Patent application title: REPLIKINS AND METHODS OF IDENTIFYING REPLIKIN-CONTAINING SEQUENCES
Samuel Bogoch (New York, NY, US)
Elenore S. Bogoch (New York, NY, US)
IPC8 Class: AC07K1600FI
Class name: Globulins immunoglobulin, antibody, or fragment thereof, other than immunoglobulin antibody, or fragment thereof that is conjugated or adsorbed binds specifically-identified amino acid sequence
Publication date: 2010-09-23
Patent application number: 20100240876
Patent application title: REPLIKINS AND METHODS OF IDENTIFYING REPLIKIN-CONTAINING SEQUENCES
Elenore S. BOGOCH
KENYON & KENYON LLP
Origin: WASHINGTON, DC US
IPC8 Class: AC07K1600FI
Publication date: 09/23/2010
Patent application number: 20100240876
The present invention provides methods for identifying a class of peptides
referred to as replikins and methods of using replikins to stimulate the
immune system of a subject. The method of identifying replikin peptides
is based on identifying amino acid sequences comprising 7 to about 50
amino acids that contain (1) at least one lysine residue located six to
ten residues from a second lysine residue; (2) at least one histidine
residue; and (3) at least 6% lysine residues.
15. An antibody that specifically binds to a non-viral peptide sequence comprising from 7 to about 50 amino acid residues with at least one lysine residue on one end of the peptide sequence and at least one lysine residue or at least one histidine residue on the other end of the peptide sequence, wherein said peptide sequence comprises: (1) at least one lysine residue located six to ten residues from a second lysine residue; (2) at least one histidine residue; and (3) at least 6% lysine residues.
16. An antibody cocktail comprising a plurality of antibodies, wherein each of the antibodies specifically binds to a peptide sequence comprising from 7 to 50 amino acids and including (1) at least one lysine residue located six to ten residues from a second lysine residue; (2) at least one histidine residue; and (3) at least 6% lysine residues.
109. The antibody cocktail of claim 16 wherein at least one antibody of said plurality of antibodies specifically binds to a peptide sequence having at least one lysine residue on one end of the peptide sequence and at least one lysine residue or at least one histidine residue on the other end of the peptide sequence.
110. The antibody cocktail of claim 16, wherein the peptide sequence is a non-viral peptide sequence.
111. The antibody cocktail of claim 109, wherein the peptide sequence is a non-viral peptide sequence.
112. The antibody of claim 15, wherein said non-viral peptide sequence is an algal peptide sequence.
113. The antibody of claim 15, wherein said non-viral peptide sequence is a yeast peptide sequence.
114. The antibody of claim 15, wherein said non-viral peptide sequence is an amoeba peptide sequence.
115. The antibody of claim 15, wherein said non-viral peptide sequence is a plant peptide sequence.
116. The antibody of claim 15, wherein said non-viral peptide sequence is a replication-associated peptide sequence.
117. The antibody of claim 15, wherein said non-viral peptide sequence is a bacterial peptide sequence.
118. The antibody of claim 15, wherein said non-viral peptide sequence is a transforming-associated peptide sequence.
119. The antibody of claim 15, wherein said non-viral peptide sequence is a cancer peptide sequence.
120. The antibody of claim 119, wherein said cancer peptide sequence is a glioma peptide sequence.
121. The antibody of claim 15 wherein said peptide sequence comprises at least one peptide sequence of SEQ ID NO(s): 1-2, 34-46, 50, 52-53, 59, 65-72, or 74-90.
122. The antibody of claim 15, wherein said peptide sequence consists of one peptide sequence of SEQ ID NO(s): 1-2, 34-46, 50, 52-53, 59, 65-72, or 74-90.
123. An antibody that specifically binds to a peptide sequence of 7 to 50 amino acid residues comprising at least one of SEQ ID NO(s): 3-18, 47-49, 51, 54-58, 60-64, or 73.
124. The antibody of claim 123, wherein said peptide sequence consists of one peptide sequence of SEQ ID NO(s): 3-18, 47-49, 51, 54-58, 60-64, or 73.
CROSS REFERENCE TO OTHER APPLICATIONS
This application is claiming priority to Provisional Application Ser. Nos. 60/303,396 filed Jul. 9, 2001 and 60/278,761 filed Mar. 27, 2001, and Continuation-in-Part application Ser. No. 09/146,755 filed Sep. 4, 1998 (issued as U.S. Pat. No. 6,242,578 B1) and 09/817,144 filed Mar. 27, 2001 (pending), the latter of which are claiming priority to application Ser. No. 08/198,139 filed Feb. 17, 1994 (abandoned), which are incorporated herein in their entirety by reference thereto.
FIELD OF THE INVENTION
This invention relates to the identification of Recognins, a class of substances identified by a new "3-point-recognition" method, and Replikins, a subtype of Recognins, which is a class of peptides, and a "3-point-recognition" method, which is used to identify the recognin and replikin sequences.
BACKGROUND OF THE INVENTION
Glycoprotein 10B is a membrane glycoprotein isolated from brain glioblastoma multiforme, lymphoma and breast cancer cells (U.S. Pat. No. 6,242,578 B1). A constituent peptide of Aglyco 1OB, malignin, is enriched in cell membranes tenfold during anaerobic replication. Hydrolysis and mass spectrometry of malignin yielded a 16-mer peptide including (SEQ ID NO.: 1) kagvaflhkk. This peptide, which is absent from the normal human genome, was assumed to be acquired.
SUMMARY OF THE INVENTION
In one aspect of the invention there is provided a 3-point-recognition method for identifying a protein or peptide containing a replikin sequence or a recognin sequence comprising scanning the amino acid sequence of the protein or peptide for a subsequence comprising 7 to 50 amino acids which includes (1) at least one lysine residue located six to ten residues from a second lysine residue; (2) at least one histidine residue; and (3) at least 6% lysine residues. In another embodiment of this aspect of the invention, a nucleotide sequence is scanned for the presence of a subsequence comprising about 10 to about 150 nucleotides containing codons for (1) at least one lysine residue located six to ten residues from a second lysine residue; (2) at least one histidine residue; and (3) at least 6% lysine residues. The method may be carried out by visually scanning the sequences or through use of a computer.
In another aspect of the invention there are provided isolated peptides comprising 7 to about 50 amino acids which include (1) at least one lysine residue located six to ten residues from a second lysine residue; (2) at least one histidine residue; and (3) at least 6% lysine residues.
