptides, wherein the epitope and the pHLIP peptide are part of a single fusion construct or fusion protein.
30. The method of claim 1, wherein said construct comprises the formula of Epitope-Linker-Peptide, wherein Peptide is a pHLIP peptide comprising the sequence AXDDQNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: ______) or AXDQDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: ______), where X is a functional group, selected from a lysine, a cysteine, or an Azido-containing amino acid, wherein Linker is a linker or an extension of the pHLIP peptide, and wherein each is a covalent bond.
31. A composition comprising the formula of Epitope-Linker-Peptide, wherein Peptide is a pHLIP peptide comprising the sequence AXDDQNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: ______) or AXDQDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: ______), where X is a functional group, selected from a lysine, a cysteine, or an Azido-containing amino acid, and wherein Linker is a linker or an extension of the pHLIP peptide, and wherein each is a covalent bond.
32. The composition of claim 31, wherein two epitopes are linked to a single pHLIP peptide.
33. The method of claim 1, wherein said construct comprises the formula of Epitope.sub.2-Linker.sub.2-Peptide, wherein Peptide is a pHLIP peptide comprising the sequence AX(Z).sub.nXPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: ______), wherein X is a functional group, selected from a lysine, a cysteine, an Azido-containing amino acid, or others, wherein Z comprises indicates any amino acid residue, wherein n is any integer between 1 and 10, wherein Linker is a linker or an extension of the pHLIP peptide, and each is a covalent bond.
34. The composition of claim 33, wherein said composition comprising the formula of Epitope.sub.2-Linker.sub.2-Pept, wherein Pept is a pHLIP peptide comprising the sequence AX(Z).sub.nXPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: ______), where X is a functional group, selected from a lysine, a cysteine, an Azido-containing amino acid, wherein Z indicates any amino acid residue, wherein n is any integer between and including 1 and 10, wherein Linker is a linker or an extension of the pHLIP peptide, and wherein each is a covalent bond.
35. A method of inducing an immune response in a diseased tissue in a subject, comprising administering to a subject a composition comprising an epitope and a pHLIP peptide.
36. The method of claim 35, wherein said subject comprises a solid tumor.
37. The method of claim 35, wherein said subject comprises an inflamed tissue.
38. The method of claim 35, wherein said composition is injected directly into a diseased tissue tumor mass.
39. The method of claim 35, wherein said composition is systemically administered.
40. The method of claim 35, wherein a biological effect of said composition is at least 20% greater than that delivered in the absence of said composition.
41. The method of claim 35, wherein said composition targets preferentially to a diseased tissue compared to a healthy tissue, thereby minimizing damage to said healthy tissue.
42. A method for promoting an immune response in a subject, comprising administering to a subject the composition of claim 1, wherein said method comprises placement of said epitope on tumor cell or a cell in inflamed tissue of said subject.
Description
DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0084] The invention features compositions and methods for decorating target cells (cancer cells) using epitope-pHLIP compounds, such that the pHLIP targets tumors by responding to cell surface acidity, inserting into cancer cell membranes, and locating (positioning) a specified epitope on the cell surface to induce cell killing.
[0085] Epitopes bind and/or attract exogenous immune cells (T-cells or NK-cells) or exogenously developed monoclonal antibodies or antibody-drug conjugates administrated into body during or after of epitope-pHLIP compound administration, and selectively promote cell killing while sparing normal tissues. Among these epitopes include epitopes for developed monoclonal antibodies.
[0086] Epitopes bind and/or attract endogenous (natural) immune cells (lymphocytes) or endogenous antibodies already present in the blood, and selectively initiate a specific immune response to attack the tumor while sparing normal tissues. Among these epitopes are epitopes associated with the surfaces of animal cells and bacteria. In many cases, humans already have developed antibodies in blood stream, which recognize such epitopes. Epitopes delivered according to the methods described herein may also bind/attract endogenous antibodies, which are produced (generated) in the course of vaccination. The epitopes (any antigen molecule used for vaccination), is linked (conjugated) to a pHLIP peptide and delivered to tumors to promote an immune reaction within target tissue, e.g., by using harnessing influenza vaccines that create antibodies to virus surface epitopes. In some examples, an individual is immunized using a chosen antigen, followed by targeting the antigen targeted to tumor cells by a pHLIP peptide.
Decorated Acidic Diseased Cells
[0087] Acidic diseased cells, e.g., cancer cells are targeted using epitope-pHLIP peptide compositions, such that the pHLIP peptide targets tumors by responding to cell surface acidity, inserting into cancer cell membranes, and locating/positioning a specific epitope on the cell surface to induce and promote cellular responses including immune stimulation and inhibition of cell proliferation. The immune stimulation leads to cytotoxicity and death of the acidic disease cell.
Epitope-pHLIP Peptide Compositions
[0088] Representations of exemplary pHLIP compositions for therapeutic use are shown in
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[0093] An exemplary composition comprises the following formula:
Epitope-Linker-Peptide
[0094] The Epitope may include a peptide, a protein or a fragment thereof, or a small molecule such as an organic molecule, to induce an immune response or promote cell killing by attracting endogenous (pre-existing) immune cells or antibodies, exogenous (administered as in passive antibody-based immunotherapy) engineered immune cells or purified antibodies, ADCs administrated into humans, or antibodies generated in the course of vaccination.
[0095] Peptide is a pHLIP peptide (a non-limiting example is a pHLIP peptide comprising the sequence AXDDQNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: 18) or AXDQDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: ______), where X is a functional group, e.g., for conjugation purposes, selected from a lysine (Lys), a cysteine (Cys), or an Azido-containing amino acid. In some cases Peptide is a pHLIP conjugate, where the pHLIP peptide is linked with a drug molecule for intracellular delivery.
[0096] Linker comprises a covalent bond, or a chemical linker, or an extension of the membrane non-inserting flanking region of pHLIP peptide. If the epitope is a peptide or protein, it may be constructed as an extension of the pHLIP peptide, and no linker may be required. Non-limiting example of linker is a polyethylene glycol (PEG) polymer in size from 200 Da to 20 kDa. Non-limiting example of an extension is a poly-Glycine polypeptide. Epitope(s) are also linked to pHLIP peptide(s) via non-cleavable link(s).
[0097] Non-limiting examples of a Linker is a mucin domain, which is a high molecular weight, heavily glycosylated protein (glycoconjugate) produced by epithelial tissues in most humans. In other examples, the linker may include biopolymers, including, for example cellulose, starch or chitin.
[0098] An exemplary composition comprises the following formula:
Epitope.sub.2-Linker.sub.2-Pept
[0099] The Epitope comprises a peptide, a protein or a fragment thereof, or a small molecule such as an organic molecule, to induce an immune response or promote cell killing by attracting endogenous (pre-existing) immune cells or antibodies, exogenous (administered as in passive antibody-based immunotherapy) engineered T-cells or NK-cells or purified antibodies, ADCs administrated into humans, or antibodies generated as a result of vaccination.
[0100] Peptide is a pHLIP peptide comprising the sequence: AX(Z).sub.nXPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: ______), wherein upper case X indicates any amino acid residue, and can include a lysine (Lys), a cysteine (Cys), or an Azido-containing amino acid. The X may be used for conjugation to another moiety. The Z indicates any amino acid residue, and n can be any integer between and including 1-10 (e.g., 1n10). For example, (Z).sub.n may be QDNDQN (SEQ ID NO: ______) or any combination of polar amino acid residues including D, E, N or Q.
[0101] A compound is characterized as polar if it has a log P of less than 0.4. The epitope compound may be moderately hydrophobic. Polar: LogP<0.4; Moderately hydrophobic: 2.5<LogP<0.4; and Hydrophobic: LogP>2.5. The polarity and/or hydrophobicity of an epitope is measured using methods known in the art, e.g., by determining LogP, in which P is the octanol-water partition coefficient. A substance is dissolved into an octanol-water mixture, mixed, and allowed to come to equilibration. The amount of substance in each (or one) phases is then measured. The measurements itself could be in a number of ways known in the art, e.g., by measuring absorbance, or determining the amount using NMR, HPLC, or other known methods. As described herein, moderately hydrophobic, for example, is defined as molecule with LogP value in the range of 2.5 to 0.4, there are a lot of examples.
[0102] Linker is a linker, wherein the linker is a polyethylene glycol, PEG. For example, the formula PEG.sub.m, includes that m may be any integer between and including 12 and 24 (e.g., 12mPEG12-24 polymer). Each may be a covalent bond.
[0103] When an epitope is conjugated to the pHLIP peptide via a PEG12-24 linker and 6-8 residues are positioned between epitope-PEG attachments to the pHLIP peptide, the distance between epitopes can be in the range of 5-25 nm. Alternatively, the distance may be about 10 nm, or 10-15 nm, which corresponds to a typical distance between the two antigen binding sites binding sites of an antibody.
[0104] The pHLIP compositions described are used for decoration of cells in targeted acidic tissue to elicit an immune response or promote delivery of cytotoxic payload by ADCs to kill the diseased cells or tissue.
[0105] Non-limiting examples of epitopes are the following:
Peptide Epitope-pHLIP Peptide Compositions
[0106] Peptide antigens, e.g., peptides less than 50 amino acid residues in length, e.g., peptide antigens greater than 5 and less than 20, less than 15, less than 10, or less than 8 amino acid residues.
HER2 (Also Referred to as Receptor Tyrosine-Protein Kinase erbB-2 or Human Epidermal Growth Factor 2)
[0107] The HER family of transmembrane tyrosine kinase receptors are composed of four members, BER1 to HER4. HER2 is a ligand-orphan receptor expressed in many human tumors and overexpressed in 25-30% of breast cancers. HER2 amplifies the signal provided by other receptors of the HER family by forming heterodimers. The role of HER2 in the HER signaling network led to the development of anti-HER2 monoclonal antibodies (MAbs) for cancer therapy. In particular, the humanized MAb trastuzumab (Herceptin) or Herceptin-drug conjugates have antitumor activity against HER2-overexpressing human breast tumor cells and are widely used for the treatment of women with HER2 overexpressing breast cancers. One of the primary effects of trastuzumab is to induce antibody-dependent cellular cytotoxicity or promote cell killing by use of ADCs. In addition, trastuzumab induces HER2 receptor down modulation, inhibits critical signaling pathways (i.e. ras-Raf-MAPK and PI3K/Akt), and blocks cell cycle progression by inducing the formation of p27/Cdk2 complexes, inhibits HER2 cleavage, preceding antibody-induced receptor down modulation, which may also contribute to its antitumor activity in some cancers. A limitation of trastuzumab is that its activity is largely restricted to breast cancers with the highest level of HER2 overexpression.
[0108] As described above, humanized antibodies against the HER2 receptor have been developed for the treatment of HER2-positive breast cancer. However, not all breast tumors are HER2-positive, some of them are HER2 negative and in some cases during treatment, HER2-positive cancers can transform into HER2 negative cancers, and then treatment is not effective. Also, many other tumors are HER2 negative. The constructs/conjugates described herein decorate all cancer cells (regardless of tissue of origin) with HER2 epitopes to enhance antibody therapy. Useful epitopes include a small peptide mimic of the receptor binding site or a protein-pHLIP peptide fusion construct, where the protein resembles the entire extracellular domain of a receptor or part of it.
[0109] Non-limiting examples of HER2 mimicking peptides, which show high affinity binding to anti-HER-2 monoclonal antibody trastuzumab (Herceptin) include the following peptides:
TABLE-US-00021 (SEQIDNO:1) QVSHWVSGLAEGSFG (SEQIDNO:2) LSHTSGRVEGSVSLL (SEQIDNO:3) QMWAPQWGPD
[0110] The constructs are useful to decorate all cancer cells with HER2 epitopes to enhance antibody therapy. A useful epitope comprises a small peptide mimic of the receptor binding site or a protein-pHLIP fusion construct, where the protein resembles the entire extracellular domain of a receptor or part of it. An additional strategy includes use a pHLIP peptide to position epitopes for therapy using ADCs, some of which have been approved for clinical uses including trastuzumab emtansine, an anti-HER2 antibody conjugated with cytotoxic maytansinois payload (DM1 or mertansine), which is a potent tubulin inhibitor. By targeting the epitopes already in use, or by developing ADCs for new epitopes, the effective use of ADCs is significantly expanded. Other monoclonal antibodies developed for cancer treatment
[0111] In addition to anti-HER2 antibodies the non-limiting examples of monoclonal antibodies directed against different receptors currently in clinical use for cancer treatment include daratumumab (binds to CD38cluster of differentiation 38); dinutuximab (binds to glycolipid GD2disialoganglioside); bevacizumab (binds to VEGF-Avascular endothelial growth factor A); cetuximab, necitumumab and panitumumab (bind to EGFRepidermal growth factor); elotuzumab (binds to CD 319cluster of differentiation 319), necitumumab (binds EGFR). ramucirumab (binds to VEGFR2). Epitope-pHLIP constructs could be developed to use with all of these antibodies and their drug conjugates.
