Methods Of Making An Antibody And Compositions Thereof
The present invention relates to new methods for making antibodies that involves chemically modifying the antigen with methylglyoxal. The present invention further relates to antibodies made in this manner, and include antibodies specific to LL-37. The present invention pertains to assays that utilize anti-LL-37 antibodies and assess compounds that stimulate LL-37 production. Such compounds are useful in antimicrobial compositions.
This application claims the benefit of U.S. Provisional Application No. 61/169,845 filed Apr. 16, 2009, entitled “Antibacterial Compositions” by Toshihisa Kawai, et al.
The entire teachings of the above application are incorporated herein by reference.
GOVERNMENT SUPPORTThe invention was supported, in whole or in part, by a grant DE-18310 from the National Institute of Dental And Craniofacial Research (NIDCR), an institute of National Institute of Health (NIH). The Government has certain rights in the invention.
BACKGROUND OF THE INVENTIONA protein, known as CAP-18 is a human cathelicidin, which was first shown to be expressed in certain epithelial cells. The protein is characterized by a conserved N-terminal cathelin domain and a variable C-terminal antimicrobial domain. This C-terminal domain can be cleaved off by proteinases, releasing the active peptide, referred to as LL-37. LL-37 is believed to have antimicrobial activity against various microorganisms.
Compounds that stimulate the antimicrobial activity of LL-37 as well as expression of LL-37 could be useful in antimicrobial compositions. Antibodies that are specific to CAP-18 and to its active form, LL-37, would be helpful in assessing compounds that stimulate expression of LL-37.
Accordingly, a need exists to develop antibodies that are specific to CAP-18 and to LL-37. Additionally, a further need exists to develop assays to identify compounds that stimulate expression of LL-37 and induce antimicrobial activity.
SUMMARY OF THE INVENTIONThe present invention relates to methods of preparing an immunizing antigen to be used in making an antibody to a native antigen by selecting a native antigen having an amino acid sequence with a high content of arginine, lysine, cysteine or a combination thereof; and contacting the antigen with methylglyoxal in an amount sufficient to chemically modify the antigen to thereby obtain a chemically modified antigen; wherein the chemically modified antigen is prepared for use in making an antibody specific to the native antigen. In an embodiment, the amino acid sequence includes one having a content of arginine, lysine, cysteine or a combination thereof in a range between about 1% and about 30% total content. The present invention includes contacting the antigen with an amount of methylglyoxal in a range between about 1 nM and about 1 mM. Once chemically modified, the present invention includes, e.g., immunizing an animal with the chemically modified antigen.
In an embodiment, the present invention includes methods of making an antibody to a native antigen by selecting a native antigen having an amino acid sequence with a high content of arginine, lysine, cysteine or a combination thereof; contacting the antigen with methylglyoxal in an amount sufficient to chemically modify the antigen to thereby obtain a chemically modified antigen; and injecting the chemically modified antigen into an animal in an amount sufficient to illicit an immune response by the animal, wherein the immune response includes the production of one or more antibody secreting cells that is specific to the native antigen; and wherein the antibody to the native antigen is made. In an aspect, the antibody secreting cell is contacted with a myeloma cell under conditions suitable for fusion thereof to thereby obtain a hybridoma that secretes an antibody specific to the native antigen. The steps include, e.g., selecting for hybridomas made during fusion and isolating the hybridoma that secretes an antibody specific to the native antigen. Accordingly, the present invention includes antibodies made from the methods described herein.
The present invention also embodies an isolated polypeptide molecule that includes any one of the following amino acid sequence an amino acid sequence encoded by a nucleic acid molecule having a sequence of SEQ ID NO: 1, 3, or combination thereof; an amino acid sequence encoded by a complement of SEQ ID NO: 1, 3, or combination thereof; an amino acid sequence encoded by a nucleic acid molecule that hybridizes to SEQ ID NO: 1, 3, or combination thereof; or an amino acid sequence set forth in SEQ ID NO: 2, 4, or combination thereof.
Similarly, the present invention pertains to an isolated nucleic acid molecule having a nucleic acid sequence including any one of the following: a nucleic acid sequence set forth in SEQ ID NO: 1, 3, or combination thereof; a nucleic acid sequence that is a complement of SEQ ID NO: 1, 3, or combination thereof; a nucleic acid sequence that hybridizes to SEQ ID NO:1, 3, or combination thereof; or a nucleic acid sequence that encodes SEQ ID NO: 2, 4, or combination thereof. The isolated nucleic acid molecule of the present invention can include a nucleic acid sequence that encodes a detectable label (e.g., luciferase). A vector, plasmid or host cell that includes or is transferred with a nucleic acid molecule described herein in also encompassed by the present invention. Compositions include the polypeptide and/or nucleic acid sequences described herein related to the present invention.
The present invention also pertains to an antibody specific to LL-37, wherein the antibody comprises a variable heavy chain having an amino acid sequence, as described herein. The antibody can include a detectable label.
Yet in another embodiment, the present invention includes methods for measuring the presence, absence or amount of LL-37 in a sample by contacting the sample with an antibody that binds to LL-37 sufficiently to allow formation of a complex between the sample and the antibody, to thereby form an antigen-antibody complex; and assessing the presence, absence or amount of the antigen-antibody complex. The method, in an aspect, includes comparing the amount of the antigen-antibody complex to a control. In an embodiment, the method includes contacting the sample with a second antibody specific to LL-37 or said antigen-antibody complex. In a particular embodiment, the antibody that binds to LL-37 has an amino acid sequence set forth in SEQ ID NO: 1,3, or combination thereof. The anti-microbial peptide or the antibody can be bound to a solid support.
The present invention further embodies methods for assessing a compound for stimulation of LL-37 in a sample by contacting the sample with a compound to be tested to allow stimulation of LL-37; and contacting the sample with an antibody that binds to LL-37 sufficiently to allow formation of a complex between the sample and the antibody, to thereby form an antigen-antibody complex; assessing the presence, absence or amount of the antigen-antibody complex; wherein the presence or an increased level of the LL-37, as compared to a control, indicates that the compound stimulates production of the LL-37, and the absence or decreased level of LL-37, as compared to a control, indicates that the compound does not stimulate of the production of the LL-37.
Embodied by the present invention also includes methods for assessing a compound for stimulation of LL-37 in a sample by contacting the sample with the compound to be tested sufficiently to allow stimulation of LL-37; contacting the sample with at least two oligonucleotide primers in a polymerase chain reaction, wherein at least one of the oligonucleotide primers is specific for the nucleic acid sequence of LL-37, sufficiently to allow amplification of the primers; and detecting in the sample the amplified nucleic acid sequence; wherein the presence the amplified nucleic acid sequence indicates that the compound stimulates production of the LL-37, and the absence of the amplified nucleic acid sequence indicates that the compound does not stimulate of the production of the LL-37. In an aspect, at least one of the oligonucleotide primers has at least about 10 contiguous bases.