In another aspect of the invention there is provided an antibody or non-immune based organic agent, e.g., lipid, carbohydrate, and the like, which binds specifically to a peptide sequence comprising from 7 to about 50 amino acids including (1) at least one lysine residue located six to ten residues from a second lysine residue; (2) at least one histidine residue; and (3) at least 6% lysine residues. In an embodiment of this aspect of the invention the antibody is included in an antibody cocktail containing other antibodies that bind specifically to other replikin or recognin sequences.
As used herein, the term "peptide" refers to a compound of two or more amino acids in which the carboxyl group of one is united with an amino group of another, forming a peptide bond. The term peptide is also used to denote the sequence encoding such a compound. Thus, a peptide sequence may be a subsequence of a larger polypeptide sequence.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a bar graph depicting the frequency of occurrence of replikins in various protein groups.
FIG. 2 is a graph depicting the percentage of malignin per milligram total membrane protein during anaerobic replication of glioblastoma cells.
FIG. 3 is a bar graph showing amount of antimalignin antibody produced in response to exposure to the recognin 16-mer.
FIG. 4A is a photograph of a blood smear taken with ordinary and fluorescent light. FIG. 4B is a photograph of a blood smear taken with and fluorescent light illustrating the presence of two leukemic cells. FIG. 4C is a photograph of a dense layer of glioma cells in the presence of antimalignin antibody. FIG. 4D and FIG. 4E are photographs of the layer of cells in FIG. 4C taken at 30 and 45 minutes following addition of antimalignin antibody.
FIG. 4F is a bar graph showing the inhibition of growth of small cell lung carcinoma cells in vitro by antimalignin antibody.
FIG. 5 is a plot of the amount of antimalignin antibody present in the serum of patients with benign or malignant breast disease pre- and post surgery.
FIG. 6 is a box diagram depicting an embodiment of the invention wherein a computer is used to carry out the 3-point-recognition method of identifying replikin and recognin sequences.
DETAILED DESCRIPTION OF THE INVENTION
In one aspect of the invention there is provided a method for identifying nucleotide or amino acid sequences that include a recognin or replikin sequence. The method is referred to herein as a 3-point-recognition method. By use of the "3-point recognition" method, described herein below, a new class of peptides was revealed in algae, yeast, fungi, amoebae, bacteria, plant and virus proteins having replication, transformation, or redox functions. This class of peptides is referred to herein as replikins.
One example of a replikin that was identified by the 3-point-recognition method, is the amino acid sequence, (SEQ ID NO.: 2) hsikrelgiifdk, which occurs in Saccharomyces cerevisiae "replication binding protein". Five replikins were found in amino acids 1-163 of the "replicating protein" of tomato leaf curl Gemini vinis. Amino acids 1-160 of this tomato virus protein bind DNA. Another replikin, (SEQ ID NO.: 3) hkqkivapvk, is highly conserved in 236 isolates of foot and mouth disease virus.
Although replikins were found to be present in only 1.5% of published sequences identified by the PubMed data bank as "virus peptides" as a whole, and in only 8.5% of sequences identified as "brain peptides" plus "neuropeptides", surprisingly, replikins were found in 100% of "tumor viruses", in 85% of "transforming proteins," and 97% of "cancer proteins" (as categorized in the PubMed data bank). The recognin, (SEQ ID NO.: 4) ykagvafihkkndide, was not found in published sequences of the human genome.
The 16-mer recognin peptide, (SEQ ID NO.: 4) ykagvaflhkkndide, when synthesized and injected as vaccine into a mammal, has been shown to produce antimalignin antibody, which is cytotoxic to malignant replicating cells of several types at picogram per cell amounts. Replikins identified in organisms such as diatom plankton, H. pylori, tomato leaf curl virus, foot and mouth disease virus, hepatitis B and C viruses, and HIV, also are thus targets for diagnosis and treatment or as vaccines for the control of replication of their respective virus source.
Table 1 illustrates the sequence of the malignin peptide, the 16-mer recognin sequence, (SEQ ID NO.: 4) ykagvaflhkkndide.
TABLE-US-00001 TABLE 1 16-mer peptide sequence ykagyaflhkkndide obtained from malignin by hydrolysis and mass spectrometry Method By Which Fragment Obtained Auto-hydroly- Auto- sis of hydrolysis malignin Seq of malignin immobilized ID Fragment MH+ free in on bromoace- Microwaved Microwaved No. Identified (mass) Sequence solution tyl cellulose 5 seconds 30 seconds 19 1-3 381.21 ( )Oyka(g) + 20 1-5 537.30 ( )ykagv(a) + 21 2-6 445.28 (y)kagva(f) + 22 2-7 592.35 (Y)kagvaf(l) + 23 4-11 899.55 (a)gvaflhkk(n) + 24 5-7 336.19 (g)vaf(l) + 25 6-7 237.12 (v)af(l) + 26 6-10 615.36 (v)aflhk(k) + 27 6-10 615.36 (v)aflhk(k) + 28 6-12 857.50 (v)aflhkkn(d) + 29 6-12 857.50 (v)afhkkn(d) + 30 7-8 279.17 (a)fl(h) + 31 10-16 861.43 (h)kkndide( ) + 32 11-14 489.27 (k)kndi(d) + 33 12-15 476.2- (k)ndid(e) +
The malignin peptide was isolated from membranes of glioblastoma multiforme (glioma) cells grown in tissue culture (U.S. Pat. No. 6,242,578 B1). The sequence of a 16-mer peptide of malignin was determined by hydrolysis and mass spectrometry: (SEQ ID NO.: 4) ykagvaflhkkndide (Table 1). A search of published human genome sequences for sequence encoding the 16-mer amino acid sequence was negative. Since this 16-mer peptide was absent from normal human genome data a search was made of sequences of other organisms for possible origins and homologues. No identical sequences were found. But, using the sequence of the 16-mer peptide as a template, and constructing a "3-point-recognition" method to visually scan protein sequences of several different organisms, a new class of peptides, the replikins, was revealed in organisms as diverse as algae, yeast and viruses. Surprisingly, these peptides were found to be concentrated in larger `replicating` and `transforming` proteins (so designated by their investigators, based on activities, see Table 2).
Table 2 illustrates several replikin sequences that were identified by the 3-point-recognition method of the invention.