Other Examples of Peptide Epitopes
[0112] Non-limiting examples of peptide epitopes for conjugation to pHLIP peptides for which monoclonal antibodies are already developed include:
[0113] MASMTGGQQMG (SEQ ID NO: 4): a T7 peptide derived from the T7 major capsid protein;
[0114] EQKLISEEDL (SEQ ID NO: 5): a Myc peptide derived from c-Myc;
[0115] YPYDVPDYA (SEQ ID NO: 6): a hemagglutinin (HA) peptide derived from hemagglutinin;
[0116] YTDIEMNRLGK (SEQ ID NO: 7): a vesiculovirus (VSV-G) peptide derived from the vesicular stomatitis viral glycoprotein;
[0117] KETAAAKFERQHMDS (SEQ ID NO: 8): an S peptide derived from pancreatic ribonuclease A;
[0118] GKPIPNPLLGLDST (SEQ ID NO: 9): a V5 peptide derived from the P and V proteins of the paramyxovirus of simian virus 5;
[0119] PDRVRAVSHWSS (SEQ ID NO: 15): a peptide derived from the protein beta-catenin and optimized for higher affinity binding to the Spot-Tag Nanobody;
[0120] DYKDDDDK (SEQ ID NO: 10): a FLAG synthetic peptide;
[0121] GAPVPYPDPLEPR (SEQ ID NO: ______) an E synthetic peptide;
[0122] HHHHHH (SEQ ID NO: 12): a Histidine synthetic peptide;
[0123] TKENPRSNQEESYDDNES (SEQ ID NO: 13): an NE synthetic peptide;
[0124] WSHPQFEK (SEQ ID NO: 14): a synthetic peptide recognized by streptavidin;
[0125] YTDIEMNRLGK (SEQ ID NO: 7): a vesicular stomatitis virus (VSV) synthetic peptide.
Protein Epitope pHLIP Peptide Compositions
[0126] In addition to peptide epitopes, larger epitope-containing cytokine proteins may be used for the production of the compositions described. Cytokines are small proteins from 5 to 20 kDa, which perform immuno-modulating role. Among cytokine proteins are IL-17, TNF, CXCL and CCL chemokines.
[0127] For example, CXCL9, CXCL10 or CXCL11 chemokine is expressed as N-terminal part of pHLIP to induce homing and activation of T-cells and NK-cells when pHLIP inserts into cellular membrane and tethers CXCL9 or CXCL10 or CXCL11 proteins to surface of membrane.
[0128] The cytokine can be expressed together with a mucin-domain (a glycosylated protein) as a linker (spacer) between cytokine and pHLIP peptide to allow the cytokine to be fully exposed to the extracellular space.
[0129] The nucleic acid sequence encoding CXCL10 chemokine and the amino acid sequence of the protein antigen is described below.
TABLE-US-00022 SEQIDNO:498 ccagtctcagcaccatgaatcaaactgccattctgatttgctgccttatct ttctgactctaagtggcattcaaggagtacctctctctagaactgtacgct gtacctgcatcagcattagtaatcaacctgttaatccaaggtctttagaaa aacttgaaattattcctgcaagccaattttgtccacgtgttgagatcattg ctacaatgaaaaagaagggtgagaagagatgtctgaatccagaatcgaagg ccatcaagaatttactgaaagcagttagcaaggaaaggtctaaaagatctc ct
[0130] The amino acid sequence of CXCL 10 is provided below.
TABLE-US-00023 (SEQIDNO:514) mnqtailicclifltlsgiqgvplsrtvrctcisisnqpv nprslekleiipasqfcprveiiatmkkkgekrclnpesk aiknllkavskerskrsp
[0131] His or myc tags are used for purification purposes and not needed for use as an antigen (e.g., for conjugation to a pHLIP peptide).
Small Molecule Antigens or Epitopes
[0132] Small molecule antigens are those that are characterized by a molecular mass of less than 2000 daltons. For example, the molecular mass of the small molecule antigen is preferably less than 1000 daltons, more preferably less than 600 daltons, e.g., the compound is less than 500 daltons, 400 daltons, 300 daltons, 200 daltons, or 100 daltons. Some small molecule antigens are characterized by their binding to endogenous antibodies in the blood or serum of many normal humans (or humans characterized as having a pathological tumor).
[0133] Exemplary small molecule epitopes are described below.
Di-Nitrophenyl (DNP)
[0134] Antibodies to DNP (O-(2,4-dinitrophenyl)hydroxylamine, shown below) have been identified in the IgG fraction of normal human sera.
##STR00002##
[0135] Thus, another small molecule antigen that binds endogenous antibodies is dinitrophenyl (DNP) and its derivatives. For example, N-(4-dimethylamino-3,5-dinitrophenyl)maleimide is shown below:
##STR00003##
[0136] Or DNP-PEG4-NHS (1-(2,4-Dinitrophenylamino)-3,6,9,12-tetraoxapentadecanoic acid succinimidyl ester) is shown below:
##STR00004##
[0137] DNP is a low molecular weight antigen known for its ability to bind antibodies in normal human serum. Binding of the antibodies to DNP delivered to the surface of tumor or otherwise diseased acidic cells leads to cytotoxicity of the antigen-labeled target cells. The DNP recruits endogenous antibodies, e.g., antibodies that exist in a subject prior to administration of the pHLIP peptide constructs described herein.
Linker
[0138] In the schematic structure, Epitope-Linker-Peptide:
[0139] Linker could be relatively small, e.g., only a few atoms, to a rather large polar (or moderately hydrophobic) polymer or an N-terminal lengthening of the pHLIP peptide by the addition of amino acids, e.g., glycine residues (poly-Gly). In some examples, a linker can be part of membrane non-inserting pHLIP peptide sequence, such as those with a poly-Gly motif. In some examples, a linker could be PEG polymer. The purpose of a polymer or pHLIP extension is to position epitopes at the surfaces of cells to enhance the access of antibodies or proteins for binding to the epitope. Non-limiting example of linker is a PEG polymer in size from 200 Da up to 20 kDa.
[0140] In some examples the following linkers and their derivatives could be used: N--maleimidoacet-oxysuccinimide ester (AMAS); N--maleimidobutyryl-oxysuccinimide ester (GMBS); N--maleimidopropyl-oxysuccinimide ester (BMPS); N--malemidocaproyl-oxysuccinimide ester (EMCS); m-maleimidobenzoyl-n-hydroxysuccinimide ester (MBS); succinimidyl 3-(bromoacetamido)propionate (SBAP); succinimidyl (4-iodoacetyl)aminobenzoate (SIAB); N--maleimidocaproic acid (EMCA); succinimidyl 4-(n-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC); succinimidyl iodoacetate (SIA); succinimidyl (4-iodoacetyl)aminobenzoate (SIAB); succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB); succinimidyl 6-((beta-maleimidopropionamido)hexanoate) (SMPH); 3-propargyloxypropanoic acid, succinimidyl ester (alkyne, succinimidyl ester); 1,4-bismaleimidobutane (BMB); bismaleimidohexane (BMH); bismaleimidoethane (BMOE); tris(2-maleimidoethyl)amine (TMEA); N--maleimidopropionic acid hydrazide; (BMPH); N--maleimidocaproic acid hydrazide (EMCH); N--maleimidoundecanoic acid hydrazide (KMUH); 4-(4-n-maleimidophenyl)butyric acid hydrazide (MBPH); or p-maleimidophenyl isocyanate (PMPI).
[0141] In some examples the flexible linker or mucin domain is positioned between epitope and pHLIP peptide.
[0142] The mucin-domain of CX3CL1 and the nucleic acid sequence encoding mucin-domain of CX3CL1 is described below:
TABLE-US-00024 SEQIDNO:500 aatggcggcaccttcgagaagcagatcggcgaggtgaagcccaggaccacc cctgccgccgggggaatggacgagtctgtggtcctggagcccgaagccaca ggcgaaagcagtagcctggagccgactccttcttcccaggaagcacagagg gccctggggacctccccagagctgccgacgggtgtgactggttcctcaggg accaggctccccccgacgccaaaggctcaggatggagggcctgtgggcacg gagcttttccgagtgcctcccgtctccactgccgccacgtggcagagttct gctccccaccaacctgggcccagcctctgggctgaggcaaagacctctgag gccccgtccacccaggacccctccacccaggcctccactgcgtcctcccca gccccagaggagaatgctccgtctgaaggccagcgtgtgtggggtcagggg cagagccccaggccagagaactctctggagcgggaggagatgggtcccgtg ccagcgcacacggatgccttccaggactgggggcctggcagcatggcccac gtctctgtggtccctgtctcctcagaagggacccccagcagggagccagtg gcttcaggcagctggacccctaaggctgaggaacccatccatgccaccatg gacccccagaggctgggcgtccttatcactcctgtccctgacgcccaggct gccacccggaggcag
[0143] The sequence of mucin-domain of the human CX3CL1 protein (UniProt P78423 or NP_002987.1) is shown below (these are residues 111-141 from the human CX3CL1 protein):
TABLE-US-00025 SEQIDNO:526 gtfekqigevkprttpaaggmdesvvlepeatgessslep tpssqeaqralgtspelptgvtgssgtrlpptpkaqdggp vgtelfrvppvstaatwqssaphqpgpslwaeaktseaps tqdpstqastasspapeenapsegqrvwgqgqsprpensl ereemgpvpahtdafqdwgpgsmahvsvvpvssegtpsre pvasgswtpkaeepihatmdpqrlgvlitpvpdaqaatrrq
[0144] The full length CXCL1 amino acid dsequence is depicted below (NP_002987.1), and incorporated herein by reference.
TABLE-US-00026 SEQIDNO:527 1 mapislswllrlatfchltvllagqhhgvtkcnitcskmtskipvallihyqqnqascgk 61 raiiletrqhrlfcadpkeqwvkdamqhldrqaaaltrnggtfekqigevkprttpaagg 121 mdesvvlepeatgesssleptpssqeaqralgtspelptgvtgssgtrlpptpkaqdggp 181 vgtelfrvppvstaatwqssaphqpgpslwaeaktseapstqdpstqastasspapeena 241 psegqrvwgqgqsprpenslereemgpvpahtdafqdwgpgsmahvsvvpvssegtpsre 301 pvasgswtpkaeepihatmdpqrlgvlitpvpdaqaatrrqavgllaflgllfclgvamf 361 tyqslqgcprkmagemaeglryiprscgsnsyvlvpv
[0145] Exemplary landmark residues, domains, and fragments of CXCL1 include, but are not limited to residues 1-24 (signal peptide), residues 25-397 (mature peptide), or residues 111-141 (as described above). A fragment (e.g., a peptide or an epitope) of a CXCL1 protein is less than the length of the full length protein, e.g., a fragment is at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200 or more residues in length, but less than e.g., 397 residues in the case of CXCL1 above.
pHLIP Peptides
[0146] In the schematic structure, Epitope-Linker-Peptide:
[0147] Peptide is a pHLIP peptide (non-limiting example is pHLIP comprising the Var3 sequence AXDDQNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: ______) or AXDQDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: ______), where X is a functional group for conjugation purposes, selected from a lysine (Lys), a cysteine (Cys), or Azido-containing amino acid or others. The membrane non-inserting N-terminal flanking sequence of pHLIP peptide can be extend. For example, the pHLIP peptide shares the sequence:
TABLE-US-00027 (SEQIDNO:112) WRAYLDLLFPTDTLLLDLLW.
[0148] Non-limiting examples of the extension can be poly-Gly motif or AX(Z).sub.nXPWRAYLDLLFPTDTLLLDLLWA, where Z indicates any amino acid residue, and n is any integer between and including 1-10 (e.g., 1n10). Additionally, (Z).sub.n could be QDNDQN (SEQ ID NO: ______) or NENENN (SEQ ID NO: 528) or NDNDNN (SEQ ID NO: 529) or NDNDNDN (SEQ ID NO:530), any combination of polar residues. D, E, N or Q.
[0149] An example of a wild type (WT) pHLIP peptide is AXEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT (SEQ ID NO: 20) where X is a functional group for conjugation purposes, selected from a lysine (Lys), a cysteine (Cys), an Azido-containing amino acid, or others, and in which AEQNPIY (SEQ ID NO: ______) represents a flanking sequence, WARYADWLFTTPLLLLDLALLV (SEQ ID NO: 21) represents a membrane-inserting sequence, and DADEGT represents a flanking sequence.
[0150] The constructs may include a pHLIP peptide with a N-terminal extension. For example, the N terminus of any of these peptide sequences can be extended by the addition of amino acids to space the epitope away from the cell surface, e.g. by including a (glycine) extension.
[0151] Other exemplary pHLIP peptides are shown in the Tables below.