The present invention includes methods for assessing a compound for stimulation of LL-37 in a sample, by contacting the sample with the compound to be tested sufficiently to allow stimulation of LL-37; contacting the sample with one or more oligonucleotide probes specific for an isolated nucleic acid molecule of LL-37 under high stringency conditions, sufficiently to allow hybridization between the sample and the probe; and detecting the nucleic acid molecule that hybridizes to the oligonucleotide probe in the sample; wherein the presence the hybridization indicates that the compound stimulates production of the LL-37, and the absence of hybridization indicates that the compound does not stimulate the production of the LL-37.
The present invention is advantage for several reasons. The present invention provides a new method for increasing antigencity of an antigen, resulting in monoclonal antibodies having higher affinities. As a result, better assays can be developed. In particular, the present invention provides for an LL-37 assay to assess a compounds ability to stimulate anti-microbial activity. This assay provides a way to assess compounds for potentially new antibacterial medications and compositions.
A description of preferred embodiments of the invention follows.
The present invention relates to novel antibody fragments that have specificity to LL-37, and new methods for making antibodies. Additionally, the present invention relates to assays (e.g., high throughput assays) using the anti-LL-37 antibodies to assess compounds that stimulate LL-37 expression.
Human CAP-18, a human cathelicidin, was first identified in neutrophils and later shown to be expressed in various squamous epithelia, surface epithelial cells of the conducting airways, and serous and mucous cells of the submucosal glands, by keratinocytes in inflamed skin and by specific lymphocyte and monocyte populations. Human CAP-18 is the only human cathelicidin identified to date. It belongs to the cathelicidin family of antimicrobial peptides that are characterized by a conserved N-terminal cathelin domain and a variable C-terminal antimicrobial domain. This C-terminal domain can be cleaved off from the precursor by proteinases, releasing the active peptide. Exocytosed material from neutrophils contains hCAP-18 that has been proteolytically cleaved by proteinase-3 yielding the 4.5 kD active alpha helical peptide LL-37. LL-37 displays antimicrobial activity against a broad spectrum of microorganisms and possesses synergistic antibacterial effects with other antimicrobial peptides, such as defensins. It is believed that cathelicidins play a role in effective host defense against infection. A study has that mice deficient in the murine cathelicidin-related antimicrobial peptide suffer from more severe bacterial skin infections. Tjabringa, Sandra, et al., J. Immunol, 171:6690-6696 (2003). For another instance, deficiency in saliva LL-37 accords with occurrence of periodontal disease in patients with morbus Kostmann. Pütsep, Katrin, et al, Lancet, 360:1144-1149 (2002).
As used herein hCAP-18 is considered to be the pro-form of LL-37. After cleavage by proteinases, a 4 kD polypeptide results as the active form of LL-37. As used herein, both forms are referred to as LL-37. The antibodies of the present invention bind to both forms of the LL-37, the pro-form and the active form.
Antibodies specific to LL-37 have often been difficult to obtain and/or make. Antibodies to the active form of LL-37 have not been able to be developed. LL-37, when injected into mice to make monoclonal antibodies, does not illicit a strong antigen response because LL-37 is highly conserved between human and animal species, and has little or no MHC-class-II binding epitopes.
It has been determined that LL-37 has antimicrobial properties. For example, LL-37 is stimulated by certain compounds such as those having trihydroxybenzoate moieties. In particular, trihydroxybenzoate derivatives including 3,4,5-trihydroxybenzoic acid (i.e., gallic acid), its isoform, 2,3,4-trihydroxybenzoic acid, C1-4alkyl 3,4,5-trihydroxybenzoate (e.g., ethyl 3,4,5-trihydroxybenzoate), C1-4alkyl 2,3,4-trihydroxybenzoate (e.g., ethyl 2,3,4-trihydroxybenzoate), EGCG and mixtures thereof. In particular, “THBC” refers to chemical structures such as trihydroxybenzoates that comprise tri-hydroxy benzoic acid and its isoforms, which are found in the polyphenol extracts of natural plant products or in antioxidant food preservatives. As such trihydroxybenzoate moieties can be used as a positive control the LL-37 assay described herein.
Compounds for stimulating production of anti-microbial peptide LL-37 can be helpful in treating bacterial infection. Particularly, such compositions are particularly useful for treating a bacterial infection wherein (a) the bacteria is antibiotic resistant, (b) the patient is allergic to antibiotics; (c) the patient is immuno-compromised (e.g., HIV/AIDS patients); or (d) the infection is of an oral cavity. Additionally, LL-37 is a potent antimicrobial against various staphylococcal species and particularly virulent antibiotic-resistant strains of pathogenic bacteria, such as those found in Methicilin-resistant Staphylococcus aureus (MRSA). Such compounds can also be useful for 1) oral health care products, 2) skin and mucosal health care products, 3) antimicrobial drugs, and 4) neutraceutical products.
Antibodies for Assessing LL-37:Antibodies that are specific to LL-37 are helpful in determining compounds that stimulate LL-37 production, and would be helpful in treating the above disease or conditions. Compounds that stimulate LL-37 production can be included in compositions in which anti-microbial properties are needed. Since it is also reported that LL-37 can down regulate the production of pro-inflammatory cytokines, the increased production of LL-37 also results in the suppression of inflammatory responses. Scott M G, J Immunol. 2002; 169:3883-389 (2002). Compounds that stimulate LL-37 can be included in health compositions (e.g., toothpaste, mouth wash, floss) and medications to treat various disease, including those related to the gut (e.g., for Crohn's disease or Colitis), and the like.
Accordingly, a need exists for antibodies that are specific for LL-37 to screen for compounds that stimulate LL-37 production. A need exists for a method of making antibodies specific to LL-37, and other antigens for which antibodies are difficult to obtain.
The present invention relates to an antibody specific to LL-37. As described in the exemplification section, two antibodies were made and isolated, and are referred to as L2, made by hybridoma cell line called AL37-2, and L7, made by hybridoma cell line called AL37-7.
The term, “antibody,” encompasses polyclonal antibodies, monoclonal antibodies, single chain antibodies, VHH antibodies, chimeric, humanized, primatized, CDR-grafted, and veneered antibodies. This term further includes portions derived from different species, human antibodies which are native or derived from combinatorial libraries, and the like. Conventional techniques can chemically join together the various portions of these antibodies. Genetic engineering techniques can also prepare the antibody as a contiguous protein. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein.
Specifically, the term “antibody fragment” refers to portion of an immunoglobulin having specificity to the LL-37 or a portion thereof. The term, “antibody fragment”, is intended to encompass fragments from both polyclonal and monoclonal antibodies including transgenically produced antibodies, single-chain antibodies (scFvs), recombinant Fabs, and recombinant camelid heavy-chain-only antibodies (VHHs). VHHs are also referred to as nanobodies.