TABLE-US-00002 TABLE 2 Examples of replikins in various organisms - prototype: Glioma rcplikin* kagvaflhkk (SEQ ID No. 1) Algae: SEQ ID 34 Caldophera prolifera kaskftkh NO. 35 Isolepisprolifera kaqaetgeikgh Yeast 36 Schizosaccharomyces pombe ksfkypkkhk 37 Oryza sativa kkaygnelhk 2 Sacch. cerevisiae replication binding protein hsikrelgiifdk Fungi: 38 Isocitrate lyase ICI 1, Penicillium marneffei kvdivthqk 39 DNA-dependent RNA polymerase 11, Diseula destructiva kleedaayhrkk 40 Ophiostoma novo-u1m 1, RNA in Dutch elm disease fungus kvilplrgnikgiffkh Amoeba: 41 Entamoeba invadens, histone H2B klilkgdlnkh Bacteria: 42 Pribosomal protein replication factor, Helicobacter pylori ksvhaflk 10 Replication-associated protein Staph. aureus kkektthnk 43 Mycoplasma pulmonis, ehromosome replication kvhffqlkk 90 Macrophage infectivity potentiator, L. legionella kihlisvkk Plants: 44 Arabidopsis thaliana, prolifera kdhdfdgdk 45 Arabidopsis thaliana, cytoplasmic ribosomal kmkglkqkkah 46 Arabidopsis thaliana, DNA binding protein kelssttqeksh Viruses: 9 Replication associated protein A [Maize streak virus] kekkpskdeimrdiish 11 Bovine herpes virus 4, DNA replication protein hkinitngqk 12 Meleagrid herpesvirus 1, replication binding protein hkdlyrllmk 47 Feline immunodeficiency hlkdyklvk 3 Foot and Mouth Disease (0) hkqkivapvk 5 HIV Type 1 kcfncgkegh 7 HIV Type 2 kcwncgkegh Tumor 48 Rous sarcoma virus tyrosine-protein kinase kklrhek Viruses: 49 v-yes, avian sarcoma kklrhdk 50 c-yes, colon cancer, malignnut melanoma kklrhdk 51 v-src, avian sarcoma kklrhek 52 c-src, colon, mammary, panercreatic cancer kklrhek 53 Neuroblastoma RAS viral (v-ras) oncogene kqahelak 54 VP1 (major capsid protein) [Polyomavirus sp.] kthrfskh 55 Sindbis knlhekik 56 E1 [Human papilloamavirus type 71] khrpllqlk 57 v-erbB from AEV and c-erb kspnhvk 58 v-fms (feline sarcoma) knihlekk 59 c-fms (acute and chronic myelomonocytic tumors) knihlekk 60 large t-antigen I [Polyomavirus sp.] kphlaqslek 61 middle t-antigen [Polyomavirus sp,]- kqhrelkdk 62 small t-antigen [Polyomavirus sp], kqhrelkdk 63 v-abl, murine acute leukemia kvpvlisptlkh 64 Human T-cell lymphotropic virus typo 2 kslllevdkdish 65 c-kit, GI tumors, small cell lung carcinoma kagitimvkreyh 18 Hepatitis C hyppkpgcivpak Trans 66 Transforming protein myb ksgkhlgk forming 67 Transforming protein myc, Burkitt lymphoma krreqlkhk Proteins: 68 Ras-related GTP-binding protein ksfevikvih 69 Transforming protein ras (teratocarcinoma) kkkhtvkk 70 TRAF-associated NF•kB activator TANK kaqkdhlsk 71 RFP transforming protein hlkrvkdlkk 72 Transforming protein D (src) kygspkhrlik 73 Papilloma virus type 11, transforming protein klkhilgkarfik 74 Protein tryrosine kinasc (EC 188.8.131.52slk kgdhvkhykirk 75 Transforming protein (axl(-)) keklrdvmvdrhk 76 Transforming protein (N-myc) klqarqqqllkkieh 77 Fibroblast growth factor 4 (Kaposi sarcoma) kkgnrvsptmkvth Cancer 78 Matrix metaloproteinase 7 (uterine) keiplhfrk Cell 79 Transcription factor 7-like kkkphikk Proteins: 80 Breast cancer antigen NY-BR-87 ktrhdplak 81 BRCA-1-Associated Ring Domain Protein (breast) khhpkdnlik 82 `Autoantigen from a breast tumor` khkrkkfrqk 83 Glioma replikin (this study) kagvaflhkk 84 Ovarian cancer antigen khkrkkfrqk 85 EE L leukemia kkkskkhkdk 86 Proto-oncogene tyrosine-protein kinase C-ABLE hksekpalprk 87 Adenomatosis polyposis coli kkkkpsrlkgdnek 88 Gastric cancer transforming protein ktkkgnrvsptmkvth 89 Transforming protein (K-RAS 2B), lung khkekmskdgkkkkkksk
Identification of an amino acid sequence as a replikin or as containing a replikin, i.e., a homologue of the malignin 16-mer peptide, requires that the three following "3-point recognition" requirements be met. The peptide sequence must have (1) at least one lysine residue located six to ten residues from another lysine residue; (2) at least one histidine residue; and (3) a composition of at least 6% lysine within an amino acid sequence of 7 to about 50 residues.
Databases were searched using the National Library of Medicine keyword "PubMed" descriptor for protein sequences containing replikin sequences. Sequences of all individual proteins within each group of PubMed-classified proteins were visually scanned for peptides meeting the three above-listed requirements. An infrequent occurrence of homologues was observed in "virus peptides" as a whole (1.5%), and in other peptides not designated as associated with malignant transformation or replication such as "brain peptides" and "neuropeptides" (together 8.5%). Surprisingly, homologues were identified in 100% of "tumor viruses", in 85% of "transforming proteins", and in 97% of "cancer cell proteins" (FIG. 1). The peptides identified by this search were named replikins, and a ten amino acid portion of the 16-mer peptide, (SEQ ID NO.: 1) "kagvaflhkk", was named the glioma replikin.
To permit classification of subtypes of replikins, additional or "auxiliary specifications" to the basic "3-point-recognition" requirements may be added: (a) on a structural basis, such as the common occurrence of adjacent di- and polylysines in cancer cell proteins (e.g., Transforming protein P21B(K-RAS 2B), lung, Table 2, SEQ ID NO.: 89), and other adjacent di-amino acids in TOLL-like receptors, orb) on a functional basis, such as exhibiting ATPase, tyrosine kinase or redox activity as seen in Table 2.