TABLE-US-00028 TABLE1 ExemplarypHLIPpeptides Name Sequence SEQIDNo. Var3-1a ACDQDNPWRAYLDLLFPTDTLLLDLLWA SEQ.IDNO. Var3-1b AKDQDNPWRAYLDLLFPTDTLLLDLLWA SEQ.IDNO. Var3-2a ACQDNDQNCPWRAYLDLLFPTDTLLLDLLWA SEQ.IDNO. Var3-2b AKQDNDQNKPWRAYLDLLFPTDTLLLDLLWA SEQ.IDNO. WT-1 GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT SEQ.IDNO.22 WT-2 AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT SEQ.IDNO.23 Var3-WT-Cys ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG SEQ.INO.24 Cys-Var3-WT ACDDQNPWRAYLDLLFPTDTLLLDLLWDADEG SEQ.INO.25 Lys-Var3-WT AKDDQNPWRAYLDLLEPTDTELLDLLWDADEG SEQ.INO.26 WT-Cys1 AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG SEQ.IDNO.27 WT-Cys2 Ac-AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGCT SEQIDNO:28 WT-Cys3 GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG SEQ.IDNO.29 Cys-WT1 Ac-ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG SEQ.IDNO.30 Var0-NT ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT SEQ.IDNO.31 Lys-WT1 AKEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT SEQ.IDNO.32 Lys-WT2 Ac-AKEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG SEQIDNO:33 WT-KC AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG SEQ.IDNO.34 K-WT-C AKEQNPIYWARYADWLFTTPLLLLDLALLVDADECT SEQ.IDNO.35 N-pHLIP ACEQNPIYWARYANWLFTTPLLLLNLALLVDADEGTG SEQ.IDNO.36 N-pHLIP-b ACEQNPIYWARYANWLFTTPLLLLNLALLVDADEGT SEQIDNO:37 K-pHLIP ACEQNPIYWARYAKWLFTTPLLLLKLALLVDADEGTG SEQ.IDNO.38 NNQ GGEQNPIYWARYADWLFTTPLLLLDLALLVNANQGT SEQ.IDNO.39 D25A AAEQNPIYWARYADWLFTTPLLLLALALLVDADEGT SEQ.IDNO.40 D25A-KC Ac-AAEQNPIYWARYADWLFTTPLLLLELALLVDADEGTKCG SEQIDNO:41 D14A AAEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGT SEQ.IDNO.42 P20A AAEQNPIYWARYADWLFTTALLLLDLALLVDADEGT SEQ.IDNO.43 D25E AAEQNPIYWARYADWLFTTPLLLLELALLVDADEGT SEQ.IDNO.44 D14E AAEQNPIYWARYAEWLFTTPLLLLDLALLVDADEGT SEQ.IDNO.45 3D AAEQNPIIYWARYADWLFTDLPLLLLDLLALLVDADEGT SEQ.IDNO.46 R11Q GEQNPIYWAQYADWLFTTPLLLLDLALLVDADEGTCG SEQ.IDNO.47 D25Up GGEQNPIYWARYADWLFTTPLLLDLLALLVDADEGTCG SEQ.IDNO.48 D25Down GGEQNPIYWARYADWLFTTPLLLLLDALLVDADEGTCG SEQ.IDNO.49 D14Up GGEQNPIYWARYDAWLFTTPLLLLDLALLVDADEGTCG SEQ.IDNO.50 D14Down GGEQNPIYWARYAWDLFTTPLLLLDLALLVDADEGTCG SEQ.IDNO.51 P20G AAEQNPIYWARYADWLFTTGLLLLDLALLVDADEGT SEQ.IDNO.52 H1-Cys DDDEDNPIYWARYADWLFTTPLLLLHGALLVDADECT SEQ.IDNO.53 H1 DDDEDNPIYWARYADWLFTTPLLLLHGALLVDADET SEQIDNO:54 H2-Cys DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADEGCT SEQ.IDNO.55 Cys-H2 CDDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADET SEQIDNO:56 H2 DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADEGT SEQIDNO:57 H2N-Cys DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT SEQ.IDNO.58 H2N DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADEGT SEQIDNO:59 H2N2-Cys DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANECT SEQ.IDNO.60 H2N2 DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANEGT SEQIDNO:61 1a-Trp AEQNPIYWARYADFLFTTPLLLLDLALLVDADET SEQ.IDNO.62 1b-Trp AEQNPIYFARYADWLFTTPLLLLDLALLVDADEGT SEQ.IDNO.63 1c-Trp AEQNPIYFARYADFLFTTPLLLLDLALLWDADET SEQ.IDNO.64 Fast-1orVar1 AKEDQNPYWARYADWLFTTPLLLLDLALLVDG SEQ.IDNO.65 Var1-2D1D ACEDQNPYWARYADWLFTTPLLLLDLALLVDG SEQ.IDNO.66 Fast1-Cysor AEDQNPYWARYADWLFTTPLLLLDLALLVDCG SEQ.IDNO.67 Var1-2D1D-Cys Fast1-E-Cysor AEDQNPYWARYADWLFTTPLLLLELALLVECG SEQ.IDNO.68 Var1E Fast1-E-Lys AKEDQNDPYWARYADWLFTTPLLLLDLALLVG SEQIDNO:69 Fast2orVar2 AKEDQNPYWRAYADLFTPLTLLDLLALWDG SEQ.IDNO.70 Fast2-E-Cysor AEDQNPYWARYADWLFTTPLLLLELALLVCG SEQIDNO:71 Var2E Var2-2D1D ACEDQNPYWRAYADLFTPLTLLDLLALWDG SEQ.IDNO.72 Var3-3D ACDDQNPWRAYLDLLFPTDTLLLDLLW SEQ.IDNO.73 Var3-3D-cys AKDDQNPWRAYLDLLFPTDTLLLDLLWC SEQIDNO:74 Var4-3E ACEEQNPWRAYLELLFPTETLLLELLW SEQIDNO:75 Var5-3Da ACDDQNPWARYLDWLFPTDTLLLDL SEQIDNO:76 Var6-3Db CDNNNPWRAYLDLLFPTDTLLLDW SEQIDNO:77 Var8-3Eb CEEQQPWAQYLELLFPTETLLLEW SEQIDNO:78 Var9-3Ec CEEQQPWRAYLELLFPTETLLLEW SEQIDNO:79 Var15-2N CDDDDDNPNYWARYANWLFTTPLLLLNGALLVEAEET SEQIDNO:80 Var16-2P CDDDDDNPNYWARYAPWLFTTPLLLLPGALLVEAEE SEQIDNO:81
TABLE-US-00029 TABLE2 ExemplarypHLIPpeptides Name Sequence SEQIDNo. Var14- Ac-TEDADVLLALDLLLLPTTFLW SEQ.IDNO.82 Rev DAYRAWYPNQECA-Am Sh AEQNPIYWARYADWLFTTPL SEQ.IDNO.83 Sh-Cys AEQNPIYWARYADWLFTTPCL SEQ.IDNO.84 Cys-Sh ACEQNPIYWARYADWLFTTPL SEQ.IDNO.85 Sh-1Trp AEQNPIYFARYADWLFTTPL SEQ.IDNO.86 Sh-W2 AEQNPIYFARYADLLFPTTLAW SEQIDNO:87 Sh-W1 AEQNPIYWARYADLLFPTTLAF SEQIDNO:88 Sh-2W AEQNPIYWARYADLLFPTTLAW SEQIDNO:89 Sh-1D KEDQNPWARYADLLFPTTLAW SEQ.IDNO.90 Sh-1Db KEDQNPWARYADLLFPTTLW SEQIDNO:91 Var12-1D ACEDQNPWARYADLLFPTTLAW SEQ.IDNO.92 Var10-2D ACEDQNPWARYADWLFPTTLLLLD SEQ.IDNO.93 Var13-1E ACEEQNPWARYAELLFPTTLAW SEQ.IDNO.94 Var11-2E ACEEQNPWARYAEWLFPTTLLLLE SEQ.IDNO.95 Var7-3E ACEEQNPWARYLEWLFPTETLLLEL SEQ.IDNO.96 Var7-3Eb ACEEQNPQAEYAEWLFPTTLLLLE SEQIDNO:97 Acmeans Acetylated N-terminus Ammeans Amidated C-terminus
TABLE-US-00030 TABLE 3 Coded and exemplary non-coded amino acids including L-isomers, D- isomers, alpha-isomers, beta-isomers, glycol-, and methyl- modifications. No. Abbrev Name 1 Ala Alanine 2 Arg Arginine 3 Asn Asparagine 4 Asp Aspartic acid 5 Cys Cysteine 6 Gln Glutamine 7 Glu Glutamic acid 8 Gly Glycine 9 His Histidine 10 Ile Isoleucine 11 Leu Leucine 12 Lys Lysine 13 Met Methionine 14 Phe Phenylalanine 15 Pro Proline 16 Ser Serine 17 Thr Threonine 18 Trp Tryptophan 19 Tyr Tyrosine 20 Val Valine 21 Sec Selenocysteine 22 Sem Selenomethionine 23 Pyl Pyrrolysine 24 Aad Alpha-aminoadipic acid 25 Acpa Amino-caprylic acid 26 Aecys Aminoethyl cysteine 27 Afa Aminophenyl acetate 28 Gaba Gamma-aminobutyric acid 29 Aiba Aminoisobutyric acid 30 Aile Alloisoleucine 31 AIg Allylglycine 32 Aba Amino-butyric acid 33 Aphe Amino-phenylalanine 34 Brphe Bromo-phenylalanine 35 Cha Cyclo-hexylalanine 36 Cit Citrulline 37 Clala Chloroalanine 38 Cie Cycloleucine 39 Clphe Fenclonine (or chlorophenylalanine) 40 Cya Cysteic acid 41 Dab Diaminobutyric acid 42 Dap Diaminopropionic acid 43 Dap Diaminopimelic acid 44 Dhp Dehydro-proline 45 Dhphe DOPA (or 3,4-dihydroxyphenylalanine) 46 Fphe Fluorophenylalanine 47 Gaa Glucosaminic acid 48 Gla Gamma-carboxyglutamic acid 49 Hag Homoarginine 50 Hlys Hydroxylysine 51 Hnvl Hydroxynorvaline 52 Hog Homoglutamine 53 Hoph Homophenylalanine 54 Has Homoserine 55 Hse Homocysteine 56 Hpr Hydroxyproline 57 Iphe Iodo-phenylalanine 58 Ise Isoserine 59 Mle Methyl-leucine 60 Msmet Methionine-methylsulfonium chloride 61 Nala Naphthyl-alanine 62 Nle Norleucine (or 2-aminohexanoic acid) 63 Nmala N-methyl-alanine 64 Nva Norvaline (or 2-aminopentanoic acid) 65 Obser O-benzyl-serine 66 Obtyr O-benzyl-tyrosine 67 Oetyr O-ethyl-tyrosine 68 Omser O-methyl-serine 69 Omthr O-methy-threonine 70 Omtyr O-methyl-tyrosine 71 Orn Ornithine 72 Pen Penicillamine 73 Pga Pyroglutamic acid 74 Pip Pipecolic acid 75 Sar Sarcosine 76 Tfa Trifluoro-alanine 77 Thphe Hydroxy-Dopa 78 Vig Vinylglycine 79 Aaspa Amino-aminoethylsulfanylpropanoic acid 80 Ahdna Amino-hydroxy-dioxanonanolic acid 81 Ahoha Amino-hydroxy-oxahexanoic acid 82 Ahsopa Amino-hydroxyethylsulfanylpropanoic acid 83 Tyr(Me) Methoxyphenyl-methylpropanyl oxycarbonylamino propanoic acid 84 MTrp Methyl-tryptophan 85 pTyr Phosphorylated Tyr 86 pSer Phosphorylated Ser 87 pThr Phosphorylated Thr 88 BLys BiotinLys 89 Hyp Hydroproline 90 Phg Phenylglycine 91 Cha Cyclohexyl-alanine 92 Chg Cyclohexylglycine 93 Nal Naphthylalanine 94 Pal Pyridyl-alanine 95 Pra Propargylglycine 96 Gly(allyl) Pentenoic acid 97 Pen Penicillamine 98 MetO Methionine sulfoxide 99 Pca Pyroglutamic acid 100 Ac-Lys Acetylation of Lys
TABLE-US-00031 TABLE 4 Non-limiting examples of protonatable residues and their substitutions including L-isomers, D- isomers, alpha-isomers, and beta-isomers. Original Residue Exemplary amino acids substitution Asp (D) Glu (E); Gla (Gla); Aad (Aad) Glu (E) Asp (D); Gla (Gla); Aad (Aad)
TABLE-US-00032 TABLE 5 Examples of coded amino acid substitutions Original Residue Substitution Ala (A) Gly; Ile; Leu; Met; Phe; Pro; Trp; Tyr; Val Arg (R) Lys Asn (N) Gln; His Asp (D) Glu Cys (C) Ser; Met Gln (Q) Asn; His Glu (E) Asp Gly (G) Ala; Ile; Leu; Met; Phe; Pro; Trp; Tyr; Val His (H) Asn; Gln Ile (I) Ala; Gly; Leu; Met; Phe; Pro; Trp; Tyr; Val Leu (L) Ala; Gly; Ile; Met; Phe; Pro; Trp; Tyr; Val Lys (K) Arg Met (M) Ala; Gly; Leu; Ile; Phe; Pro; Trp; Tyr; Val Phe (F) Ala; Gly; Leu; Ile; Met; Pro; Trp; Tyr; Val Pro (P) Ala; Gly; Leu; Ile; Met; Phe; Trp; Tyr; Val Ser (S) Thr Thr (T) Ser Trp (W) Ala; Gly; Leu; Ile; Met; Pro; Phe; Tyr; Val Tyr (Y) Ala; Gly; Leu; Ile; Met; Pro; Phe; Trp; Val Val (V) Ala; Gly; Leu; Ile; Met; Pro; Phe; Trp; Tyr