Functional fragments of antibodies, including fragments of chimeric, humanized, primatized, veneered or single chain antibodies, can also be produced. Functional fragments or portions of the foregoing antibodies include those which are reactive with the LL-37. For example, antibody fragments capable of binding to LL-37 or portion thereof, including, but not limited to scFvs, Fabs, VHHs, Fv, Fab, Fab′ and F(ab′)2 are encompassed by the invention. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For instance, papain or pepsin cleavage can generate Fab or F(ab′)2 fragments, respectively. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons has been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab′)2 heavy chain portion can be designed to include DNA sequences encoding the CH1 domain and hinge region of the heavy chain. Accordingly, the present invention encompasses a polynucleic acid that encodes the anti-LL-37 antibody fragments including heavy chain variable regions, which can optionally further nucleic acid that encodes a tag or detector (e.g., luciferase report gene).
The term “antibody” also includes various forms of modified antibodies. For example, modification may occur by directly or indirectly attaching a detectable label. The detectable labels may include a radioisotope, spin label, antigen label such as a FLAG tag, enzyme label, fluorescent or chemiluminescent group and the like.
The term “sample” includes fluid and/or cells from an oral swab (e.g., containing saliva and/or epithelial cells from the oral cavity), tissue, fluid, whole blood, plasma, serum and aqueous blood components from a patient.
Methods of Assessment of LL-37 and Methods of Making an Anti-LL-37 Antibody:Method for assessing the presence, absence or level of LL-37, in a sample, is encompassed by the present invention. Suitable assays include immunological methods, such as high throughput assays, radioimmunoassay, enzyme-linked immunosorbent assays (ELISA), chemiluminescence assays, and rapid immunochromatographic assays. A high throughput assays is a preferred embodiment of the present invention. Any method known now or developed later can be used for measuring LL-37 using the antibodies described herein.
The antibodies described herein are monoclonal antibodies. However, one or more variable regions of the monoclonal antibody can be constructed into a synthetic antibody having an antibody backbone, or variable fragments of the antibody can be used to detect LL-37 expression. Single chain antibody using one or more variable regions can be used. Accordingly, the term antibody is intended to encompass monoclonal antibodies, and functional fragments thereof.
In several of the preferred embodiments, immunological techniques detect the presence, absence, or levels of LL-37 described herein by means of an anti-LL-37 antibody (i.e., one or more antibodies). The term “anti-LL-37” includes one or more monoclonal antibodies or fragments thereof, and mixtures or cocktails thereof, and refers to antibodies specific to polypeptides having a sequence set forth in SEQ ID Nos: 2, 4, or combination thereof, or portions thereof.
Anti-LL-37 antibodies can be raised against appropriate immunogens, such as isolated and/or recombinant LL-37 polypeptides described herein, analogs or portion thereof (including synthetic molecules, such as synthetic peptides). Developing an antigenic response in the mice and obtaining high affinity antibodies to LL-37 was a challenge. Conserved amino acid sequences of LL-37 between human and animals in the range of about 70% to about 80% exist, and so mice injected with LL-37, even with an adjuvant, did not illicit a significant antigenic response that allowed one to obtain an specific and high affinity LL-37 antibody. Additionally, LL-37 has little or no MHC-class-II binding epitopes in its sequence, and therefore, does not easily allow absorption by the antigen presenting cells, making it more difficult to obtain the desired antigenic response in the animal. No MHC-class-II binding motif in LL-37 peptide was detected by RankPep algorism that evaluates the peptide binding capacity to mouse MHC-class-II [mouse strains testes, BALB/c and C57BL/6]: http://bio.dfci.harvard.edu/RANKPEP/.
To increase immunogenicity, the LL-37 protein underwent a chemical modification prior to being injected into an animal (e.g., mouse, rodent, rabbit, goat, monkey, camel, and the like) during the monoclonal antibody procedures. The chemical modification allows for a reaction with certain amino acids present in the protein. In this case, methylglyoxal was used to react with Arginine (R) and Lysine (K) present in LL-37. When methylglyoxal was combined with LL-37, the absorption by antigen presenting cells increased to thereby increase immunogenicity and develop a LL-37 specific antibody having a high affinity. Antigen presenting cells express the specific receptor (RAGE; the receptor for advanced glycation end products) for methylglyoxal-modified peptide and thereby allow for better absorption.
Chemical modification of proteins prior to injection into an animal can be applied to proteins other than LL-37 for the production of monoclonal antibodies. In particular, any protein having arginine (R), lysine (K) or cysteine (C) can be chemically modified with methylglyoxal for purposes of increasing an antigenic response.
Methylglyoxal is contacted with the antigen to be modified in an amount between about 1 nM and about 1 mM. See Example 1.
Advanced Glycation End products (AGEs) are the result of nonenzymatic glycation of proteins and lipids, as a consequence of a chain of chemical reactions after an initial glycation reaction. Many cells in the body, especially immune cells express the Receptor for Advanced Glycation End products (RAGE) that, when binding AGEs, contributes to immune and inflammatory responses. Recent research has shown that physiological glycation processes also involve the formation of advanced glycation endproducts (AGEs) by reactive alpha-oxoaldehydes particularly glyoxal, methylglyoxal and 3-deoxyglucosone (3-DG), and others.
Accordingly, a native antigen can be chemically modified, as described herein, by AGEs including by reactive alpha-oxoaldehydes particularly glyoxal, methylglyoxal and 3-deoxyglucosone (3-DG),
In an embodiment, the amino acid content of the protein to be chemically modified includes at least about 5% arginine (R), lysine (K), cysteine (C), or combination of both of its total amino acid content (e.g., about 1%, 5%, 10%, 15%, 20%, 25%, 30% or 40%). The LL-37 protein is abundant in both arginine and lysine, and specifically has about 5 arginines and 6 lysines, while lacking cyctein among a total 37 amino acids. The full amino acid sequence of LL-37 is LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (SEQ ID NO: 5). Using this method, two MAbs were generated and bind at high affinity to different epitopes present in LL-37. Chemically modifying LL-37 to increase antigencity allowed mAbs specific to the active form of LL-37, in addition to the pro-form of LL-37.
In one embodiment, antibodies are raised against an isolated, recombinant, active, chemically modified LL-37 polypeptide described herein or portion thereof (e.g., a peptide) or against a host cell which expresses recombinant or chemically modified LL-37. In addition, cells expressing recombinant or modified antigenic LL-37 polypeptides described herein, such as transfected cells, can be used as immunogens or in a screen for antibody which binds receptor.