Whether replikin structures are conserved or are subject to extensive natural mutation was examined by scanning the protein sequences of various isolates of foot and mouth disease virus (FMDV), where mutations in proteins of these viruses have been well documented worldwide for decades. Protein sequences of FMDV isolates were visually examined for the presence of both the entire replikin and each of the component replikin amino acid residues observed in a particular replikin. For example, in the protein VP1 of FMDV type 0, the replikin (SEQ ID NO.: 3) "hkqkivapvk" was found to be conserved in 78% of the 236 isolates reported in PubMed, and each amino acid was found to be conserved in individual isolates as follows: his, 95.6%; lys, 91.8%; gln 92.3%; lys, 84.1%; ile, 90.7%; val, 91.8%; ala, 97.3%; pro, 96.2%; ala, 75.4%; and lys, 88.4%. The high rate of conservation suggests structural and functional stability of the replikin structure. Similarly, sequence conservation was observed in different isolates of HIV for its replikins, such as (SEQ ID NO.: 5) "kcfncgkegh" or (SEQ ID NO.: 6) "kvylawvpahk" in HIV Type 1 and (SEQ ID NO.: 7) "kcwncgkegh" in HIV Type 2 (Table 2). Other examples of conservation are seen in the constant presence of malignin in successive generations, over 10 years of tissue culture of glioma cells, and by the constancy of affinity of the glioma replikin for antimalignin antibody isolated by immunoadsorption from 8,090 human sera from the U.S., U.K., Europe and Asia (e.g., FIG. 5 and U.S. Pat. No. 6,242,578 B 1.).
As seen in FIG. 2, during anaerobic respiration when the rate of cell replication is increased, malignin is enriched. That is, malignin is found to increase not simply in proportion to the increase in cell number and total membrane proteins, but is enriched as much as tenfold in concentration, starting with 3% at rest and reaching 30% of total membrane protein. This clear demonstration of a marked increase in replikin concentration with glioma cell replication points to and is consistent with the presence of replikins. These replikins were sought by the 3-point recognition method and found in the proteins of various organisms which had been shown by mutation studies and other previous studies to be critical to replication. For example, replikins were identified in such proteins as "Saccharomyces cerevisiae replication binding protein" (SEQ ID NO.: 2) (hsikrelgiifdk); the "replication associated protein A of maize streak virus" (SEQ ID NO.: 8) (kyivcareahk and (SEQ ID NO.: 9) kekkpskdeimrdiish); the "replication-associated protein of Staphylococcus aureus" (SEQ ID NO.: 10) (kkektthnk); the "DNA replication protein of bovine herpes virus 4" (SEQ ID NO.: 11) (hkinitngqk); and the "Mealigrid herpes virus 1 replication binding protein" (SEQ ID NO.: 12) (hkdlyrllmk). Previous studies of tomato leaf curl gemini virus show that the regulation of virus accumulation appears to involve binding of amino acids 1-160 of the "replicating protein" of that virus to leaf DNA and to other replication protein molecules during virus replication. Analysis of this sequence showed that amino acids 1-163 of this "replicating protein" contain five replikins, namely: (SEQ ID NO.: 13) kfrinaknyfltyph, (SEQ ID NO.: 14) knletpvnklfiricrefh, (SEQ ID NO.: 15) hpniqaaksstdvk, (SEQ ID NO.: 16) ksstdvkaymdkdgdvldh, and (SEQ ID NO.: 17) kasalnilrekapkdfvlqfh.
Table 2 shows that replikin-containing proteins also are associated frequently with redox functions, and protein synthesis or elongation, as well as with cell replication. The association with metal-based redox functions, the enrichment of the replikin-containing glioma malignin concentration during anaerobic replication, and the cytotoxicity of antimalignin at low concentrations (picograms/cell) (FIG. 4c-f), all suggest that the replikins are related to central respiratory functions, which are perhaps less often subjected to the mutations characteristic of proteins of more superficial location or less central survival function.
Data on anti-replikin antibodies support replikin class unity. An anti-replikin antibody response has been quantified by immunoadsorption of serum antimalignin antibody to immobilized malignin (see Methods in U.S. Pat. No. 5,866,690). The abundant production of antimalignin antibody by administration to rabbits of the synthetic version of the 16-mer peptide whose sequence was derived from malignin, absent carbohydrate or other groups, has established rigorously that this peptide alone is an epitope, that is, it is a sufficient basis for this immune response (FIG. 3). The 16-mer peptide produced both IgM and IgG forms of the antibody. Antimalignin antibody was found to be increased in concentration in serum in 37% of 79 cases in the U.S. and Asia of hepatitis B and C, early, in the first five years of infection, long before the usual observance of liver cancer, which develops about fifteen to twenty-five years after infection. Relevant to both infectious hepatitis and HIV infections, transformed cells may be one form of safe haven for the virus: prolonging cell life and avoiding virus eviction, so that the virus remains inaccessible to anti-viral treatment.
A synthetic replikin vaccine such as the glioma replikin (SEQ ID NO.: 1) "kagvaflhkk" or the hepatitis C replikin (SEQ ID NO.: 18) "hyppkpgcivpak", or HIV replikins such as (SEQ ID NO.: 5) "kcfncgkegh" or (SEQ ID NO.: 6) "kvylawvpahk" may be used to augment antibody concentration in order to lyse the respective virus infected cells and release virus extracellularly where chemical treatment can then be effective. Recognin and/or replikin peptides may be administered to a subject to induce the immune system of the subject to produce anti-replikin and/or anti-recognin antibodies. Generally, a 0.5 to about 2 mg dosage, preferably a 1 mg dosage of each peptide is administered to the subject to induce an immune response. Subsequent dosages may be administered if desired.
In another embodiment of the invention, isolated recognin or replikin peptides may be used to generate antibodies. Various procedures known in the art may be used for the production of antibodies to replikin sequences or recognin sequences. Such antibodies include but are not limited polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments and fragments produced by an Fab expression library. Antibodies that are linked to a cytotoxic agent may also be generated.
For the production of antibodies various host animals may be immunized by injection with a replikin or recognin peptide, including but not limited to rabbits, mice, rats, and larger mammals. Various adjuvants may be used to enhance the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels, such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, key limpet hemocyanin, dintrophenol, and potentially useful human adjuvants such as BCG and Corynebacterium parvum.
Monoclonal antibodies to replikins or recognins may be prepared by using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein, (Nature, 1975, 256:495-497), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today, 4:72), and the EBV hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In addition, techniques developed for the production of chimeric antibodies (Morrison et al., 1984, Proc. Nat. Acad. Sci USA, 81:6851-6855) may be used. Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce replikin- or recognin-specific single chain antibodies.