TABLE-US-00033 TABLE6 Non-limitingexamplesofmembrane-inserting sequencesbelongingtodifferentgroupsof pHLIPpeptides.Eachprotonatableresidue (showninbold)couldbereplacedbyits substitutionfromTable4.Eachnon-polar residuecouldbereplacedbyitscodedamino acidsubstitutionfromTable5,and/ornon- codedaminoacidsubstitutionsfromTable3. Groups Sequences WT-BRC WARYADWLFTTPLLLLDLALL(SEQIDNO:98) YARYADWLFTTPLLLLDLALL(SEQIDNO:99) WARYSDWLFTTPLLLYDLGLL(SEQIDNO:100) WARYTDWFTTPLLLYDLALLA(SEQIDNO:101) WARYTDWLFTTPLLLYDLGLL(SEQIDNO:102) WARYADWLFTTPLLLLDLSLL(SEQIDNO:103) WT-BRC LLALDLLLLPTTFLWDAYRAW(SEQIDNO:104) Reverse LLALDLLLLPTTFLWDAYRAY(SEQIDNO:105) LLGLDYLLLPTTFLWDSYRAW(SEQIDNO:106) ALLALDYLLLPTTFWDTYRAW(SEQIDNO:107) LLGLDYLLLPTTFLWDTYRAW(SEQIDNO:108) LLSLDLLLLPTTFLWDAYRAW(SEQIDNO:109) ATRAM GLAGLLGLEGLLGLPLGLLEGLWLGL(SEQID NO:110) ATRAM LGLWLGELLGLPLGLLGELGLLGALG(SEQID Reverse NO:111) Var3 WRAYLDLLFPTDTLLLDLLW(SEQIDNO:112) Var3 WLLDLLLTDTPFLLDLYARW(SEQIDNO:113) Reverse Var7 WARYLEWLFPTETLLLEL(SEQIDNO:114) WAQYLELLFPTETLLLEW(SEQIDNO:115) Var7 LELLLTETPFLWELYRAW(SEQIDNO:116) Reverse WELLLTETPFLLELYQAW(SEQIDNO:117) Single WLFTTPLLLLNGALLVE(SEQIDNO:118) D/E WLFTTPLLLLPGALLVE(SEQIDNO:119) WARYADLLFPTTLAW(SEQIDNO:120) Single EVLLAGNLLLLPTTFLW(SEQIDNO:121) D/E EVLLAGPLLLLPTTFLW(SEQIDNO:122) Reverse WALTTPFLLDAYRAW(SEQIDNO:123) pHLIP- NLEGFFATLGGEIALWSLVVLAIE(SEQIDNO:124) Rho EGFFATLGGEIALWSDVVLAIE(SEQIDNO:125) EGFFATLGGEIPLWSDVVLAIE(SEQIDNO:126) pHLIP- EIALVVLSWLAIEGGLTAFFGELN(SEQIDNO:127) Rho EIALVVDSWLAIEGGLTAFFGE(SEQIDNO:128) Reverse EIALVVDSWLPIEGGLTAFFGE(SEQIDNO:129) pHLIP- ILDLVFGLLFAVTSVDFLVQW(SEQIDNO:130) CA9 pHLIP- WQVLFDVSTVAFLLGFVLDLI(SEQIDNO:131) CA9 Reverse
TABLE-US-00034 TABLE7 Non-limitingexamplesofpHLIPsequences.Acysteine,alysine, anazido-modifiedaminoacid,oranalkynylmodifiedaminoacid canbeincorporatedattheN-terminal(first6residues)orC- terminal(last6residues)partsofthepeptidesforconjugation withacargo,andalinker. SEQIDNO Name Sequence SEQIDNO:132 WT-2D AEQNPIYWARYADWLFTTPLLLLDLALLVDADET SEQIDNO:133 WT-6E AEQNPIYWARYAEWLFTTPLLLLELALLVEAEET SEQIDNO:134 WT-3D ADDQNPWRAYLDLLFPDTTDLLLLDLLWDADET SEQIDNO:135 WT-9E AEEQNPWRAYLELLFPETTELLLLELLWEAEET SEQIDNO:136 WT-GlaD AEQNPIYWARYAGlaWLFTTPLLLLDLALLVDADET SEQIDNO:137 WT-DGla AEQNPIYWARYADWLFTTPLLLLGlaLALLVDADET SEQIDNO:138 WT-2G1a AEQNPIYWARYAGlaWLFTTPLLLLGlaLALLVDADET SEQIDNO:139 WT-AadD AEQNPIYWARYAAadWLFTTPLLLLDLALLVDADET SEQIDNO:140 WT-DAad AEQNPIYWARYADWLFTTPLLLLAadLALLVDADET SEQIDNO:141 WT-2Aad AEQNPIYWARYAAadWLFTTPLLLLAadLALLVDADET SEQIDNO:142 WT-GlaAad AEQNPIYWARYAGlaWLFTTPLLLLAadLALLVDADET SEQIDNO:143 WT-AadGla AEQNPIYWARYAAadWLFTTPLLLLGlaLALLVDADET SEQIDNO:144 WT-1 GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT SEQIDNO:145 WT-2 GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT SEQIDNO:146 WT-3 AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT SEQIDNO:147 WT-4 AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT SEQIDNO:148 WT-2N AEQNPIYWARYANWLFTTPLLLLNLALLVDADEGT SEQIDNO:149 WT-2K AEQNPIYWARYAKWLFTTPLLLLKLALLVDADEGT SEQIDNO:150 WT-2DNANQ GGEQNPIYWARYADWLFTTPLLLLDLALLVNANQGT SEQIDNO:151 WT-D25A AAEQNPIYWARYADWLFTTPLLLLALALLVDADEGT SEQIDNO:152 WT-D14A AAEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGT SEQIDNO:153 WT-P20A AAEQNPIYWARYADWLFTTALLLLDLALLVDADEGT SEQIDNO:154 WT-D25E AAEQNPIYWARYADWLFTTPLLLLELALLVDADEGT SEQIDNO:155 WT-D14E AAEQNPIYWARYAEWLFTTPLLLLDLALLVDADEGT SEQIDNO:156 WT-3D-2 AAEQNPIIYWARYADWLFTDLPLLLLDLLALLVDADEGT SEQIDNO:157 WT-R11Q GEQNPIYWAQYADWLFTTPLLLLDLALLVDADEG SEQIDNO:158 WT-D25Up GGEQNPIYWARYADWLFTTPLLLDLLALLVDADEG SEQIDNO:159 WT-D25Down GGEQNPIYWARYADWLFTTPLLLLLDALLVDADEG SEQIDNO:160 WT-D14Up GGEQNPIYWARYDAWLFTTPLLLLDLALLVDADEGT SEQIDNO:161 WT-D14Down GGEQNPIYWARYAWDLFTTPLLLLDLALLVDADEG SEQIDNO:162 WT-P20G AAEQNPIYWARYADWLFTTGLLLLDLALLVDADEGT SEQIDNO:163 WT-DH DDDEDNPIYWARYADWLFTTPLLLLHGALLVDAD SEQIDNO:164 WT-2H DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADE SEQIDNO:165 WT-L16H CEQNPIYWARYADWHFTTPLLLLDLALLVDADE SEQIDNO:166 WT-1Wa AEQNPIYWARYADFLFTTPLLLLDLALLVDADET SEQIDNO:167 WT-1Wb AEQNPIYFARYADWLFTTPLLLLDLALLVDADE SEQIDNO:168 WT-1Wc AEQNPIYFARYADFLFTTPLLLLDLALLWDADET SEQIDNO:169 WT-W6 ADNNPWIYARYADLTTFPLLLLDLALLVDFDD SEQIDNO:170 WT-W17 ADNNPFIYARYADLTTWPLLLLDLALLVDFDD SEQIDNO:171 WT-W30 ADNNPFIYARYADLTTFPLLLLDLALLVDWDD SEQIDNO:172 WT-W17-P7 ADNNPFPYARYADLTTWILLLLDLALLVDFDD SEQIDNO:173 WT-W39-R11 ADNNPFIYAYRADLTTFPLLLLDLALLVDWDD SEQIDNO:174 WT-W30-R15 ADNNPFIYATYADLRTFPLLLLDLALLVDWDD SEQIDNO:175 WT-Rev Ac-TEDADVLLALDLLLLPTTFLWDAYRAWYPNQEA-Am SEQIDNO:176 Var1-3D AEDQNPYWARYADWLFTTPLLLLDLALLVD SEQIDNO:177 Var1-1D2E AEDQNPYWARYADWLFTTPLLLLELALLVE SEQIDNO:178 Var2-3D AEDQNPYWRAYADLFTPLTLLDLLALWD SEQIDNO:179 Var3-3D ADDQNPWRAYLDLLFPTDTLLLDLLW SEQIDNO:180 Var3-WT ADDQNPWRAYLDLLFPTDTLLLDLLWDADE SEQIDNO:181 Var3-Gla2D ADDQNPWRAYLGlaLLFPTDTLLLDLLW SEQIDNO:182 Var3-DGlaD ADDQNPWRAYLDLLFPTGlaTLLLDLLW SEQIDNO:183 Var3-2DGla ADDQNPWRAYLDLLFPTDTLLLGlaLLW SEQIDNO:184 Var3-2GlaD ADDQNPWRAYLGlaLLFPTGlaTLLLDLLW SEQIDNO:185 Var3-GlaDGla ADDQNPWRAYLGlaLLFPTDTLLLGlaLLW SEQIDNO:186 Var3-D2Gla ADDQNPWRAYLDLLFPTGlaTLLLGlaLLW SEQIDNO:187 Var3-3Gla ADDQNPWRAYLGlaLLFPTGlaTLLLGlaLLW SEQIDNO:188 Var3-Aad2D ADDQNPWRAYLAadLLFPTDTLLLDLLW SEQIDNO:189 Var3-DAadD ADDQNPWRAYLDLLFPTAadTLLLDLLW SEQIDNO:190 Var3-2DAad ADDQNPWRAYLDLLFPTDTLLLAadLLW SEQIDNO:191 Var3-2AadD ADDQNPWRAYLAadLLFPTAadTLLLDLLW SEQIDNO:191 Var3-AadDAad ADDQNPWRAYLAadLLFPTDTLLLAadLLW SEQIDNO:192 Var3-D2Aad ADDQNPWRAYLDLLFPTAadTLLLAadLLW SEQIDNO:193 Var3-3Aad ADDQNPWRAYLAadLLFPTAadTLLLAadLLW SEQIDNO:194 Var3-GlaAadD ADDQNPWRAYLGlaLLFPTAadTLLLDLLW SEQIDNO:195 Var3-GlaDAad ADDQNPWRAYLGlaLLFPTDTLLLAadLLW SEQIDNO:196 Var3-2GlaAad ADDQNPWRAYL LLFPT
TLLL
LLW SEQIDNO:197 Var3-AadGlaD ADDQNPWRAYL
LLFPT
TLLLDLLW SEQIDNO:198 Var3-AadDGla ADDQNPWRAYL
LLFPTDTLLL
LLW SEQIDNO:199 Var3-GlaAadGla ADDQNPWRAYL
LLFPT
TLLL
LLW SEQIDNO:200 Var3-GLL GEEQNPWLGAYLDLLFPLELLGLLELGLW SEQIDNO:201 Var3-M ADDDDDDPWQAYLDLLFPTDTLLLDLLW SEQIDNO:202 Var4-3E AEEQNPWRAYLELLFPTETLLLELLW SEQIDNO:203 Var5-3Da ADDQNPWARYLDWLFPTDTLLLDL SEQIDNO:204 Var6-3Db DNNNPWRAYLDLLFPTDTLLLDW SEQIDNO:205 Var7-3E AEEQNPWARYLEWLFPTETLLLEL SEQIDNO:206 Var7-M DDDDDDPWQAYLDLFPTDTLALDLW SEQIDNO:207 Var8-3E EEQQPWAQYLELLFPTETLLLEW SEQIDNO:208 Var9-3E EEQQPWRAYLELLFPTETLLLEW SEQIDNO:209 Var10-2D AEDQNPWARYADWLFPTTLLLLD SEQIDNO:210 Var11-2E AEEQNPWARYAEWLFPTTLLLLE SEQIDNO:211 Var12-1D AEDQNPWARYADLLFPTTLAW SEQIDNO:212 Var13-1E AEEQNPWARYAELLFPTTLAW SEQIDNO:213 Var15-2N DDDDDNPNYWARYANWLFTTPLLLLNGALLVEAEET SEQIDNO:214 Var16-2P DDDDDNPNYWARYAPWLFTTPLLLLPGALLVEAEET SEQIDNO:215 Var17 AEQNPIYFARYADFLFTTPLLLLDLALLWDADET SEQIDNO:216 Var18 AEQNPIYWARYADFLFTTPLLLLDLALLVDADET SEQIDNO:217 Var19a AEQNPIYWARYADWLFTTPL SEQIDNO:218 Var20 AEQNPIYFARYADLLFPTTLAW SEQIDNO:219 Var21 AEQNPIYWARYADLLFPTTLAF SEQIDNO:220 Var22 AEQNPIYWARYADLLFPTTLAW SEQIDNO:221 Var23 AEQNPIYFARYADWLFTTPL SEQIDNO:222 Var24 EDQNPWARYADLLFPTTLAW SEQIDNO:223 ATRAM GLAGLAGLLGLEGLLGLPLGLLEGLWLGLELEGN SEQIDNO:224 pHLIP-CA9 EQNPIYILDLVFGLLFAVTSVDFLVQWDDAGD SEQIDNO:225 pHLIP-Rho NLEGFFATLGGEIALWSLVVLAIE SEQIDNO:226 pHLIP-RhoM1 NNEGFFATLGGEIALWSDVVLAIE SEQIDNO:227 pHLIP-RhoM2 DNNEGFFATLGGEIPLWSDVVLAIE
[0152] Epitopes may also be delivered to the cell surface of target cells (tumor cells and other diseased tissues/cells) using cyclic pHLIP peptides. A cyclic peptide is one that comprises a circle geometry or structure. For example, the entire structure of the peptide is circular or a portion of the structure is circular. For example, in the latter case the peptide comprises a cyclic portion and a linear (or tail) portion. In various embodiments, a pH triggered peptide comprises at least 4 amino acids, where (a) at least 2 of the at least 4 amino acids of the peptide are non-polar amino acids, (b) at least 1 of the at least 4 amino acids of the peptide is a protonatable amino acid, and (c) the peptide has a higher affinity to a membrane lipid bilayer at pH 5.0 compared to at pH 8.0. Such pHLIP peptides are described in International Patent Application No. PCT/US2017/023458 (PCT publication no. WO2017/165452A1, hereby incorporated by reference.
[0153] Exemplary cyclic pHLIP peptides are described and shown below. A lowercase c at the beginning of a sequence herein denotes a cyclic peptide (e.g., as in c[WE).sub.3WC]) (SEQ ID NO: 1), and a lowercase l denotes a linear peptide (e.g., as in l(CW(EW).sub.4)) (SEQ ID NO: 188). In the case of cyclic structures that comprise a tail, the cyclic portion of the compound is within brackets, and the tail portion follows (is to the right of) the brackets. For example, in the compound c[E.sub.5K]W.sub.5C, c[E.sub.5K] is the cyclic peptide portion, and W.sub.5C is the peptide tail portion. As another example, in c[E.sub.5K]W.sub.4C, the cyclic peptide portion is c[E.sub.5K] and the peptide tail portion is W.sub.4C.