Preparing the immunizing antigen can be done, as described above and any suitable technique, now known or later developed, can be used to produce polyclonal or monoclonal antibodies. The art contains a variety of these methods (see e.g., Kohler et al., Nature, 256: 495-497 (1975) and Eur. J. Immunol. 6: 511-519 (1976); Milstein et al., Nature 266: 550-552 (1977); Koprowski et al., U.S. Pat. No. 4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.); Current Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer '94), Ausubel, F. M. et al., Eds., (John Wiley & Sons: New York, N.Y.), Chapter 11, (1991)). Generally, fusing a suitable immortal or myeloma cell line, such as SP2/0, with antibody producing cells can produce a hybridoma. Animals immunized with the antigen of interest, after chemical modification, provide a cell that produces an antibody. Surprisingly, one or more antibodies specific for the native antigen are made and have a high binding affinity to the native antigen, even though the immunizing antigen was chemically modified. The immunized animal generally produces cells that make antibody that is specific to the antigen and these cells are typically cells from the spleen or lymph nodes. Cells from the spleen or lymph node are teased or separated from one another and cultured. Polyethylene glycol (PEG) or similar compound is used to fuse the myeloma cell line with the spleen or lymph node cells and a selective medium is used in which only fused cells can grow. Selective culture conditions isolate antibody producing hybridoma cells while limiting dilution techniques produce them. This mixture of cells is diluted and clones are grown from single parent cells generally in wells of a plate. The antibodies secreted by the different clones are then assayed for their ability to bind to the antigen. Researchers can use suitable assays such as ELISA to select antibody producing cells with the desired specificity. The hybridomas can be grown indefinitely in a suitable cell culture media, or they can be injected in mice into the peritoneal cavity, wherein they produce tumors containing an antibody-rich fluid called ascites fluid. The ascites that contains the antibody can be drained, bled or otherwise withdrawn from the animal. The antibody can be purified using standard purification techniques (e.g., ultra-filtration, dialysis, and chromatography).
Other suitable methods can produce or isolate antibodies of the requisite specificity. Examples of other methods include selecting recombinant antibody from a library or relying upon immunization of transgenic animals such as mice to make human monoclonal antibodies.
According to the method, an assay can determine the presence, absence or level of LL-37 peptides in a biological sample. Such an assay includes combining the sample to be tested with an antibody having specificity for LL-37 described herein, under conditions suitable for formation of a complex between antibody and LL-37, and detecting or measuring (directly or indirectly) the formation of a complex. In the event that a compound is being assessed, the sample can be combined with the compound to be tested and incubated under conditions to allow for expression of LL-37. The sample can be obtained directly or indirectly (e.g., provided by a healthcare provider), and can be prepared by a method suitable for the particular sample (e.g., saliva, epithelial cells from the oral cavity, urine, sputum, fecal matter, cerebral spinal fluid, whole blood, platelet rich plasma, platelet poor plasma, serum) and select an assay format. Methods of combining sample and antibody, and methods of detecting complex formation are also selected to be compatible with the assay format.
The assays described herein can be modified to assess the effect of a compound on LL-37 expression. In such a case, the compound can be introduced to the assay by contacting the compound with cells or samples that can express LL-37. The cells or sample can be subjected to compound to be tested to determine if the compound has an effect on LL-37 expression. Alternatively, the subject, from whom the sample is taken, can be subjected to the compound and then a sample is taken. The sample can then be assessed for LL-37 expression, as described herein. Since it has been determined that trihydroxybenzoate stimulates LL-37 expression, a trihydroxybenzoate moiety can be used as a positive control in the assay.
Suitable labels can be detected directly, such as radioactive, fluorescent or chemiluminescent labels. They can also be indirectly detected using labels such as enzyme labels and other antigenic or specific binding partners like biotin. Examples of such labels include fluorescent labels such as fluorescein, rhodamine, chemiluminescent labels such as luciferase, radioisotope labels such as 32P, 125I, 131I, enzyme labels such as horseradish peroxidase, and alkaline phosphatase, galactosidase, biotin, avidin, spin labels and the like. The detection of antibodies in a complex can also be done immunologically with a second antibody, which is then detected (e.g., by means of a label). Conventional methods or other suitable methods can directly or indirectly label an antibody. Labeled primary and secondary antibodies can be obtained commercially or prepared using methods know to one of skill in the art (see Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.).
In a preferred embodiment, the presence, absence, or level of LL-37 in a sample is determined using a high throughput assay, an ELISA assay, a sandwich ELISA assay, or immunochromatographic assay.
For detection of LL-37 in a suitable sample, a sample (e.g., saliva) is collected. Samples can be processed as known in the art. The compound to be assessed can be contacted with the sample, or in contact with the subject prior to taking the sample. For example, the compound can be part of an oral composition that the subject uses.
In an embodiment, LL-37 is determined using an ELISA assay or a sandwich ELISA assay. In one embodiment, murine L2 is used as capture antibody and murine L7 is used as detector antibody.
In an embodiment, the assay further includes combining a suitable sample, and optionally the compound to be tested, with a composition having an anti-LL-37 polypeptide antibody as detector (e.g., biotinylated anti-LL-37 polypeptides MAb and HRP-streptavidin, or HRP-conjugated anti-LL-37 polypeptides Mab), and a solid support, such as a microtiter plate, having an anti-LL-37 polypeptide capture antibody bound (directly or indirectly) thereto. The detector antibody binds to a different LL-37 polypeptide epitope from that recognized by the capture antibody, under conditions suitable for the formation of the complex. The assay then involves determining the formation of complex in the samples. The presence of or increase in LL-37 in a sample of an individual indicates the presence of a compound that increased expression or production of LL-37, whereas the absence of or decrease in a LL-37 polypeptide indicates the compound to be tested that does not increase expression or production of LL-37.
The solid support, such as a microtiter plate, dipstick, bead, pad, strip, or other suitable support, can be coated directly or indirectly with an anti-LL-37 polypeptide antibody or LL-37 specific antigen. For example, an anti-LL-37 polypeptide antibody can coat a microtiter well, or a biotinylated anti-LL-37 polypeptide Mab can be added to a streptavidin coated support. With respect to a immunochromatographic assay, a pad or strip can be coated with an antibody specific for the antigen, and when a sample having the one or more of antigens described herein comes into contact with the antibody, the complex can turn a color with aid of a detector, as further described herein. A variety of immobilizing or coating methods as well as a number of solid supports can be used, and can be selected according to the desired format.
In another embodiment, the sample (or an LL-37 polypeptide standard) is combined with the solid support simultaneously with the detector antibody, and optionally with a one or more reagents by which detection is monitored. For example, the sample can be combined with the solid support simultaneously with (a) HRP-conjugated anti-LL-37 polypeptide Mab, or (b) a biotinylated anti-LL-37 polypeptide Mab and HRP-streptavidin.