Antibody fragments which contain binding sites for a replikin or recognin may be generated by known techniques. For example, such fragments include but are not limited to F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecules and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries can be generated (Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
The fact that antimalignin antibody is increased in concentration in human malignancy regardless of cancer cell type (FIG. 5), and that this antibody binds to malignant cells regardless of cell type now may be explained by the presence of the replikin structures present in most malignancies (FIG. 1 and Table 2). Population studies have shown that antimalignin antibody increases in concentration in healthy adults with age, and more so in high-risk families, as the frequency of cancer increases. An additional two-fold or greater antibody increase which occurs in early malignancy has been independently confirmed with a sensitivity of 97% in breast cancers 1-10 mm in size. Shown to localize preferentially in malignant cells in vivo, histochemically the antibody does not bind to normal cells but selectively binds to (FIGS. 4a,b) and is highly cytotoxic to transformed cells in vitro (FIG. 4c-f). Since in these examples the same antibody is bound by several cell types, that is, brain glioma, hematopoietic cells (leukemia), and small cell carcinoma of lung, malignant replikin class unity is again supported.
Antimalignin does not increase with benign proliferation, but specifically increases only with malignant transformation and replication in breast in vivo and returns from elevated to normal values upon elimination of malignant cells (FIG. 5). Antimalignin antibody concentration has been shown to relate quantitatively to the survival of cancer patients, that is, the more antibody, the longer the survival. Taken together, these results suggest that antireplikin antibodies may be a part of a mechanism of control of cell transformation and replication. Augmentation of this immune response may be useful in the control of replication, either actively with synthetic replikins as vaccines, or passively by the administration of anti-replikin antibodies, or by the introduction of non-immune based organic agents, such as for example, carbohydrates, lipids and the like, which are similarly designed to target the replikin specifically. For organisms such as diatom plankton, foot and mouth disease virus, tomato leaf curl gemini virus, hepatitis B and C, and HIV, and malignant cells, identified constituent replikins are useful as vaccines, and also may be usefully targeted for diagnostic purposes.
The replikin sequence structure is associated with the function of replication. Thus, whether the replikins of this invention are used for targeting sequences that contain replikins for the purpose of diagnostic identification, promoting replication, or inhibiting or attacking replication, for example, the structure-function relationship of the replikin is fundamental. Thus, while the structure of the replikin may be a part of a larger protein sequence, which may have been previously identified, it is necessary to utilize only the specific replikin structure when seeking to induce antibodies that will recognize and attach to the replikin fragment and thereby cause destruction of the cell. Even though the larger protein sequence may be known in the art as having a "replication associated function," vaccines using the larger protein often have failed or proven ineffective, even though they contain one or more replikin sequences.
Although the present inventor does not wish to be held to a single theory, the studies herein suggest that the prior art vaccines are ineffective because they are based on the use of the larger protein sequence. The larger protein sequence invariably has one or more epitopes (independent antigenic sequences that can induce specific antibody formation); replikin structures usually comprise one of these potential epitopes. The presence of other epitopes within the larger protein may interfere with adequate formation of antibodies to the replikin, See, e.g., Webster, R. G., J. Immunol., 97(2):177-183 (1966); and Webster et al., J. Infect. Dis., 134:48-58, 1976; Klenerman et al, Nature 394:421-422 (1998) for a discussion of the well-known phenomenon "original antigenic sin"). The formation of an antibody to a non-replikin epitope may allow binding to the cell, but not necessarily lead to cell destruction.
It is well known in the art that in the course of antibody production against a "foreign" protein, the protein is first hydrolyzed into smaller fragments. Usually fragments containing from about six to ten amino acids are selected for antibody formation. Thus, if hydrolysis of a protein does not result in replikin-containing fragments, anti-replikin antibodies will not be produced. In this regard, it is interesting that replikins contain lysine residues located six to ten amino acids apart, since lysine residues are known to bind to membranes.
Furthermore, replikin sequences contain at least one histidine residue. Histidine is frequently involved in binding to redox centers. Thus, an antibody that specifically recognizes a replikin sequence has a better chance of inactivating or destroying the cell in which the replikin is located, as seen with anti-malignin antibody, which is perhaps the most cytotoxic antibody yet described, being active at picograms per cell.
One of the reasons that vaccines directed towards a particular protein antigen of a disease causing agent have not been fully effective in providing protection against the disease (such as foot and mouth vaccine which has been developed against the VP1 protein or large segments of the VP1 protein) is that antibody to the replikins have not been produced. That is, either epitopes other than replikins present in the larger protein fragments may interfere according to the phenomenon of "original antigenic sin", and/or because the hydrolysis of larger protein sequences into smaller sequences for processing to produce antibodies results in loss of integrity of any replikin structure that is present, e.g., the replikin is cut in two and/or the histidine residue is lost in the hydrolytic processing. The present studies suggest that for an effective vaccine to be produced, the replikin sequences, and no other epitope, should be used as the vaccine.
Replikin or recognin DNA or RNA may have a number of uses for the diagnosis of diseases resulting from infection with a virus, bacterium or other replikin or recognin encoding agent. For example, replikin or recognin nucleotide sequences may be used in hybridization assays of biopsied tissue to diagnose the presence of a particular organism, e.g., Southern or Northern analysis, including in situ hybridization assays.
Also within the scope of the invention are oligoribonucleotide sequences, that include antisense RNA and DNA molecules and ribozymes that function to inhibit the translation of replikin- or recognin-containing mRNA. Both antisense RNA and DNA molecules and ribozymes may be prepared by any method known in the art. The antisense molecules can be incorporated into a wide variety of vectors for delivery to a subject.
Visual scanning of over three thousand sequences was performed in developing the present 3-point-recognition methods. However, data banks comprising nucleotide and/or amino acid sequences can also be scanned by computer for the presence of sequences meeting the 3 point recognition requirements.