[0154] With respect to cyclic peptides, the amino acids within brackets may be present in the order listed in brackets from left to right, or in any order. For example, a cyclic peptide c[X.sub.2Y.sub.2] may have the corresponding linear sequence: XXYY, XYXY, YXXY, XYYX, or YXYX. In some cases, multiple examples of corresponding linear sequences for an exemplary cyclic peptide are listed in Table 3.
Table 8 provides a summary of peptide sequences.
TABLE-US-00035 Peptide Sequence LinearSequence SEQIDNO 1 c[(WE).sub.3WC] WEWEWEWC 228 2 c[(WE).sub.4WC] WEWEWEWEWC 229 3 c[(WE).sub.5WC] WEWEWEWEWEWC 230 4 c[(LE).sub.4WC] LELELELEWC 231 5 c[E.sub.4W.sub.5C] EEEEWWWWWC 232 6 l(CW(EW).sub.4) CWEWEWEWEW 233 7 c[R.sub.4W.sub.5C] RRRRWWWWWC 234
[0155] In column 2 (Sequence, the lower case c indicates circular peptide, and the lower case l indicated linear peptide.
Table 9 provides additional non-limiting examples of peptide sequences.
TABLE-US-00036 Cy- clic Pep- Circular Linear tide Sequence Sequence SEQIDNO 1 c[E.sub.3W.sub.5C] EEEWWWWWC 235 2 c[E.sub.3W.sub.5C] EWEWWWWEC 236 3 c[E.sub.3W.sub.5C] EWWEWWWEC 237 4 c[E.sub.3W.sub.5C] EWWWEWWEC 238 5 c[E.sub.3W.sub.5C] EWWWWEWEC 239 6 c[E.sub.3W.sub.5C] EWWWWWEEC 240 7 c[E.sub.3W.sub.5C] EWEEWWWWC 241 8 c[E.sub.3W.sub.5C] EWWEEWWWC 242 9 c[E.sub.3W.sub.5C] EWWWEEWWC 243 10 c[E.sub.3W.sub.5C] EWWWWEEWC 244 11 c[E.sub.3W.sub.5C] WEEEWWWWC 245 12 c[E.sub.3W.sub.5C] WWEEEWWWC 246 13 c[E.sub.3W.sub.5C] WWWEEEWWC 247 14 c[E.sub.3W.sub.5C] WWWWEEEWC 248 15 c[E.sub.3W.sub.5C] WEWEEWWWC 249 16 c[E.sub.3W.sub.5C] WEWWEEWWC 250 17 c[E.sub.3W.sub.5C] WEWWWEEWC 251 18 c[E.sub.3W.sub.5C] WEWWWWEEC 252 19 c[E.sub.3W.sub.5] EEEWWWWW 253 20 c[E.sub.3W.sub.5] EWEWWWWE 254 21 c[E.sub.3W.sub.5] EWWEWWWE 255 22 c[E.sub.3W.sub.5] EWWWEWWE 256 23 c[E.sub.3W.sub.5] EWWWWEWE 257 24 c[E.sub.3W.sub.5] EWWWWWEE 258 25 c[E.sub.3W.sub.5] EWEEWWWW 259 26 c[E.sub.3W.sub.5] EWWEEWWW 260 27 c[E.sub.3W.sub.5] EWWWEEWW 261 28 c[E.sub.3W.sub.5] EWWWWEEW 262 29 c[E.sub.3W.sub.5] WEEEWWWW 263 30 c[E.sub.3W.sub.5] WWEEEWWW 264 31 c[E.sub.3W.sub.5] WWWEEEWW 265 32 c[E.sub.3W.sub.5] WWWWEEEW 266 33 c[E.sub.3W.sub.5] WEWEEWWW 267 34 c[E.sub.3W.sub.5] WEWWEEWW 268 35 c[E.sub.3W.sub.5] WEWWWEEW 269 36 c[E.sub.3W.sub.5] WEWWWWEE 270 37 c[D.sub.3W.sub.5C] DDDWWWWWC 271 38 c[D.sub.3W.sub.5C] DWDWWWWDC 272 39 c[D.sub.3W.sub.5C] DWWDWWWDC 273 40 c[D.sub.3W.sub.5C] DWWWDWWDC 274 41 c[D.sub.3W.sub.5C] DWWWWDWDC 275 42 c[D.sub.3W.sub.5C] DWWWWWDDC 276 43 c[D.sub.3W.sub.5C] DWDDWWWWC 277 44 c[D.sub.3W.sub.5C] DWWDDWWWC 278 45 c[D.sub.3W.sub.5C] DWWWDDWWC 279 46 c[D.sub.3W.sub.5C] DWWWWDDWC 280 47 c[D.sub.3W.sub.5C] WDDDWWWWC 281 48 c[D.sub.3W.sub.5C] WWDDDWWWC 282 49 c[D.sub.3W.sub.5C] WWWDDDWWC 283 50 c[D.sub.3W.sub.5C] WWWWDDDWC 284 51 c[D.sub.3W.sub.5C] WDWDDWWWC 285 52 c[D.sub.3W.sub.5C] WDWWDDWWC 286 53 c[D.sub.3W.sub.5C] WDWWWDDWC 287 54 c[D.sub.3W.sub.5C] WDWWWWDDC 288 55 c[D.sub.3W.sub.5] DDDWWWWW 289 56 c[D.sub.3W.sub.5] DWDWWWWD 290 57 c[D.sub.3W.sub.5] DWWDWWWD 291 58 c[D.sub.3W.sub.5] DWWWDWWD 292 59 c[D.sub.3W.sub.5] DWWWWDWD 293 60 c[D.sub.3W.sub.5] DWWWWWDD 294 61 c[D.sub.3W.sub.5] DWDDWWWW 295 62 c[D.sub.3W.sub.5] DWWDDWWW 296 63 c[D.sub.3W.sub.5] DWWWDDWW 297 64 c[D.sub.3W.sub.5] DWWWWDDW 298 65 c[D.sub.3W.sub.5] WDDDWWWW 299 66 c[D.sub.3W.sub.5] WWDDDWWW 300 67 c[D.sub.3W.sub.5] WWWDDDWW 301 68 c[D.sub.3W.sub.5] WWWWDDDW 302 69 c[D.sub.3W.sub.5] WDWDDWWW 303 70 c[D.sub.3W.sub.5] WDWWDDWW 304 71 c[D.sub.3W.sub.5] WDWWWDDW 305 72 c[D.sub.3W.sub.5] WDWWWWDD 306 73 c[Gla.sub.3W.sub.5] GlaGlaGlaWWWWW 307 74 c[Gla.sub.3W.sub.5] GlaWGlaWWWWGla 308 75 c[Gla.sub.3W.sub.5] GlaWWGlaWWWGla 309 76 c[Gla.sub.3W.sub.5] GlaWWWGlaWWGla 310 77 c[Gla.sub.3W.sub.5] GlaWWWWGlaWGla 311 78 c[Gla.sub.3W.sub.5] GlaWWWWWGlaGla 312 79 c[Gla.sub.3W.sub.5] GlaWGlaGlaWWWW 313 80 c[Gla.sub.3W.sub.5] GlaWWGlaGlaWWW 314 81 c[Gla.sub.3W.sub.5] GlaWWWGlaGlaWW 315 82 c[Gla.sub.3W.sub.5] GlaWWWWGlaGlaW 316 83 c[Gla.sub.3W.sub.5] WGlaGlaGlaWWWW 317 84 c[Gla.sub.3W.sub.5] WWGlaGlaGlaWWW 318 85 c[Gla.sub.3W.sub.5] WWWGlaGlaGlaWW 319 86 c[Gla.sub.3W.sub.5] WWWWGlaGlaGlaW 320 87 c[Gla.sub.3W.sub.5] WGlaWGlaGlaWWW 321 88 c[Gla.sub.3W.sub.5] WGlaWWGlaGlaWW 322 89 c[Gla.sub.3W.sub.5] WGlaWWWGlaGlaW 323 90 c[Gla.sub.3W.sub.5] WGlaWWWWGlaGla 324 91 c[E.sub.3W.sub.4C] EEEWWWWC 325 92 c[E.sub.3W.sub.4C] EWEWWWEC 326 93 c[E.sub.3W.sub.4C] EWWEWWEC 327 94 c[E.sub.3W.sub.4C] EWWWEWEC 328 95 c[E.sub.3W.sub.4C] EWWWWEEC 329 96 c[E.sub.3W.sub.4C] EWEEWWWC 330 97 c[E.sub.3W.sub.4C] EWWEEWWC 331 98 c[E.sub.3W.sub.4C] EWWWEEWC 332 99 c[E.sub.3W.sub.4C] EWWWWEEC 333 100 c[E.sub.3W.sub.4C] WEEEWWWC 334 101 c[E.sub.3W.sub.4C] WWEEEWWC 335 102 c[E.sub.3W.sub.4C] WWWEEEWC 336 103 c[E.sub.3W.sub.4C] WWWWEEEC 337 104 c[E.sub.3W.sub.4C] WEWEEWWC 338 105 c[E.sub.3W.sub.4C] WEWWEEWC 339 106 c[E.sub.3W.sub.4C] WEWWWEEC 340 107 c[E.sub.3W.sub.4] EEEWWWW 341 108 c[E.sub.3W.sub.4] EWEWWWE 342 119 c[E.sub.3W.sub.4] EWWEWWE 343 110 c[E.sub.3W.sub.4] EWWWEWE 344 111 c[E.sub.3W.sub.4] EWWWWEE 345 112 c[E.sub.3W.sub.4] EWEEWWW 346 113 c[E.sub.3W.sub.4] EWWEEWW 347 114 c[E.sub.3W.sub.4] EWWWEEW 348 115 c[E.sub.3W.sub.4] EWWWWEE 349 116 c[E.sub.3W.sub.4] WEEEWWW 350 117 c[E.sub.3W.sub.4] WWEEEWW 351 118 c[E.sub.3W.sub.4] WWWEEEW 352 119 c[E.sub.3W.sub.4] WWWWEEE 353 120 c[E.sub.3W.sub.4] WEWEEWW 354 121 c[E.sub.3W.sub.4] WEWWEEW 355 122 c[E.sub.3W.sub.4] WEWWWEE 356 123 c[D.sub.3W.sub.4C] DDDWWWWC 357 124 c[D.sub.3W.sub.4C] DWDWWWDC 358 125 c[D.sub.3W.sub.4C] DWWDWWDC 359 126 c[D.sub.3W.sub.4C] DWWWDWDC 360 127 c[D.sub.3W.sub.4C] DWWWWDDC 361 128 c[D.sub.3W.sub.4C] DWDDWWWC 362 129 c[D.sub.3W.sub.4C] DWWDDWWC 363 130 c[D.sub.3W.sub.4C] DWWWDDWC 364 131 c[D.sub.3W.sub.4C] DWWWWDDC 365 132 c[D.sub.3W.sub.4C] WDDDWWWC 366 133 c[D.sub.3W.sub.4C] WWDDDWWC 367 134 c[D.sub.3W.sub.4C] WWWDDDWC 368 135 c[D.sub.3W.sub.4C] WWWWDDDC 369 136 c[D.sub.3W.sub.4C] WDWDDWWC 370 137 c[D.sub.3W.sub.4C] WDWWDDWC 371 138 c[D.sub.3W.sub.4C] WDWWWDDC 372 139 c[D.sub.3W.sub.4] DDDWWWW 373 140 c[D.sub.3W.sub.4] DWDWWWD 374 141 c[D.sub.3W.sub.4] DWWDWWD 375 142 c[D.sub.3W.sub.4] DWWWDWD 376 143 c[D.sub.3W.sub.4] DWWWWDD 377 144 c[D.sub.3W.sub.4] DWDDWWW 378 145 c[D.sub.3W.sub.4] DWWDDWW 379 146 c[D.sub.3W.sub.4] DWWWDDW 380 147 c[D.sub.3W.sub.4] DWWWWDD 381 148 c[D.sub.3W.sub.4] WDDDWWW 382 149 c[D.sub.3W.sub.4] WWDDDWW 383 150 c[D.sub.3W.sub.4] WWWDDDW 385 151 c[D.sub.3W.sub.4] WWWWDDD 386 152 c[D.sub.3W.sub.4] WDWDDWW 387 153 c[D.sub.3W.sub.4] WDWWDDW 388 154 c[D.sub.3W.sub.4] WDWWWDD 399 155 c[Gla.sub.3W.sub.4] GlaGlaGlaWWWW 400 156 c[Gla.sub.3W.sub.4] GlaWGlaWWWGla 401 157 c[Gla.sub.3W.sub.4] GlaWWGlaWWGla 402 158 c[Gla.sub.3W.sub.4] GlaWWWGlaWGla 403 159 c[Gla.sub.3W.sub.4] GlaWWWWGlaGla 404 160 c[Gla.sub.3W.sub.4] GlaWGlaGlaWWW 405 161 c[Gla.sub.3W.sub.4] GlaWWGlaGlaWW 406 162 c[Gla.sub.3W.sub.4] GlaWWWGlaGlaW 407 163 c[Gla.sub.3W.sub.4] GlaWWWWGlaGla 408 164 c[Gla.sub.3W.sub.4] WGlaGlaGlaWWW 409 165 c[Gla.sub.3W.sub.4] WWGlaGlaGlaWW 410 166 c[Gla.sub.3W.sub.4] WWWGlaGlaGlaW 411 167 c[Gla.sub.3W.sub.4] WWWWGlaGlaGla 412 168 c[Gla.sub.3W.sub.4] WGlaWGlaGlaWW 413 169 c[Gla.sub.3W.sub.4] WGlaWWGlaGlaW 414 170 c[Gla.sub.3W.sub.4] WGlaWWWGlaGla 415 171 c[(WE).sub.3WC] WEWEWEWC 416 172 c[(EW).sub.3WC] EWEWEWWC 417 173 c[(WD).sub.3WC] WDWDWDWC 418 174 c[(DW).sub.3WC] DWDWDWWC 419 175 c[(WGla).sub.3WC] WGlaWGlaWDWC 420 176 c[(GlaW).sub.3WC] DWDWDWDC 421 177 c[(WE).sub.4] WEWEWEWE 422 178 c[(EW).sub.4] EWEWEWEW 423 179 c[(WD).sub.4] WDWDWDWD 424 180 c[(DW).sub.4] DWDWDWDW 425 181 c[(WGla).sub.4] WGlaWGlaWGlaW 426 Gla 182 c[(GlaW).sub.4] GlaWGlaWGlaWG 427 laW 183 c[CW(EW).sub.4] CWEWEWEWEW 428 184 c[(WGla).sub.2WDWC] WGlaWGlaWDWC 429 185 c[(EW).sub.3EC] EWEWEWEC 430 186 c[(DW).sub.3DC] DWDWDWDC 431 187 c[E.sub.5K]W.sub.5C Cyclic:EEEEEK 432(cyclic Tail:WWWWWC portion), 433(Tail) 188 c[E.sub.4K]W.sub.5C Cyclic:EEEEK 434(cyclic Tail:WWWWWC portion), 435(Tail) 189 c[E.sub.5K]W.sub.4C Cyclic:EEEEEK 436(cyclic Tail:WWWWC portion), 437(Tail) 190 c[E.sub.4K]W.sub.4C Cyclic:EEEEK 438(cyclic Tail:WWWWC portion), 439(Tail) 191 c[E.sub.5K]W.sub.5 Cyclic:EEEEEK 440(cyclic Tail:WWWWW portion), 441(Tail) 192 c[E.sub.4K]W.sub.5 Cyclic:EEEEK 442(cyclic Tail:WWWWW portion), 443(Tail) 193 c[E.sub.5K]W.sub.4 Cyclic:EEEEEK 444(cyclic Tail:WWWW portion), 445(Tail) 194 c[E.sub.4K]W.sub.4 Cyclic:EEEEK 446(cyclic Tail:WWWW portion), 447(Tail) 195 c[D.sub.5K]W.sub.5C Cyclic:DDDDDK 448(cyclic Tail:WWWWWC portion), 449(Tail) 196 c[D.sub.4K]W.sub.5C Cyclic:DDDDK 450(cyclic Tail:WWWWWC portion), 451(Tail) 197 c[D.sub.5K]W.sub.4C Cyclic:DDDDDK 452(cyclic Tail:WWWWC portion), 453(Tail) 198 c[D.sub.4K]W.sub.4C Cyclic:DDDDK 454(cyclic Tail:WWWWC portion), 455(Tail) 199 c[D.sub.5K]W.sub.5 Cyclic:DDDDDK 456(cyclic Tail:WWWWW portion), 457(Tail) 200 c[D.sub.4K]W.sub.5 Cyclic:DDDDK 458(cyclic Tail:WWWWW portion), 459(Tail) 201 c[D.sub.5K]W.sub.4 Cyclic:DDDDDK 460(cyclic Tail:WWWW portion), 461(Tail) 202 c[D.sub.4K]W.sub.4 Cyclic:DDDDK 462(cyclic Tail:WWWW portion), 463(Tail) 203 c[Gla.sub.5K]W.sub.5C Cyclic:GlaGla 464(cyclic GlaGlaGlaK portion), Tail:WWWWWC 465(Tail) 204 c[Gla.sub.4K]W.sub.5C Cyclic:GlaGla 466(cyclic GlaGlaK portion), Tail:WWWWWC 467(Tail) 205 c[Gla.sub.5K]W.sub.4C Cyclic:GlaGla 468(cyclic GlaGlaGlaK portion), Tail:WWWWC 469(Tail) 206 c[Gla.sub.4K]W.sub.4C Cyclic:GlaGla 470(cyclic GlaGlaK portion), Tail:WWWWC 471(Tail) 207 c[Gla.sub.5K]W.sub.5 Cyclic:GlaGla 472(cyclic GlaGlaGlaK portion), Tail:WWWWW 473(Tail) 208 c[Gla.sub.4K]W.sub.5 Cyclic:GlaGla 474(cyclic GlaGlaK portion), Tail:WWWWW 475(Tail) 209 c[Gla.sub.5K]W.sub.4 Cyclic:GlaGla 476(cyclic GlaGlaGlaK portion), Tail:WWWW 477(Tail) 210 c[Gla.sub.4K]W.sub.4 Cyclic:GlaGla 478(cyclic GlaGlaK portion), Tail:WWWW 479(Tail) 211 c[E.sub.5W.sub.5C] EEEEEWWWWWC 480 212 c[E.sub.4W.sub.4C] EEEEWWWWC 481 213 c[(WE).sub.4CW] WEWEWEWECW 482 214 c[(WR).sub.4WC] WRWRWRWRWC 483