A known amount of an LL-37 polypeptide standard can be prepared and processed as described above for a suitable sample. This LL-37 polypeptide standard assists in quantifying the amount of LL-37 detected by comparing the level of LL-37 in the sample relative to that in the standard. In one embodiment, active LL-37 is used as a standard.
A physician, technician, apparatus or a qualified person can compare the amount of detected complex with a suitable control to determine if the LL-37 levels are elevated or not. A control can be the level of LL-37 in a sample take from the subject, but not subjected to the compound to be tested. A positive control can be the level of LL-37 subjected to a known compound that stimulates LL-37 express (e.g., trihydroxybenzoate moiety, or EGCG). A control can also be the average level of LL-37 for the particular sample, in a healthy population.
Typical assays for LL-37 are sequential assays in which a plate is coated with first antibody, sample is added, the plate is washed, second tagged antibody is added, and the plate is washed and bound second antibody is quantified. In another embodiment, a format in which antibodies and the sample are added simultaneously, in a competitive ELISA format, can achieve greater sensitivity.
A variety of methods can determine the amount of LL-37 in complexes. For example, when HRP is used as a label, a suitable substrate such as OPD can be added to produce color intensity directly proportional to the bound anti-LL-37 polypeptides mAb (assessed e.g., by optical density), and therefore to the LL-37 in the sample.
A technician, physician, qualified person or apparatus can compare the results to a suitable control such as a standard, or baseline levels of LL-37 in a sample from the same donor. For example, the assay can be performed using a known amount of LL-37 standard in lieu of a sample, and a standard curved established. One can relatively compare known amounts of the LL-37 standard to the amount of complex formed or detected.
The Anti-LL-37 Antibody Polypeptides and their Function
The present invention relates to isolated polypeptide molecules including anti-LL-37 antibodies or portions thereof. The present invention includes polypeptide molecules that contain any one of the anti-LL-37 antibody amino acid sequences (SEQ ID NO: 2, 4, or combinations thereof). The present invention also pertains to polypeptide molecules that are encoded by nucleic acid sequences, SEQ ID NO: 1, 3, or combinations thereof).
As used herein, the term “polypeptide” encompasses amino acid chains of any length (e.g., heavy chains of variable regions), including full length proteins, wherein the amino acid residues are linked by covalent peptide bonds. Thus, a polypeptide comprising a heavy chain of any of the sequences can consist entirely of the heavy chain, or can contain additional sequences. The additional sequences can be derived from the antibody backbones, or can be heterologous. In general, the polypeptides disclosed herein are prepared such that they are in substantially pure form. Preferably, the polypeptides are at least about 80% pure, more preferably at least about 90% pure and most preferably at least about 99% pure.
Polypeptides of the present invention referred to herein as “isolated” are polypeptides that separated away from other proteins and cellular material of their source of origin. Anti-LL-37 antibody polypeptides include peptides derived by the monoclonal/polyclonal process described herein, essentially pure protein, proteins produced by chemical synthesis, by combinations of biological and chemical synthesis and by recombinant methods. The proteins of the present invention have been isolated and characterized as to its physical characteristics using the procedures described herein and in the Exemplification. Such techniques include, for example, the monoclonal/polyclonal antibody making process, salting out, immunoprecipation, column chromatography, high pressure liquid chromatography or electrophoresis.
The compositions and methods of the present invention also encompass variants of the above polypeptides and DNA molecules. A polypeptide “variant,” as used herein, is a polypeptide that differs from the recited polypeptide only in conservative substitutions and/or modifications, such that the polypeptide binding and/or affinity pertaining to LL-37 properties (e.g., specificity, affinity, or combination thereof) of the polypeptide are retained. Polypeptide variants preferably exhibit at least about 60%, more preferably at least about 70% and most preferably at least about 80% homology to the identified polypeptides (e.g., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% homology). For polypeptides with LL-37 binding and/or affinity properties, variants can, alternatively, be identified by modifying the amino acid sequence of one of the above polypeptides, and evaluating the binding and/or affinity of the modified polypeptide. Such modified sequences can be prepared and tested using, for example, the representative procedures described herein (e.g., an ELISA). The homology between the two isolated antibody sequences is about 63%. Most of homologous sequence is derived from the frame work of hypervariable region of immunoglobulin. Please see below).
The polypeptides of the present invention, including a full length sequence, partial sequences, functional fragments and homologues, that allow for or assist in binding to or having affinity for LL-37. “Binding”, as used herein, refers to the ability to for the antibody to attach to or otherwise be specific for portion of LL-37, e.g., through hydrogen bonds. “Affinity” refers to the degree to which the antibody of the present invention binds to LL-37.
As used herein, a “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. In general, the following groups of amino acids represent conservative changes: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.
Variants can also, or alternatively, contain other modifications, including the deletion or addition of amino acids that have minimal influence on the antigenic properties, secondary structure and hydropathic nature of the polypeptide. For example, a polypeptide can be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post-translationally directs transfer of the protein. The polypeptide can also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide can be conjugated to an immunoglobulin Fc region.
The present invention also encompasses anti-LL-37 antibodies, chains thereof, polypeptides, variants thereof, or those having amino acid sequences analogous to the amino acid sequences of anti-LL-37 antibodies described herein. Such polypeptides are defined herein as anti-LL-37 antibody analogs (e.g., homologues), or mutants or derivatives. “Analogous” or “homologous” amino acid sequences refer to amino acid sequences with sufficient identity of any one of the anti-LL-37 antibody sequences so as to possess the biological activity (e.g., the ability to bind to LL-37 and/or have affinity for LL-37) of any one of the native anti-LL-37 antibodies. For example, an analog polypeptide can be produced with “silent” changes in the amino acid sequence wherein one, or more, amino acid residues differ from the amino acid residues of any one of the anti-LL-37 antibodies, yet still possess the function or biological activity of the anti-LL-37 antibodies. Examples of such differences include additions, deletions or substitutions of residues of the amino acid sequence of anti-LL-37 antibodies described herein. Also encompassed by the present invention are analogous polypeptides that exhibit greater, or lesser, biological activity of any one of the anti-LL-37 antibodies of the present invention. Such polypeptides can be made by mutating (e.g., substituting, deleting or adding) one or more amino acid or nucleic acid residues to any of the isolated anti-LL-37 antibodies described herein. Such mutations can be performed using methods described herein and those known in the art. In particular, the present invention relates to homologous polypeptide molecules having at least about 70% (e.g., 75%, 80%, 85%, 90% or 95%) identity or similarity with SEQ ID NO: 2, 4, or combination thereof. Percent “identity” refers to the amount of identical nucleotides or amino acids between two nucleotides or amino acid sequences, respectfully. As used herein, “percent similarity” refers to the amount of similar or conservative amino acids between two amino acid sequences.