The three point recognition method may also be modified to identify other useful compounds of covalently linked organic molecules, including other covalently linked amino acids, nucleotides, carbohydrates, lipids or combinations thereof. In this embodiment of the invention a sequence is screened for subsequences containing three or more desired structural characteristics. In the case of screening compounds composed of covalently linked amino acids, lipids or carbohydrates the subsequence of 7 to about 50 covalently linked units should contain (1) at least one first amino acid, carbohydrate or lipid residue located six to ten residues from a second of the first amino acid, carbohydrate or lipid residue; (2) at least one second amino acid, lipid or carbohydrate residue; and (3) at least 6% of the first amino acid, carbohydrate or lipid residue. In the case of screening nucleotide sequences, the subsequence of about 21 to about 150 nucleotides should contain (1) at least one first amino acid residue located within eighteen to thirty nucleotides from a second codon encoding the first amino acid residue; (2) at least one second amino acid residue; and (3) encodes at least 6% of said first amino acid residue.
According to another embodiment of the invention, the methods described herein may be performed by a computer. FIG. 6 is a block diagram of a computer available for use with the foregoing embodiments of the present invention. The computer may include a processor, an input/output device and a memory storing executable program instructions representing the 3-point-recognition methods of the foregoing embodiments. The memory may include a static memory, volatile memory and/or a nonvolatile memory. The static memory conventionally may be a read only memory ("ROM") provided on a magnetic, or an electrical or optical storage medium. The volatile memory conventionally may be a random access memory ("RAM") and may be integrated as a cache within the processor or provided externally from the processor as a separate integrated circuit. The non-volatile memory may be an electrical, magnetic or optical storage medium.
Process for Extraction, Isolation and Identification of Replikins and the Use of Replikins to Target, Label or Destroy Replikin-Containing Organisms
The following algae were collected from Bermuda water sites and either extracted on the same day or frozen at -20 degrees C. and extracted the next day. The algae were homogenized in a cold room (at 0 to 5 degrees C.) in 1 gram aliquots in neutral buffer, for example 100 cc. of 0.005M phosphate buffer solution, pH7 ("phosphate buffer") for 15 minutes in a Waring blender, centrifuged at 3000 rpm, and the supernatant concentrated by perevaporation and dialyzed against phosphate buffer in the cold to produce a volume of approximately 15 ml. The volume of this extract solution was noted and an aliquot taken for protein analysis, and the remainder was fractionated to obtain the protein fraction having a pK range between 1 and 4. The preferred method of fractionation is chromatography as follows:
The extract solution is fractionated in the cold room (4 degrees C.) on a DEAF cellulose (Cellex-D) column 2.5×11.0 cm, which has been equilibrated with 0.005M phosphate buffer. Stepwise eluting solvent changes are made with the following solutions:
Solution 1--4.04 g. NaH2PO4 and 0.5 g NaH2PO4 are dissolved in 15 litres of distilled water (0.005 molar, pH7);
Solution 2--8.57 g. NaH2PO4 is dissolved in 2,480 ml of distilled water;
Solution 3--17.1 g. of NaH2PO4 is dissolved in 2480 ml of distilled water (0.05 molar, pH 4.7);
Solution 4--59.65 g. of NaH2PO4 is dissolved in 2470 ml distilled water (0.175 molar);
Solution 5--101.6 g. of NaH2PO4 is dissolved in 2455 ml distilled water (pH 4.3);
Solution 6--340.2 g. of NaH2PO4 is dissolved in 2465 of distilled water (1.0 molar, pX-i 4.1);
Solution 7--283.63 g. of 80% phosphoric acid (H3PO4) is made up in 2460 ml of distilled water (1.0 molar, pH 1.0).
The extract solution, in 6 to 10 ml volume, is passed onto the column and overlayed with Solution 1, and a reservoir of 300 ml of Solution 1 is attached and allowed to drip by gravity onto the column. Three ml aliquots of eluant are collected and analyzed for protein content at OD 280 until all of the protein to be removed with Solution 1 has been removed from the column. Solution 2 is then applied to the column, followed in succession by Solutions 3, 4, 5, 6 aid 7 until all of the protein which can, be removed with each Solution is removed from the column. The eluates from Solution 7 are combined, dialyzed against phosphate buffer, the protein content determined of both dialysand and dialyzate, and both analyzed by gel electrophoresis. One or two bands of peptide or protein of molecular weight between 3,000 and 25,000 Daltons are obtained in Solution 7. For example the algae Caulerpa mexicana, Laurencia obtura, Cladophexa prolifera, Sargassum natans, Caulerpa verticillata, Halimeda tuna, and Penicillos capitatus, after extraction and treatment as above, all demonstrated in Solution 7 eluates sharp peptide bands in this molecular weight region with no contaminants. These Solution 7 proteins or their eluted bands are hydrolyzed, and the amino acid composition determined. The peptides so obtained, which have a lysine composition of 6% or greater are Replikin precursors. These Replikin peptide precursors are then determined for amino acid sequence by hydrolysis and mass spectrometry as detailed in U.S. Pat. No. 6,242,578 B1. Those which fulfill the criteria defined by the "3-point-recognition" method are identified as Replikins. This procedure can also be applied to obtain yeast, bacterial and any plant Replikins.
Using the same extraction and column chromatography separation methods as above in a) for algae, Replikens in virus-infected cells are isolated and identified.
c) Tumor Cells In Vivo and In Vitro Tissue Culture
Using the same extraction and column chromatography separation methods as above in a) for algae, Replikins in tumor cells are isolated and identified. For example, Replikin precursors of Astrocytin isolated from malignant brain tumors, Malignin (Aglyco 1OB) isolated from glioblastoma tumor cells in tissue culture, MCF7 mammary carcinoma cells in tissue culture, and P3J Lymphoma cells in tissue culture each treated as above in a) yielded Replikin precursors with lysine content of 9.1%, 6.7%, 6.7%, and 6.5% respectively. Hydrolysis and mass spectrometry of Aglyco 1OB as described in Example 10 U.S. Pat. No. 6,242,578 B1 produced the amino acid sequence, ykagvaflhkkndiide the 16-mer Replikin.
As an example of diagnostic use of Replikins: Aglyco 1OB or the 16-mer Repliken may be used as antigen to capture and quantify the amount of its corresponding antibody present in serum for diagnostic purposes are as shown in FIGS. 2,3,4 and 7 of U.S. Pat. No. 6,242,578 B1,
As an example of the production of agents to attach to Replikins for labeling, nutritional or destructive purposes: Injection of the 16-mer Replikin into rabbits to produce the specific antibody to the 16-mer Replikin is shown in Example 6 and FIGS. 9A and 9B of U.S. Pat. No. 6,242,578 B 1.
As an example of the use of agents to label Replikins: The use of antibodies to the 16-mer Replikin to label specific cells which contain this Replikin is shown in FIG. 5 and Example 6 of U.S. Pat. No. 6,242,578 B1.