Production of Epitope-pHLIP Peptide Compositions
[0156] To manufacture the constructs or compositions to decorate the cell surfaces of diseased cell, a variety of methods known in the art can be used, e.g.: [0157] i) epitope synthesized and linked to the membrane non-inserting part of pHLIP peptide; or [0158] ii) epitope synthesized as membrane non-inserting part of pHLIP as a single peptide; or [0159] iii) epitope expressed as membrane non-inserting part of pHLIP fusion protein.
EXAMPLES
[0160] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.
Example 1: Tethering Small Molecule Epitope Di-nitrophenyl (DNP) to Cancer Cells by pHLIP Promoted Cell Killing
[0161] Three different pHLIP constructs were synthesized with a DNP(O(2,4-dinitrophenyl)hydroxylamine):
i) DNP-pHLIP, where DNP-malemide was conjugated with a single Cys residue at the N-terminal of the pHLIP peptide;
ii) DNP-PEG4-pHLIP, where DNP-PEG4-NHS was conjugated with a single Lys residue at the N-terminal of the pHLIP peptide; and
iii) DNP-PEG12-pHLIP, where DNP-PEG12-NHS as conjugated with a single Lys residue at the N-terminal of pHLIP peptide.
[0162] pHLIP peptide with a single Cys residues used in the study for conjugation with DNP-malemide is the following: (ACDDQNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: ______) pHLIP peptide with single Lys residue and acetylated N-terminus used in the study for conjugation with DNP-PEG4-NHS and DNP-PEG12-NHS is the following: Ac-AKDDQNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: ______)). Peptides were prepared by solid-phase synthesis. Progressions of coupling reactions and purifications were performed using reverse-phase HPLC (RP-HPLC) (the gradient: water and acetonitrile with 0.05% trifluoroacetic acid (TFA) followed by lyophilization. The purity and identity of the constructs were established by analytical RP-HPLC and surface-enhanced laser desorption/ionization time of flight (SELDI-TOF) mass spectroscopy, respectively. Constructs concentrations were calculated by absorbance at 280 nm using pHLIP peptide extinction coefficient.
[0163] The key for induction of immunological response is a proper positioning of epitope at the surface of tumor cells, which was verified on 3-D tumor cancer cell culture (tumor spheroids). Briefly, a 2% agarose solution was made by dissolving in pH 7.4 PBS. 150 L of the solution was pipetted into each well of a 48-well flat bottom tissue culture plate. After the agarose gel sufficiently settled (1 h), 150 L of DMEM supplemented with 10% FBS and ciprofloxacin.Math.HCl was added to each well. The covered plate was left in a humidified atmosphere at 37 C. and 5% CO.sub.2 in cell culture incubator for 24 h. On the next day, the excess medium was removed from the agarose layer. HeLa cells (10,000 cells) in 200 L of DMEM containing 2% matrigel were added into each well and incubated for 3-4 days to allow the formation of spheroids. Matrigel was dissolved on ice overnight and added in ice cold DMEM at a concentration of 2.5% (to obtain a final concentration of 2% once added to the wells). Then the mixture was heated to 37 C. before being combined with the cells. Tumor spheroids were incubated in 50 L of PBS buffer, pH 6.0-6.5 containing 0-2 M DNP-pHLIP, DNP-PEG4-pHLIP or DNP-PEG12-pHLIP in a humidified atmosphere of 5% CO.sub.2 at 37 C. for 30 min. After treatment, the spheroids were washed several times in 1 mL of PBS. Next, spheroids were treated with anti-DNP antibody labeled with 647 nm fluorescent dye at pH 7.4 followed by washing. Spheroids were also stained with DAPI to mark cell nucleus.
[0164] The spheroids were imaged using a fluorescent inverted confocal microscope. The representative images are shown in
[0165] Images presented in
[0166] To establish a biological effect, the tumor speroids were treated with 5 M of DNP-pHLIP, DNP-PEG4-pHLIP or DNP-PEG12-pHLIP for 1 hour in PBS pH6.5, washed followed by incubation with anti-DNP antibody or human IgM antibody for 1 hr in PBS pH7.4. Then human serum with active complement and propidium iodine (PI) were added, washed and spheroids were imaged. Cell impermeable PI dye stains only dead (or dying) cells with the compromised membrane. Therefore presence of red color on
Example 2: Tethering Two Peptide Epitopes by pHLIP to Cancer Cells to Bind Two Heads of Ig Antibody
[0167] To enhance performance of antibodies and enhance immune response, it is important to promote binding of both heads of IgG with 2 epitopes coupled to the same pHLIP peptide (see, e.g.,
[0168] PEG12 and PEG24 are be stretched for 5 nm and 10 nm, respectively. The six residues (QNDDQN (SEQ ID NO: ______)) between points of PEG conjugation to pHLIP provides additional space of few nanometers, for example, from 5 to 25 nm, or, from about 10-15 nm. Thus, two epitopes at the single pHLIP construct bind two heads of Ig antibody, since the distance between heads is 10-15 nm.
[0169] HeLa cancer cells in 2D and 3D cell culture are stained with (HA).sub.2-(PEG12).sub.2-pHLIP, (HA).sub.2-(PEG24).sub.2-pHLIP, HA-PEG12-pHLIP and HA-PEG24-pHLIP. Affinity of fluorescent antibody against HA peptide epitope is evaluated.
Example 3: Tethering CXCL10 Protein Chemokine Epitope by pHLIP to Cancer Cells to Promote NK-Cells Binding
[0170] Two fusion proteins with 2 different tags (His and cMyc) are expressed and purified:
TABLE-US-00037 CXCL10-mucin-2x-Myc-pHLIP SEQIDNO:531 mnqtailicclifltlsgiqgvplsrtvrctcisisnqpvnprslekleii pasqfcprveiiatmkkkgekrclnpeskaiknllkavskerskrspgtfe kqigevkprttpaaggmdesvvlepeatgesssleptpssqeaqralgtsp elptgvtgssgtrlpptpkaqdggpvgtelfrvppvstaatwqssaphqpg pslwaeaktseapstqdpstqastasspapeenapsegqrvwgqgqsprpe nslereemgpvpahtdafqdwgpgsmahvsvvpvssegtpsrepvasgswt pkaeepihatmdpqrlgvlitpvpdaqaatrrqeqkliseedleqklisee dladdqnpwrayidllfptdtllldllw CXCL10-mucin-6x-His-pHLIP SEQIDNO:532 mnqtailicclifltlsgiqgvplsrtvrctcisisnqpvnprslekleii pasqfcprveiiatmkkkgekrclnpeskaiknllkavskerskrspgtfe kqigevkprttpaaggmdesvvlepeatgesssleptpssqeaqralgtsp elptgvtgssgtrlpptpkaqdggpvgtelfrvppvstaatwqssaphqpg pslwaeaktseapstqdpstqastasspapeenapsegqrvwgqgqsprpe nslereemgpvpahtdafqdwgpgsmahvsvvpvssegtpsrepvasgswt pkaeepihatmdpqrlgvlitpvpdaqaatrrqhhhhhhaddqnpwrayld llfptdtllldllw
[0171] Both fusion proteins are treated with HeLa or HeLa-GFP cancer cells at pH 6.0-6.5 followed by washing and applying NK-cells loaded with red fluorescent dye DiI (1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine) for 30 min followed by gentle washing. Binding and adhesion of NK-cells to cancer cells decorated with CXCL10 chemokine pHLIP constructs and non-decorated are compared under fluorescent microscope by analyzing green and red fluorescent signals coming from cancer cells and immune NK-cells, respectively.
pHLIP Peptide-Mediated Epitope Tethering at Cell Surfaces: Summary I
[0172] The invention features compositions and methods for eliciting an immune response in a subject by administering to the subject a pHLIP construct comprising an antibody recruiting molecule or an immune cell recruiting molecule. The antibody recruiting molecule or an immune cell recruiting molecule is linked to one or more pHLIP peptides and wherein The construct increases the amount of antibody recruiting molecule or immune cell recruiting molecule on the surface of a diseased cell.
[0173] For example, the composition comprises the formula of:
Epitope-Linker-Pept
[0174] wherein Epitope is an antibody or immune cell recruiting molecule;
[0175] wherein Linker is a non-cleavable linker compound or a membrane non-inserting end of the pHLIP peptide further comprises an amino acid extension;
[0176] wherein Pept is a pHLIP peptide comprising the sequence
[0177] AXDDQNPWRAYLDLLFPTDTLLLDLLW (SEQ ID NO: ______) or
[0178] AXDQDNPWRAYLDLLFPTDTLLLDLLW (SEQ ID NO: ______), where X is a functional group, selected from a lysine, a cysteine, or an Azido-containing amino acid;
[0179] wherein each is a covalent bond;
[0180] In some examples, the construct comprises an antibody recruiting molecule; in other examples, the construct comprises an immune cell recruiting molecule. Optionally, the construct comprises both an antibody recruiting molecule or an immune cell recruiting molecule
[0181] In some embodiments, 2 antibody recruiting molecules are linked to pHLIP peptide. Such an exemplary construct and method is described below. For example, the composition or method includes a construct that comprises the formula of
Epitope1-Linker-Pept-Linker-Epitope1
[0182] wherein Epitope1 is an antibody recruiting molecule;
[0183] wherein Linker is a polyethylene glycol linker;
[0184] wherein Pept is a pHLIP peptide comprising the sequence
TABLE-US-00038 (SEQIDNO:_) Ac-AKQNDDQNKPWRAYLDLLFPTDTLLLDLLWA or (SEQIDNO:_) Ac-AKQNDNDNKPWRAYLDLLFPTDTLLLDLLWA or (SEQIDNO:_) ACQNDDQNCPWRAYLDLLFPTDTLLLDLLWA or (SEQIDNO:_) ACQNDNDNCPWRAYLDLLFPTDTLLLDLLWA
[0185] wherein each is a covalent bond.