Homologous polypeptides can be determined using methods known to those of skill in the art. Initial homology searches can be performed at NCBI against the GenBank, EMBL and SwissProt databases using, for example, the BLAST network service. Altschuler, S. F., et al., J. Mol. Biol., 215:403 (1990), Altschuler, S. F., Nucleic Acids Res., 25:3389-3402 (1998). Computer analysis of nucleotide sequences can be performed using the MOTIFS and the FindPatterns subroutines of the Genetics Computing Group (GCG, version 8.0) software. Protein and/or nucleotide comparisons were performed according to Higgins and Sharp (Higgins, D. G. and Sharp, P. M., Gene, 73:237-244 (1988) e.g., using default parameters).
Additionally, the individual isolated polypeptides of the present invention are biologically active or functional fragments (e.g., fragments of heavy chains). The present invention includes fragments of these isolated amino acid sequences, yet possesses the function or biological activity of the sequence. For example, polypeptide fragments comprising variable regions of the anti-LL-37 antibodies can be designed and expressed by well-known laboratory methods. Fragments, homologues, or analogous polypeptides can be evaluated for biological activity, as described herein.
The present invention also encompasses biologically active derivatives or analogs of the above described anti-LL-37 antibodies, referred to herein as peptide mimetics. Mimetics can be designed and produced by techniques known to those of skill in the art. (see e.g., U.S. Pat. Nos. 4,612,132; 5,643,873 and 5,654,276). These mimetics can be based, for example, on an anti-LL-37 antibody amino acid sequence and maintain the relative position in space of the corresponding amino acid sequence. These peptide mimetics possess biological activity similar to the biological activity of the corresponding peptide compound, but possess a “biological advantage” over the corresponding anti-LL-37 antibody amino acid sequence with respect to one, or more, of the following properties: solubility, stability and susceptibility to hydrolysis and proteolysis.
Methods for preparing peptide mimetics include modifying the N-terminal amino group, the C-terminal carboxyl group, and/or changing one or more of the amino linkages in the peptide to a non-amino linkage. Two or more such modifications can be coupled in one peptide mimetic molecule. Modifications of peptides to produce peptide mimetics are described in U.S. Pat. Nos. 5,643,873 and 5,654,276. Other forms of the anti-LL-37 antibodies, encompassed by the present invention, include those which are “functionally equivalent.” This term, as used herein, refers to any nucleic acid sequence and its encoded amino acid, which mimics the biological activity of the anti-LL-37 antibody and/or functional domains thereof.
Anti-LL-37 antibody Sequences, Plasmids, Vectors and Host Cells
The present invention, in one embodiment, includes an isolated nucleic acid molecule having a sequence of SEQ ID NO: 1, 3, or combinations thereof. The present invention includes sequences of the variable heavy chain, as recited in
As used herein, the terms “DNA molecule” or “nucleic acid molecule” include both sense and anti-sense strands, cDNA, genomic DNA, recombinant DNA, RNA, and wholly or partially synthesized nucleic acid molecules. A nucleotide “variant” is a sequence that differs from the recited nucleotide sequence in having one or more nucleotide deletions, substitutions or additions. Such modifications can be readily introduced using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis as taught, for example, by Adelman et al. (DNA 2:183, 1983). Nucleotide variants can be naturally occurring allelic variants, or non-naturally occurring variants. Variant nucleotide sequences preferably exhibit at least about 70%, more preferably at least about 80% and most preferably at least about 90% homology to the recited sequence. Such variant nucleotide sequences will generally hybridize to the recited nucleotide sequence under stringent conditions. In one embodiment, “stringent conditions” refers to prewashing in a solution of 6×SSC, 0.2% SDS; hybridizing at 65° Celsius, 6×SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in 1×SSC, 0.1% SDS at 65° C. and two washes of 30 minutes each in 0.2×SSC, 0.1% SDS at 65° C.
The present invention also encompasses isolated nucleic acid sequences that encode anti-LL-37 antibodies, and in particular, those which encode a polypeptide molecule having an amino acid sequence of SEQ ID NO: 2, 4, or combinations thereof. These nucleic acid sequences encode polypeptides that bind LL-37 and/or are involved the functions further described herein.
As used herein, an “isolated” gene or nucleotide sequence which is not flanked by nucleotide sequences which normally (e.g., in nature) flank the gene or nucleotide sequence (e.g., as in genomic sequences) and/or has been completely or partially purified from other transcribed sequences (e.g., as in a cDNA or RNA library). Thus, an isolated gene or nucleotide sequence can include a gene or nucleotide sequence which is synthesized chemically or by recombinant means. Nucleic acid constructs contained in a vector are included in the definition of “isolated” as used herein. Also, isolated nucleotide sequences include recombinant nucleic acid molecules and heterologous host cells, as well as partially or substantially or purified nucleic acid molecules in solution. In vivo and in vitro RNA transcripts of the present invention are also encompassed by “isolated” nucleotide sequences.
The nucleic acid sequences of the present invention include homologues nucleic acid sequences. “Analogous” or “homologous” nucleic acid sequences refer to nucleic acid sequences with sufficient identity of any one of the anti-LL-37 antibody nucleic acid sequences, such that once encoded into polypeptides, they possess the biological activity of any one of the anti-LL-37 antibodies described herein. For example, an analogous nucleic acid molecule can be produced with “silent” changes in the sequence wherein one, or more, nucleotides differ from the nucleotides of any one of the anti-LL-37 antibodies described herein, yet, once encoded into a polypeptide, still possesses its function or biological activity. Examples of such differences include additions, deletions or substitutions. Also encompassed by the present invention are nucleic acid sequences that encode analogous polypeptides that exhibit greater, or lesser, biological activity of the anti-LL-37 antibodies of the present invention. In particular, the present invention is directed to nucleic acid molecules having at least about 70% (e.g., 75%, 80%, 85%, 90% or 95%) identity with SEQ ID NO: 1, 3, or combinations thereof.
The nucleic acid molecules of the present invention, including the full length sequences, the partial sequences, functional fragments and homologues, once encoded into polypeptides, bind to LL-37, or has the function of the polypeptide, as further described herein. The homologous nucleic acid sequences can be determined using methods known to those of skill in the art, and by methods described herein including those described for determining homologous polypeptide sequences. Immunogenic antigens can then be sequenced using techniques such as Edman chemistry. See Edman and Berg, Eur. J. Biochem. 80:116-132, 1967.