As an example of the use of agents to destroy Replikins: The use of antibodies to the 16-mer Replikin to inhibit or destroy specific cells which contain this Replikin is shown in FIG. 6 of U.S. Pat. No. 6,242,578 B1.
From a proteomic point of view the construction of a "3-point recognition" template based on the new glioma peptide sequence led directly to identification of a biology-wide class of proteins having related structures and functions. The operation of the 3-point-recognition method resembles identification by the use of a "keyword" search; but instead of using the exact spelling of the keyword "kagvafihkk" as in a typical sequence homology search, or in the nucleotide specification of an amino acid, an abstraction of the keyword delimited by the "3-point-recognition" parameters is used. This delimited abstraction, although derived from a single relatively short amino acid sequence leads to identification of a class of proteins with structures that are defined by the same specifications. That particular functions, in this case transformation and replication, in addition to structures, turn out also to be shared by members of the exposed class suggests that these structures and functions are related. Thus, from this newly identified short peptide sequence, a molecular recognition `language` has been formulated, which previously has not been described. Further, the sharing of immunological specificity by diverse members of the class, as here demonstrated for the cancer replikins, suggests that B cells and their product antibodies recognize replikins by means of a similar recognition language. Since "3-point-recognition" is a proteomic method that specifies a particular class of proteins, using three or more different recognition points for other peptides similarly should provide useful information concerning other proteins classes. Further, the "3-point-recognition" method is applicable to other recognins, for example to the TOLL `innate` recognition of lipopolyssacharides of organisms.
Several embodiments of the present invention are specifically illustrated and described herein. However, it will be appreciated that modifications and variations of the present invention are encompassed by the above teachings and within the purview of the appended claims without departing from the spirit and intended scope of the invention.
90110PRTArtificial SequenceDescription of Artificial Sequence Synthetic glioma replikin peptide 1Lys Ala Gly Val Ala Phe Leu His Lys Lys1 5 10213PRTSaccharomyces cerevisiae 2His Ser Ile Lys Arg Glu Leu Gly Ile Ile Phe Asp Lys1 5 10310PRTGemini vinis virus 3His Lys Gln Lys Ile Val Ala Pro Val Lys1 5 10416PRTUnknownDescription of Unknown Virus recognin peptide 4Tyr Lys Ala Gly Val Ala Phe Leu His Lys Lys Asn Asp Ile Asp Glu1 5 10 15510PRTHuman immunodeficiency virus type 1 5Lys Cys Phe Asn Cys Gly Lys Glu Gly His1 5 10611PRTHuman immunodeficiency virus type 1 6Lys Val Tyr Leu Ala Trp Val Pro Ala His Lys1 5 10710PRTHuman immunodeficiency virus type 2 7Lys Cys Trp Asn Cys Gly Lys Glu Gly His1 5 10811PRTMaize streak virus 8Lys Tyr Ile Val Cys Ala Arg Glu Ala His Lys1 5 10917PRTMaize streak virus 9Lys Glu Lys Lys Pro Ser Lys Asp Glu Ile Met Arg Asp Ile Ile Ser1 5 10 15His109PRTStaphylococcus aureus 10Lys Lys Glu Lys Thr Thr His Asn Lys1 51110PRTBovine herpesvirus 4 11His Lys Ile Asn Ile Thr Asn Gly Gln Lys1 5 101210PRTMeleagrid herpesvirus 1 12His Lys Asp Leu Tyr Arg Leu Leu Met Lys1 5 101315PRTUnknownDescription of Unknown Virus recognin peptide 13Lys Phe Arg Ile Asn Ala Lys Asn Tyr Phe Leu Thr Tyr Pro His1 5 10 151419PRTUnknownDescription of Unknown Virus recognin peptide 14Lys Asn Leu Glu Thr Pro Val Asn Lys Leu Phe Ile Arg Ile Cys Arg1 5 10 15Glu Phe His1514PRTUnknownDescription of Unknown Virus recognin peptide 15His Pro Asn Ile Gln Ala Ala Lys Ser Ser Thr Asp Val Lys1 5 101619PRTUnknownDescription of Unknown Virus recognin peptide 16Lys Ser Ser Thr Asp Val Lys Ala Tyr Met Asp Lys Asp Gly Asp Val1 5 10 15Leu Asp His1721PRTUnknownDescription of Unknown Virus recognin peptide 17Lys Ala Ser Ala Leu Asn Ile Leu Arg Glu Lys Ala Pro Lys Asp Phe1 5 10 15Val Leu Gln Phe His201813PRTHepatitis C virus 18His Tyr Pro Pro Lys Pro Cys Gly Ile Val Pro Ala Lys1 5 10194PRTHomo sapiens 19Tyr Lys Ala Gly1206PRTHomo sapiens 20Tyr Lys Ala Gly Val Ala1 5217PRTHomo sapiens 21Tyr Lys Ala Gly Val Ala Phe1 5228PRTHomo sapiens 22Tyr Lys Ala Gly Val Ala Phe Leu1 52310PRTHomo sapiens 23Ala Gly Val Ala Phe Leu His Lys Lys Asn1 5 10245PRTHomo sapiens 24Gly Val Ala Phe Leu1 5254PRTHomo sapiens 25Val Ala Phe Leu1267PRTHomo sapiens 26Val Ala Phe Leu