[0186] As described above, exemplary recruiting molecules include one or more epitopes. For example, the epitope comprises a peptide with a length less than 50 amino acids.
[0187] The method of claim 1, wherein said antibody recruiting molecule or immune cell recruiting molecule comprises an epitope. In examples, the epitope comprises a peptide with a length less than 50 amino acids, e.g., the epitope comprises a length of between 5 to 20 amino acids. An exemplary epitope comprises an HA peptide. For example, the peptide comprises the amino acid sequence of YPYDVPDYA (SEQ ID NO: ______). Additional examples of epitopes include QVSHWVSGLAEGSFG (SEQ ID NO: ______), LSHTSGRVEGSVSLL (SEQ ID NO: ______), QMWAPQWGPD (SEQ ID NO: ______); MASMTGGQQMG (SEQ ID NO: 4); EQKLISEEDL (SEQ ID NO: 5); YTDIEMNRLGK (SEQ ID NO: 7); KETAAAKFERQHMDS (SEQ ID NO: 8); GKPIPNPLLGLDST (SEQ ID NO: 9); DYKDDDDK (SEQ ID NO: 10); GAPVPYPDPLEPR (SEQ ID NO: 11); HHHHHH (SEQ ID NO: 12); TKENPRSNQEESYDDNES (SEQ ID NO: 13); WSHPQFEK (SEQ ID NO: 14); or PDRVRAVSHWSS (SEQ ID NO: 15).
[0188] In examples, an epitope comprises a protein epitope with a length of 200 or less amino acids. For example, the protein epitope comprises a cytokine such as an interleukin (IL), e.g., IL-1, IL-2, IL-6, IL-7, IL-12, or IL-17. In some embodiments, the cytokine comprises tumor necrosis factor (TNF). In some some embodiments, the cytokine comprises a chemokine (CXC). Examples of chemokines include CXCL9, CXL10, or CXL11. For example, the chemokine comprises CXCL10 comprises the amino acid sequence:
TABLE-US-00039 (SEQIDNO:) MNQTAILICCLIFLTLSGIQGVPLSRTVRCTCISISNQPVNPRSLEKLEII PASQFCPRVEIIATMKKKGEKRCLNPESKAIKNLLKAVSKERSKRSP.
[0189] In yet other examples, the epitope comprises a small molecule. For example, the small molecule comprises a dinitrophenyl (DNP) or a derivative thereof.
[0190] The methods are useful to methods for eliciting an immune response in a subject. This clinically beneficial effect is accomplished by the pHLIP construct that increases the amount of antibody recruiting molecule or immune cell recruiting molecule on the surface of a diseased cell. For example, the diseased cell comprises a tumor cell. In another example, the diseased cell comprises a cell in inflamed tissue.
[0191] Also within the invention is a composition comprising an antibody or immune cell recruiting molecule linked to one or more pHLIP peptides by a non-cleavable linker compound. A composition comprising an epitope linked to one or more pHLIP peptides, wherein the epitope is a protein epitope and is an extension of the non-inserting end of the pHLIP peptide is also within the invention. For example, the non-inserting end of the pHLIP peptide further comprises an amino acid extension, wherein the extension comprises a protein epitope. In another aspect, the invention encompasses a composition comprising an epitope linked to one or more pHLIP peptides, wherein the epitope and the pHLIP peptide are part of a single fusion construct.
[0192] In the methods or compositions, the pHLIP construct comprises the formula of Epitope-Linker-Peptide, wherein Peptide is a pHLIP peptide comprising the sequence AXDDQNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: ______) or AXDQDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: ______), where X is a functional group, selected from a lysine, a cysteine, or an Azido-containing amino acid, wherein Linker is a linker or an extension of the pHLIP peptide, and wherein each is a covalent bond.
[0193] An exemplary composition comprises the formula of Epitope-Linker-Peptide, wherein Peptide is a pHLIP peptide comprising the sequence AXDDQNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: ______) or AXDQDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: ______), where X is a functional group, selected from a lysine, a cysteine, or an Azido-containing amino acid, and wherein Linker is a linker or an extension of the pHLIP peptide, and wherein each is a covalent bond. In some examples, two epitopes are linked to a single pHLIP peptide.
[0194] As described above, the construct may comprise the formula of Epitope2-Linker2-Peptide, wherein Peptide is a pHLIP peptide comprising the sequence AX(Z)nXPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: ______), wherein X is a functional group, selected from a lysine, a cysteine, an Azido-containing amino acid, or others, wherein Z comprises indicates any amino acid residue, wherein n is any integer between 1 and 10, wherein Linker is a linker or an extension of the pHLIP peptide, and each is a covalent bond. In another example, the composition or construct comprises the formula of Epitope2-Linker2-Pept, wherein Pept is a pHLIP peptide comprising the sequence AX(Z)nXPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: ______), where X is a functional group, selected from a lysine, a cysteine, an Azido-containing amino acid, wherein Z indicates any amino acid residue, wherein n is any integer between and including 1 and 10, wherein Linker is a linker or an extension of the pHLIP peptide, and wherein each is a covalent bond.
[0195] A method of inducing an immune response in a diseased tissue in a subject, which method comprises the step of administering to a subject a composition comprising an epitope and a pHLIP peptide is also within the invention. For example, the subject comprises a solid tumor or the subject comprises an inflamed tissue. The pHLIP compositions or constructs are administered in a variety of clinically-acceptable methods, e.g, the composition is injected directly into a diseased tissue tumor mass. In another example, the composition is systemically administered. The method is associated with numerous advantages, e.g., the biological effect of the composition in eliciting or promoting an antigen-specific (epitope-specific) immune response is at least 20% greater than that delivered in the absence of said composition. The response may be at least 25, 50, 75, 90% and even 2-fold, 3-fold, 5-fold, 10-fold or more greater than that delivered in the absence of pHLIP composition or construct. Another significant advantage is that the composition targets preferentially to a diseased tissue compared to a healthy tissue, thereby minimizing damage to said healthy tissue.
[0196] Also within the invention is a method for promoting an immune response in a subject, comprising administering to a subject the pHLIP compositions and constructs described herein, wherein the method comprises placement of the epitope on tumor cell or a cell in inflamed tissue of said subject.
pHLIP Peptide-Mediated Epitope Tethering at Cell Surfaces: Summary II
[0197] Exemplary pHLIP compositions comprise an epitope and a pHLIP peptide.
In one example, the composition comprises the formula of
Epitope-Linker-Pept
wherein Epitope is an antibody or immune cell recruiting molecule;
wherein Linker is a non-cleavable linker compound or a membrane non-inserting end of the pHLIP peptide further comprises an amino acid extension;
wherein Pept is a pHLIP peptide comprising the sequence AXDDQNPWRAYLDLLFPTDTLLLDLLW (SEQ ID NO: ______) or
AXDQDNPWRAYLDLLFPTDTLLLDLLW (SEQ ID NO: ______), where X is a functional group, selected from a lysine, a cysteine, or an Azido-containing amino acid; wherein each is a covalent bond.
Antibody Recruitment
[0198] In preferred embodiments, the epitope is an antibody recruiting molecule. For example, 2 antibody recruiting molecules are linked to pHLIP peptide. An example of such a composition for recruiting antibodies includes a composition comprising the formula of
Epitope1-Linker-Pept-Linker-Epitope1
wherein Epitope1 is an antibody recruiting molecule;
wherein Linker is a polyethylene glycol linker;
wherein Pept is a pHLIP peptide comprising the sequence
TABLE-US-00040 (SEQIDNO:) Ac-AKQNDDQNKPWRAYLDLLFPTDTLLLDLLWA or (SEQIDNO:) Ac-AKQNDNDNKPWRAYLDLLFPTDTLLLDLLWA or (SEQIDNO:) ACQNDDQNCPWRAYLDLLFPTDTLLLDLLWA or (SEQIDNO:) ACQNDNDNCPWRAYLDLLFPTDTLLLDLLWA
wherein each is a covalent bond;
[0199] For example, an epitope comprises a peptide with a length less than 50 amino acids, e.g., a length of between 5 to 20 amino acids. An exemplary epitope comprises an HA peptide, e.g., the peptide comprises the amino acid sequence of YPYDVPDYA (SEQ ID NO: ______).
[0200] Other epitopes include the following peptides: QVSHWVSGLAEGSFG (SEQ ID NO: ______), LSHTSGRVEGSVSLL (SEQ ID NO: ______), QMWAPQWGPD (SEQ ID NO: ______); MASMTGGQQMG (SEQ ID NO: 4); EQKLISEEDL (SEQ ID NO: 5); KETAAAKFERQHMDS (SEQ ID NO: 8); GKPIPNPLLGLDST (SEQ ID NO: 9); DYKDDDDK (SEQ ID NO: 10); GAPVPYPDPLEPR (SEQ ID NO: 11); HHHHHH (SEQ ID NO: 12); TKENPRSNQEESYDDNES (SEQ ID NO: 13); WSHPQFEK (SEQ ID NO: 14); and/or PDRVRAVSHWSS (SEQ ID NO: 15).
[0201] In some examples, the epitope comprises a small molecule such as dinitrophenyl (DNP) or a derivative thereof.
Immune Cell Recruitment
[0202] In some examples the epitope is an immune cell recruiting molecule.
An exemplary composition, e.g. for recruiting immune cells, comprises the formula of
Epitope2-Pept,
wherein Epitope2 is an immune cell recruiting molecule;
wherein Pept is a pHLIP peptide comprising the sequence
TABLE-US-00041 (SEQIDNO:) ADDQNPWRAYLDLLFPTDTLLLDLLW;
wherein is a covalent bond. In such examples, the epitope comprises a protein epitope with a length of 350 or less amino acids. An exemplary protein epitope comprises a cytokine. For example, the cytokine comprises an interleukin (IL) such as IL-2, IL-6, IL-7, or IL-12. In other examples, the cytokine comprises tumor necrosis factor (TNF).
[0203] In some embodiments, the cytokine comprises a chemokine such as CXCL9, CXCL10, or CXCL11. For example, chemokine comprises CXCL10 comprising the amino acid sequence:
TABLE-US-00042 (SEQIDNO:) MNQTAILICCLIFLTLSGIQGVPLSRTVRCTCISISNQPVNPRSLEKLEII PASQFCPRVEIIATMKKKGEKRCLNPESKAIKNLLKAVSKERSKRSP.
Compositions with Amino Acid Extension to pHLIP Peptide
[0204] In some aspect, the invention features compositions, constructs, and methods comprising an epitope wherein the membrane non-inserting end of the pHLIP peptide further comprises an amino acid extension. For example, the composition comprises an epitope and pHLIP peptide comprises a fusion protein. For example, fusion protein comprises an epitope and a pHLIP peptide. In other examples, a composition comprises an epitope linked to one or more pHLIP peptides by a non-cleavable linker compound.
[0205] A variety of linkers may be used. For example, the linker is a chemical polymer, e.g., polyethylene glycol. In other examples, the linker is a biopolymer. Exemplary linkers include mucin domain, dextran, cellulose, chitin or starch.
Methods of Treatment
[0206] The invention also includes a method of inducing an immune response in a diseased tissue in a subject, comprising administering to a subject a composition comprising an epitope and a pHLIP peptide. For example, the subject comprises a solid tumor. Alternatively, the subject comprises an inflamed tissue. In some cases, both conditions are present in the subject.
[0207] The subject is treated using a variety of clinically acceptable procedures, e.g, the composition is injected directly into a diseased tissue tumor mass. In another example, the composition is systemically administered. As described above, an advantage of the methods is that a biological effect of said composition is at least 20% or more greater than that delivered in the absence of said composition. Another advantage that contributes to the clinical safety and efficacy is that the composition targets preferentially to a diseased tissue compared to a healthy tissue, thereby minimizing damage to healthy tissue.
[0208] An exemplary method for promoting an immune response in a subject is carried out by administering to a subject the compositions and constructs described above. Such methods comprise, e.g., lead to, the placement of the epitope(s) on tumor cell or a cell in inflamed tissue of the subject. The increase amount or concentration of the epitope on the sureface of the tumor cell or cell in an inflamed tissue leads to a more robust immune response, e.g., antibody-binding or immune cell binding, and subsequent killing and/or elimination of the diseased (or otherwise undesirable) cell. For example, the diseased cell comprises a tumor cell or a cell in inflamed tissue.
General Definitions
[0209] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, and biochemistry).
[0210] As used herein, the term about in the context of a numerical value or range means 10% of the numerical value or range recited or claimed, unless the context requires a more limited range.
[0211] In the descriptions above and in the claims, phrases such as at least one of or one or more of may occur followed by a conjunctive list of elements or features. The term and/or may also occur in a list of two or more elements or features. Unless otherwise implicitly or explicitly contradicted by the context in which it is used, such a phrase is intended to mean any of the listed elements or features individually or any of the recited elements or features in combination with any of the other recited elements or features. For example, the phrases at least one of A and B; one or more of A and B; and A and/or B are each intended to mean A alone, B alone, or A and B together. A similar interpretation is also intended for lists including three or more items. For example, the phrases at least one of A, B, and C; one or more of A, B, and C; and A, B, and/or C are each intended to mean A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together. In addition, use of the term based on, above and in the claims is intended to mean, based at least in part on, such that an unrecited feature or element is also permissible.