Also encompassed by the present invention are nucleic acid sequences, DNA or RNA, which are substantially complementary to the DNA sequences encoding the anti-LL-37 antibodies of the present invention, and which specifically hybridize with their DNA sequences under conditions of stringency known to those of skill in the art. As defined herein, substantially complementary means that the nucleic acid need not reflect the exact sequence of the anti-LL-37 antibody sequences, but must be sufficiently similar in sequence to permit hybridization with nucleic acid sequence under high stringency conditions. For example, non-complementary bases can be interspersed in a nucleotide sequence, or the sequences can be longer or shorter than the nucleic acid sequence, provided that the sequence has a sufficient number of bases complementary to the sequence to allow hybridization therewith. Conditions for stringency are described in e.g., Ausubel, F. M., et al., Current Protocols in Molecular Biology, (Current Protocol, 1994), and Brown, et al., Nature, 366:575 (1993); and further defined in conjunction with certain assays.
Also encompassed by the present invention are nucleic acid sequences, genomic DNA, cDNA, RNA or a combination thereof, which are substantially complementary to the DNA sequences of the present invention and which specifically hybridize with the anti-LL-37 antibody nucleic acid sequences under conditions of sufficient stringency (e.g., high stringency) to identify DNA sequences with substantial nucleic acid identity.
The present invention also includes portions and other variants of anti-LL-37 antibodies that are generated by synthetic or recombinant means. Synthetic polypeptides having fewer than about 100 amino acids, and generally fewer than about 50 amino acids, can be generated using techniques well known to those of ordinary skill in the art. For example, such polypeptides can be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Applied BioSystems, Inc., Foster City, Calif., and can be operated according to the manufacturer's instructions. Variants of a native antigen can generally be prepared using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis. Sections of the DNA sequence can also be removed using standard techniques to permit preparation of truncated polypeptides.
In another embodiment, the present invention includes nucleic acid molecules (e.g., probes or primers) that hybridize to the anti-LL-37 antibody sequences, SEQ ID NO:1, 3, or combinations thereof under high or moderate stringency conditions. In one aspect, the present invention includes molecules that are or hybridize to at least about 20 contiguous nucleotides or longer in length (e.g., 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000). Such molecules hybridize to one of anti-LL-37 antibody nucleic acid sequences under high stringency conditions. The present invention includes such molecules and those that encode a polypeptide that has the functions or biological activity described herein.
Typically the nucleic acid probe comprises a nucleic acid sequence (e.g. SEQ ID NO: 1, 3, or combinations thereof) and is of sufficient length and complementarity to specifically hybridize to a nucleic acid sequence that encodes anti-LL-37 polypeptide. For example, a nucleic acid probe can be at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% the length of the anti-LL-37 antibody nucleic acid sequence. The requirements of sufficient length and complementarity can be easily determined by one of skill in the art. Suitable hybridization conditions (e.g., high stringency conditions) are also described herein. Additionally, the present invention encompasses fragments of the polypeptides of the present invention or nucleic acid sequences that encodes a polypeptide wherein the polypeptide has the biologically activity of the anti-LL-37 polypeptides recited herein.
Such fragments are useful in making polypeptide fragments that encode the variable region of the anti-LL-37 antibody. Antibody fragments that bind LL-37 are useful in blocking or inhibiting LL-37 function. They are also useful in performing assays to detect or determine levels of LL-37. With such fragments, compounds that affect LL-37 activity can be assayed. For example, nucleic acid fragments which encode any one of the domains described herein are also implicated by the present invention.
Stringency conditions for hybridization refers to conditions of temperature and buffer composition which permit hybridization of a first nucleic acid sequence to a second nucleic acid sequence, wherein the conditions determine the degree of identity between those sequences which hybridize to each other. Therefore, “high stringency conditions” are those conditions wherein only nucleic acid sequences which are very similar to each other will hybridize. The sequences can be less similar to each other if they hybridize under moderate stringency conditions. Still less similarity is needed for two sequences to hybridize under low stringency conditions. By varying the hybridization conditions from a stringency level at which no hybridization occurs, to a level at which hybridization is first observed, conditions can be determined at which a given sequence will hybridize to those sequences that are most similar to it. The precise conditions determining the stringency of a particular hybridization include not only the ionic strength, temperature, and the concentration of destabilizing agents such as formamide, but also factors such as the length of the nucleic acid sequences, their base composition, the percent of mismatched base pairs between the two sequences, and the frequency of occurrence of subsets of the sequences (e.g., small stretches of repeats) within other non-identical sequences. Washing is the step in which conditions are set so as to determine a minimum level of similarity between the sequences hybridizing with each other. Generally, from the lowest temperature at which only homologous hybridization occurs, a 1% mismatch between two sequences results in a 1° C. decrease in the melting temperature (Tm) for any chosen SSC concentration. Generally, a doubling of the concentration of SSC results in an increase in the Tm of about 17° C. Using these guidelines, the washing temperature can be determined empirically, depending on the level of mismatch sought. Hybridization and wash conditions are explained in Current Protocols in Molecular Biology (Ausubel, F. M. et al., eds., John Wiley & Sons, Inc., 1995, with supplemental updates) on pages 2.10.1 to 2.10.16, and 6.3.1 to 6.3.6.
High stringency conditions can employ hybridization at either (1) 1×SSC (10×SSC=3 M NaCl, 0.3 M Na3-citrate . . . 2H2O (88 g/liter), pH to 7.0 with 1 M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denatured calf thymus DNA at 65° C., (2) 1×SSC, 50% formamide, 1% SDS, 0.1-2 mg/ml denatured calf thymus DNA at 42° C., (3) 1% bovine serum albumin (fraction V), 1 mM Na2 . . . EDTA, 0.5 M NaHPO4 (pH 7.2) (1 M NaHPO4=134 g Na2HPO4 . . . 7H2O, 4 ml 85% H3PO4 per liter), 7% SDS, 0.1-2 mg/ml denatured calf thymus DNA at 65° C., (4) 50% formamide, 5×SSC, 0.02 M Tris-HCl (pH 7.6), 1×Denhardt's solution (100X=10 g Ficoll 400, 10 g polyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to 500 ml), 10% dextran sulfate, 1% SDS, 0.1-2 mg/ml denatured calf thymus DNA at 42° C., (5) 5×SSC, 5×Denhardt's solution, 1% SDS, 100 μg/ml denatured calf thymus DNA at 65° C., or (6) 5×SSC, 5×Denhardt's solution, 50% formamide, 1% SDS, 100 μg/ml denatured calf thymus DNA at 42° C., with high stringency washes of either (1) 0.3-0.1×SSC, 0.1% SDS at 65° C., or (2) 1 mM Na2EDTA, 40 mM NaHPO4 (pH 7.2), 1% SDS at 65° C. The above conditions are intended to be used for DNA-DNA hybrids of 50 base pairs or longer. Where the hybrid is believed to be less than 18 base pairs in length, the hybridization and wash temperatures should be 5-10° C. below that of the calculated Tm of the hybrid, where Tm in ° C.=(2× the number of A and T bases)+(4× the number of G and C bases). For hybrids believed to be about 18 to about 49 base pairs in length, the Tm in ° C.=(81.5° C.+16.6(log 10M)+0.41(% G+C)−0.61 (% formamide)−500/L), where “M” is the molarity of monovalent cations (e.g., Na+), and “L” is the length of the hybrid in base pairs.