His Lys Lys1 5277PRTHomo sapiens 27Val Ala Phe Leu His Lys Lys1 5289PRTHomo sapiens 28Val Ala Phe Leu His Lys Lys Asn Asp1 5298PRTHomo sapiens 29Val Ala Phe His Lys Lys Asn Asp1 5304PRTHomo sapiens 30Ala Phe Leu His1318PRTHomo sapiens 31His Lys Lys Asn Asp Ile Asp Glu1 5326PRTHomo sapiens 32Lys Lys Asn Asp Ile Asp1 5336PRTHomo sapiens 33Lys Asn Asp Ile Asp Glu1 5348PRTCaldophera prolifera 34Lys Ala Ser Lys Phe Thr Lys His1 53512PRTIsolepis prolifera 35Lys Ala Gln Ala Glu Thr Gly Glu Ile Lys Gly His1 5 103610PRTSchizosaccharomyces pombe 36Lys Ser Phe Lys Tyr Pro Lys Lys His Lys1 5 103710PRTOryza sativa 37Lys Lys Ala Tyr Gly Asn Glu Leu His Lys1 5 10389PRTPenicillium marneffei 38Lys Val Asp Ile Val Thr His Gln Lys1 53912PRTDiseula dcstructiva 39Lys Leu Glu Glu Asp Ala Ala Tyr His Arg Lys Lys1 5 104017PRTOphiostoma novo-ulmi 40Lys Val Ile Leu Pro Leu Arg Gly Asn Ile Lys Gly Ile Phe Phe Lys1 5 10 15His4111PRTEntamoeba invadens 41Lys Leu Ile Leu Lys Gly Asp Leu Asn Lys His1 5 10428PRTHelicobacter pylori 42Lys Ser Val His Ala Phe Leu Lys1 5439PRTMycoplasma pulmonis 43Lys Val His Phe Phe Gln Leu Lys Lys1 5449PRTArabidopsis thaliana 44Lys Asp His Asp Phe Asp Gly Asp Lys1 54511PRTArabidopsis thaliana 45Lys Met Lys Gly Leu Lys Gln Lys Lys Ala His1 5 104612PRTArabidopsis thaliana 46Lys Glu Leu Ser Ser Thr Thr Gln Glu Lys Ser His1 5 10479PRTFeline immunodeficiency virus 47His Leu Lys Asp Tyr Lys Leu Val Lys1 5487PRTRous sarcoma virus 48Lys Lys Leu Arg His Glu Lys1 5497PRTAvian sarcoma virus 49Lys Lys Leu Arg His Asp Lys1 5507PRTHomo sapiens 50Lys Lys Leu Arg His Asp Lys1 5517PRTAvian sarcoma virus 51Lys Lys Leu Arg His Glu Lys1 5527PRTHomo sapiens 52Lys Lys Leu Arg His Glu Lys1 5538PRTHomo sapiens 53Lys Gln Ala His Glu Leu Ala Lys1 5548PRTPolyama virus 54Lys Thr His Arg Phe Ser Lys His1 5558PRTSindbis virus 55Lys Asn Leu His Glu Lys Ile Lys1 5569PRTHuman papilloamavirus type 71 56Lys His Arg Pro Leu Leu Gln Leu Lys1 5577PRTAvian encephalomyelitis virus 57Lys Ser Pro Asn His Val Lys1 5588PRTFeline sarcoma virus 58Lys Asn Ile His Leu Glu Lys Lys1 5598PRTHomo sapiens 59Lys Asn Ile His Leu Glu Lys Lys1 56010PRTPolyoma virus 60Lys Pro His Leu Ala Gln Ser Leu Glu Lys1 5 10619PRTPolyoma virus 61Lys Gln His Arg Glu Leu Lys Asp Lys1 5629PRTPolyoma virus 62Lys Gln His Arg Glu Leu Lys Asp Lys1 56312PRTMurine leukemia virus 63Lys Val Pro Val Leu Ile Ser Pro Thr Leu Lys His1 5 106413PRTHuman T-cell lymphotropic virus type 2 64Lys Ser Leu Leu Leu Glu Val Asp Lys Asp Ile Ser His1 5 106513PRTHomo sapiens 65Lys Ala Gly Ile Thr Ile Met Val Lys Arg Glu Tyr His1 5 10668PRTHomo sapiens 66Lys Ser Gly Lys His Leu Gly Lys1 5679PRTHomo sapiens 67Lys Arg Arg Glu Gln Leu Lys His Lys1 56810PRTHomo sapiens 68Lys Ser Phe Glu Val Ile Lys Val Ile His1 5 10698PRTHomo sapiens 69Lys Lys Lys His Thr Val Lys Lys1 5709PRTHomo sapiens 70Lys Ala Gln Lys Asp His Leu Ser Lys1 57110PRTHomo sapiens 71His Leu Lys Arg Val Lys Asp Leu Lys Lys1 5 107211PRTHomo sapiens 72Lys Tyr Gly Ser Pro Lys His Arg Leu Ile Lys1 5 107313PRTPapilloma virus type 11 73Lys Leu Lys His Ile Leu Gly Lys Ala Arg Phe Ile Lys1 5 107412PRTHomo sapiens 74Lys Gly Asp His Val Lys His Tyr Lys Ile Arg Lys1 5 107513PRTHomo sapiens 75Lys Glu Lys Leu Arg Asp Val Met Val Asp Arg His Lys1 5 107615PRTHomo sapiens 76Lys Leu Gln Ala Arg Gln Gln Gln Leu Leu Lys Lys Ile Glu His1 5 10 157714PRTHomo sapiens 77Lys Lys Gly Asn Arg Val Ser Pro Thr Met Lys Val Thr His1 5 10789PRTHomo sapiens 78Lys Glu Ile Pro Leu His Phe Arg Lys1 5798PRTHomo sapiens 79Lys Lys Lys Pro His Ile Lys Lys1 5809PRTHomo sapiens 80Lys Thr Arg His Asp Pro Leu Ala Lys1 58110PRTHomo sapiens 81Lys His His Pro Lys Asp Asn Leu Ile Lys1 5 108210PRTHomo sapiens 82Lys His Lys Arg Lys Lys Phe Arg Gln Lys1 5 108310PRTHomo sapiens 83Lys Ala Gly Val Ala Phe Leu His Lys Lys1 5 108410PRTHomo sapiens 84Lys His Lys Arg Lys Lys Phe Arg Gln Lys1 5 108510PRTHomo sapiens 85Lys Lys Lys Ser Lys Lys His Lys Asp Lys1 5 108611PRTHomo sapiens 86His Lys Ser Glu Lys Pro Ala Leu Pro Arg Lys1 5 108714PRTHomo sapiens 87Lys Lys Lys Lys Pro Ser Arg Leu Lys Gly Asp Asn Glu Lys1 5 108816PRTHomo sapiens 88Lys Thr Lys Lys Gly Asn Arg Val Ser Pro Thr Met Lys Val Thr His1 5 10 158918PRTHomo sapiens 89Lys His Lys Glu Lys Met Ser Lys Asp Gly Lys Lys Lys Lys Lys Lys1 5 10 15Ser Lys909PRTLegionella sp. 90Lys Ile His Leu Ile Ser Val Lys Lys1 5
Patent applications by Elenore S. Bogoch, New York, NY US
Patent applications by Samuel Bogoch, New York, NY US
Patent applications in class Binds specifically-identified amino acid sequence
Patent applications in all subclasses Binds specifically-identified amino acid sequence