[0212] It is understood that where a parameter range is provided, all integers within that range, and tenths thereof, are also provided by the invention. For example, 0.2-5 mg is a disclosure of 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg etc. up to and including 5.0 mg.
[0213] A small molecule is a compound that is less than 2000 daltons in mass. The molecular mass of the small molecule is preferably less than 1000 daltons, more preferably less than 600 daltons, e.g., the compound is less than 500 daltons, 400 daltons, 300 daltons, 200 daltons, or 100 daltons.
[0214] As used herein, an isolated or purified nucleic acid molecule, polynucleotide, polypeptide, or protein, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. Purified compounds are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. For example, a purified compound is one that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desired compound by weight. Purity is measured by any appropriate standard method, for example, by column chromatography, thin layer chromatography, or high-performance liquid chromatography (HPLC) analysis. A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) is free of the genes or sequences that flank it in its naturally-occurring state. Purified also defines a degree of sterility that is safe for administration to a human subject, e.g., lacking infectious or toxic agents. A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) is free of the genes or sequences that flank it in its naturally-occurring state. A purified or isolated polypeptide is free of the amino acids or sequences that flank it in its naturally-occurring state.
[0215] Similarly, by substantially pure is meant a nucleotide or polypeptide that has been separated from the components that naturally accompany it. Typically, the nucleotides and polypeptides are substantially pure when they are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, free from the proteins and naturally-occurring organic molecules with they are naturally associated.
[0216] The term alkyl, by itself or as part of another substituent, means, unless otherwise stated, a non-cyclic straight (i.e., unbranched) or branched chain, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e., C.sub.1-C.sub.10 means one to ten carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (O).
[0217] The term alkylene, by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkyl, as exemplified, but not limited by, CH.sub.2CH.sub.2CH.sub.2CH.sub.2. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms. A lower alkyl or lower alkylene is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
[0218] The term heteroalkyl, by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or combinations thereof, consisting of at least one carbon atom and at least one heteroatom (e.g. selected from the group consisting of O, N, P, S, Se and Si, and wherein the nitrogen, selenium, and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized). The heteroatom(s) O, N, P, S, Se, and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to: CH.sub.2CH.sub.2OCH.sub.3, CH.sub.2CH.sub.2NHCH.sub.3, CH.sub.2CH.sub.2N(CH.sub.3)CH.sub.3, CH.sub.2SCH.sub.2CH.sub.3, CH.sub.2CH.sub.2, S(O)CH.sub.3, CH.sub.2CH.sub.2S(O).sub.2CH.sub.3, CHCHOCH.sub.3, Si(CH.sub.3).sub.3, CH.sub.2CHNOCH.sub.3, CHCHN(CH.sub.3)CH.sub.3, OCH.sub.3, OCH.sub.2CH.sub.3, and CN. Up to two heteroatoms may be consecutive, such as, for example, CH.sub.2NHOCH.sub.3.
[0219] Similarly, the term heteroalkylene, by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from heteroalkyl, as exemplified, but not limited by, CH.sub.2CH.sub.2SCH.sub.2CH.sub.2 and CH.sub.2SCH.sub.2CH.sub.2NHCH.sub.2. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula C(O).sub.2R represents both C(O).sub.2R and RC(O).sub.2. As described above, heteroalkyl groups, as used herein, include those groups that are attached to the remainder of the molecule through a heteroatom, such as C(O)R, C(O)NR, NRR, OR, SeR, SR, and/or SO.sub.2R. Where heteroalkyl is recited, followed by recitations of specific heteroalkyl groups, such as NRR or the like, it will be understood that the terms heteroalkyl and NRR are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term heteroalkyl should not be interpreted herein as excluding specific heteroalkyl groups, such as NRR or the like.
[0220] The terms cycloalkyl and heterocycloalkyl, by themselves or in combination with other terms, mean, unless otherwise stated, cyclic versions of alkyl and heteroalkyl, respectively. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. A cycloalkylene and a heterocycloalkylene, alone or as part of another substituent, means a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively.
[0221] The term aryl means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent, which can be a single ring or multiple rings (preferably from 1 to 3 rings) that are fused together (i.e., a fused ring aryl) or linked covalently. A fused ring aryl refers to multiple rings fused together wherein at least one of the fused rings is an aryl ring. The term heteroaryl refers to aryl groups (or rings) that contain from one to four heteroatoms (e.g. selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized). Thus, the term heteroaryl includes fused ring heteroaryl groups (i.e., multiple rings fused together wherein at least one of the fused rings is a heteroaromatic ring). A 5,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 5 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. Likewise, a 6,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 5 members, and wherein at least one ring is a heteroaryl ring. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. An arylene and a heteroarylene, alone or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively.
[0222] A fused ring heterocyloalkyl-aryl is an aryl fused to a heterocycloalkyl. A fused ring heterocycloalkyl-heteroaryl is a heteroaryl fused to a heterocycloalkyl. A fused ring heterocycloalkyl-cycloalkyl is a heterocycloalkyl fused to a cycloalkyl. A fused ring heterocycloalkyl-heterocycloalkyl is a heterocycloalkyl fused to another heterocycloalkyl. Fused ring heterocycloalkyl-aryl, fused ring heterocycloalkyl-heteroaryl, fused ring heterocycloalkyl-cycloalkyl, or fused ring heterocycloalkyl-heterocycloalkyl may each independently be unsubstituted or substituted with one or more of the substituents described herein. Spirocyclic rings are two or more rings wherein adjacent rings are attached through a single atom. The individual rings within spirocyclic rings may be identical or different. Individual rings in spirocyclic rings may be substituted or unsubstituted and may have different substituents from other individual rings within a set of spirocyclic rings. Possible substituents for individual rings within spirocyclic rings are the possible substituents for the same ring when not part of spirocyclic rings (e.g., substituents for cycloalkyl or heterocycloalkyl rings). Spirocylic rings may be substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heterocycloalkylene and individual rings within a spirocyclic ring group may be any of the immediately previous list, including having all rings of one type (e.g. all rings being substituted heterocycloalkylene wherein each ring may be the same or different substituted heterocycloalkylene). When referring to a spirocyclic ring system, heterocyclic spirocyclic rings means a spirocyclic rings wherein at least one ring is a heterocyclic ring and wherein each ring may be a different ring. When referring to a spirocyclic ring system, substituted spirocyclic rings means that at least one ring is substituted and each substituent may optionally be different.
[0223] Each of the above terms (e.g., alkyl, heteroalkyl, aryl, and heteroaryl) includes both substituted and unsubstituted forms of the indicated radical.
[0224] The transitional term comprising, which is synonymous with including, containing, or characterized by, is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase consisting of excludes any element, step, or ingredient not specified in the claim. The transitional phrase consisting essentially of limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.
[0225] The terms subject, patient, individual, and the like as used herein are not intended to be limiting and can be generally interchanged. That is, an individual described as a patient does not necessarily have a given disease, but may be merely seeking medical advice.
[0226] As used herein, the singular forms a, an, and the include the plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to a disease, a disease state, or a nucleic acid is a reference to one or more such embodiments, and includes equivalents thereof known to those skilled in the art and so forth.
[0227] As used herein, treating encompasses, e.g., inhibition, regression, or stasis of the progression of a disorder. Treating also encompasses the prevention or amelioration of any symptom or symptoms of the disorder. As used herein, inhibition of disease progression or a disease complication in a subject means preventing or reducing the disease progression and/or disease complication in the subject.
[0228] As used herein, a symptom associated with a disorder includes any clinical or laboratory manifestation associated with the disorder, and is not limited to what the subject can feel or observe.
[0229] As used herein, effective when referring to an amount of a therapeutic compound refers to the quantity of the compound that is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this disclosure.
[0230] An epitope is a molecular region of an antigen capable of eliciting an immune response and of combining with a specific antibody or immune cell produced by such a response. An epitope is also know as an antigenic determinant. For example, an epitope is a part of an antigen molecule to which an antibody attaches or to which an immune cell attaches.
[0231] As used herein, the term cancer refers to all types of cancer, neoplasm or malignant tumors found in mammals, including leukemias, lymphomas, melanomas, neuroendocrine tumors, carcinomas and sarcomas. Exemplary cancers that may be treated with a compound, pharmaceutical composition, or method provided herein include lymphoma (cutaneous T-cell lymphoma), sarcoma, bladder cancer, bone cancer, brain tumor, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, myeloma, thyroid cancer, leukemia, prostate cancer, breast cancer (e.g. triple negative, ER positive, ER negative, chemotherapy resistant, herceptin resistant, HER2 positive, doxorubicin resistant, tamoxifen resistant, ductal carcinoma, lobular carcinoma, primary, metastatic), ovarian cancer, pancreatic cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g. non-small cell lung carcinoma, squamous cell lung carcinoma, adenocarcinoma, large cell lung carcinoma, small cell lung carcinoma, carcinoid, sarcoma), glioblastoma multiforme, glioma, melanoma, prostate cancer, castration-resistant prostate cancer, breast cancer, triple negative breast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cell carcinoma (e.g., head, neck, or esophagus), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B cell lymphoma, or multiple myeloma. Additional examples include, cancer of the thyroid, endocrine system, brain, breast, cervix, colon, head & neck, esophagus, liver, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus or Medulloblastoma, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, cancer, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, neoplasms of the endocrine or exocrine pancreas, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, Paget's Disease of the Nipple, Phyllodes Tumors, Lobular Carcinoma, Ductal Carcinoma, cancer of the pancreatic stellate cells, cancer of the hepatic stellate cells, or prostate cancer.
[0232] By, small molecule may be referred to broadly as an organic, inorganic or organometallic compound with a low molecular weight compound (e.g., a molecular weight of less than about 2,000 Da or less than about 1,000 Da). The small molecule may have a molecular weight of less than about 2,000 Da, a molecular weight of less than about 1,500 Da, a molecular weight of less than about 1,000 Da, a molecular weight of less than about 900 Da, a molecular weight of less than about 800 Da, a molecular weight of less than about 700 Da, a molecular weight of less than about 600 Da, a molecular weight of less than about 500 Da, a molecular weight of less than about 400 Da, a molecular weight of less than about 300 Da, a molecular weight of less than about 200 Da, a molecular weight of less than about 100 Da, or a molecular weight of less than about 50 Da.
[0233] Small molecules are organic or inorganic. Exemplary organic small molecules include, but are not limited to, aliphatic hydrocarbons, alcohols, aldehydes, ketones, organic acids, esters, mono- and disaccharides, aromatic hydrocarbons, amino acids, and lipids. Exemplary inorganic small molecules comprise trace minerals, ions, free radicals, and metabolites. Alternatively, small molecules can be synthetically engineered to consist of a fragment, or small portion, or a longer amino acid chain to fill a binding pocket of an enzyme. Typically small molecules are less than one kilodalton.
[0234] As used herein, the term stereoisomers refers to compounds made up of the same atoms having the same bond order but having different three-dimensional arrangements of atoms that are not interchangeable. The three-dimensional structures are called configurations. As used herein, the term enantiomers refers to two stereoisomers that are non-superimposable mirror images of one another. As used herein, the term optical isomer is equivalent to the term enantiomer. As used herein the term diastereomer refers to two stereoisomers which are not mirror images but also not superimposable. The terms racemate, racemic mixture or racemic modification refer to a mixture of equal parts of enantiomers. The term chiral center refers to a carbon atom to which four different groups are attached. Choice of the appropriate chiral column, eluent, and conditions necessary to effect separation of the pair of enantiomers is well known to one of ordinary skill in the art using standard techniques (see e.g. Jacques, J. et al., Enantiomers, Racemates, and Resolutions, John Wiley and Sons, Inc. 1981).
[0235] As used herein, pharmaceutically acceptable carrier or excipient refers to a carrier or excipient that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio. It can be, e.g., a pharmaceutically acceptable solvent, suspending agent or vehicle, for delivering the instant compounds to the subject.
[0236] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.
[0237] Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
[0238] The term identical or percent identity, in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity over a specified region, e.g., of an entire polypeptide sequence or an individual domain thereof), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection. Such sequences that are at least about 80% identical are said to be substantially identical. In some embodiments, two sequences are 100% identical. In certain embodiments, two sequences are 100% identical over the entire length of one of the sequences (e.g., the shorter of the two sequences where the sequences have different lengths). In various embodiments, identity may refer to the complement of a test sequence. In some embodiments, the identity exists over a region that is at least about 10 to about 100, about 20 to about 75, about 30 to about 50 amino acids or nucleotides in length. In certain embodiments, the identity exists over a region that is at least about 50 amino acids in length, or more preferably over a region that is 100 to 500, 100 to 200, 150 to 200, 175 to 200, 175 to 225, 175 to 250, 200 to 225, 200 to 250 or more amino acids in length.
[0239] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. In various embodiments, when using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
[0240] A the comparison window refers to a segment of any one of the number of contiguous positions (e.g., least about 10 to about 100, about 20 to about 75, about 30 to about 50, 100 to 500, 100 to 200, 150 to 200, 175 to 200, 175 to 225, 175 to 250, 200 to 225, 200 to 250) in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. In various embodiments, a comparison window is the entire length of one or both of two aligned sequences. In some embodiments, two sequences being compared different lengths, and the comparison window is the entire length of the longer or the shorter of the two sequences. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).
[0241] In various embodiments, an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 may be used, with the parameters described herein, to determine percent sequence identity for nucleic acids and proteins. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information, as known in the art. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.
Other Embodiments
[0242] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
[0243] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.
[0244] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.