Moderate stringency conditions can employ hybridization at either (1) 4×SSC, (10×SSC=3 M NaCl, 0.3 M Na3-citrate. 2H2O (88 g/liter), pH to 7.0 with 1 M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denatured calf thymus DNA at 65° C., (2) 4×SSC, 50% formamide, 1% SDS, 0.1-2 mg/ml denatured calf thymus DNA at 42° C., (3) 1% bovine serum albumin (fraction V), 1 mM Na2 . . . EDTA, 0.5 M NaHPO4 (pH 7.2) (1 M NaHPO4=134 g Na2HPO4 . . . 7H2O, 4 ml 85% H3PO4 per liter), 7% SDS, 0.1-2 mg/ml denatured calf thymus DNA at 65° C., (4) 50% formamide, 5×SSC, 0.02 M Tris-HCl (pH 7.6), 1×Denhardt's solution (100X=10 g Ficoll 400, 10 g polyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to 500 ml), 10% dextran sulfate, 1% SDS, 0.1-2 mg/ml denatured calf thymus DNA at 42° C., (5) 5×SSC, 5×Denhardt's solution, 1% SDS, 100 μg/ml denatured calf thymus DNA at 65° C., or (6) 5×SSC, 5×Denhardt's solution, 50% formamide, 1% SDS, 100 μg/ml denatured calf thymus DNA at 42° C., with moderate stringency washes of 1×SSC, 0.1% SDS at 65° C. The above conditions are intended to be used for DNA-DNA hybrids of 50 base pairs or longer. Where the hybrid is believed to be less than 18 base pairs in length, the hybridization and wash temperatures should be 5-10° C. below that of the calculated Tm of the hybrid, where Tm in ° C.=(2× the number of A and T bases)+(4× the number of G and C bases). For hybrids believed to be about 18 to about 49 base pairs in length, the Tm in ° C.=(81.5° C.+16.6(log 10M)+0.41(% G+C)−0.61 (% formamide)−500/L), where “M” is the molarity of monovalent cations (e.g., Na+), and “L” is the length of the hybrid in base pairs.
Low stringency conditions can employ hybridization at either (1) 4×SSC, (10×SSC=3 M NaCl, 0.3 M Na3-citrate . . . 2H2O (88 g/liter), pH to 7.0 with 1 M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denatured calf thymus DNA at 50° C., (2) 6×SSC, 50% formamide, 1% SDS, 0.1-2 mg/ml denatured calf thymus DNA at 40° C., (3) 1% bovine serum albumin (fraction V), 1 mM Na2 . . . EDTA, 0.5 M NaHPO4 (pH 7.2) (1 M NaHPO4=134 g Na2HPO4 . . . 7H2O, 4 ml 85% H3PO4 per liter), 7% SDS, 0.1-2 mg/ml denatured calf thymus DNA at 50° C., (4) 50% formamide, 5×SSC, 0.02 M Tris-HCl (pH 7.6), 1×Denhardt's solution (100X=10 g Ficoll 400, 10 g polyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to 500 ml), 10% dextran sulfate, 1% SDS, 0.1-2 mg/ml denatured calf thymus DNA at 40° C., (5) 5×SSC, 5×Denhardt's solution, 1% SDS, 100 μg/ml denatured calf thymus DNA at 50° C., or (6) 5×SSC, 5×Denhardt's solution, 50% formamide, 1% SDS, 100 μg/ml denatured calf thymus DNA at 40° C., with low stringency washes of either 2×SSC, 0.1% SDS at 50° C., or (2) 0.5% bovine serum albumin (fraction V), 1 mM Na2EDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS. The above conditions are intended to be used for DNA-DNA hybrids of 50 base pairs or longer. Where the hybrid is believed to be less than 18 base pairs in length, the hybridization and wash temperatures should be 5-10° C. below that of the calculated Tm of the hybrid, where Tm in ° C.=(2× the number of A and T bases)+(4× the number of G and C bases). For hybrids believed to be about 18 to about 49 base pairs in length, the Tm in ° C.=(81.5° C.+16.6(log 10M)+0.41(% G+C)−0.61 (% formamide)−500/L), where “M” is the molarity of monovalent cations (e.g., Na.+), and “L” is the length of the hybrid in base pairs.
Anti-LL-37 antibody can be produced recombinantly using a DNA sequence that encodes the antigen, which has been inserted into an expression vector and expressed in an appropriate host cell. DNA sequences encoding an anti-LL-37 antibody can, for example, be identified by assaying the antibody or fragment thereof against LL-37 and assessing binding and/or affinity characteristics.
The invention also provides vectors, plasmids or viruses containing one or more of the anti-LL-37 antibody nucleic acid molecules (e.g., having the sequence of SEQ ID NO:1, 3, or combinations thereof). Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available or readily prepared by a skilled artisan. Additional vectors can also be found, for example, in Ausubel, F. M., et al., Current Protocols in Molecular Biology, (Current Protocol, 1994) and Sambrook et al., “Molecular Cloning: A Laboratory Manual,” 2nd ED. (1989).
Recombinant polypeptides containing portions and/or variants of a native antigen can be readily prepared from a DNA sequence encoding the polypeptide using a variety of techniques well known to those of ordinary skill in the art. For example, supernatants from suitable host/vector systems which secrete recombinant protein into culture media can be first concentrated using a commercially available filter. Following concentration, the concentrate can be applied to a suitable purification matrix such as an affinity matrix or an ion exchange resin. Finally, one or more reverse phase HPLC steps can be employed to further purify a recombinant protein.
Any of a variety of expression vectors known to those of ordinary skill in the art can be employed to express recombinant polypeptides of this invention. Expression can be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells. Preferably, the host cells employed are E. coli, yeast or a mammalian cell line such as COS or CHO. The DNA sequences expressed in this manner can encode naturally occurring antigens, portions of naturally occurring antigens, or other variants thereof.
Uses of plasmids, vectors or viruses containing the cloned anti-LL-37 antibody sequences or fragments include one or more of the following; (1) generation of hybridization probes for detection and measuring level of LL-37 in tissue; (2) generation of mRNA or protein in vitro or in vivo; and (3) generation of transgenic non-human animals or recombinant host cells.
In one embodiment, the present invention encompasses host cells transformed with the plasmids, vectors or viruses described above. Nucleic acid molecules can be inserted into a construct which can, optionally, replicate and/or integrate into a recombinant host cell, by known methods. The host cell can be a eukaryote or prokaryote and includes, for example, yeast (such as Pichia pastorius or Saccharomyces cerevisiae), bacteria (such as E. coli, or Bacillus subtilis), animal cells or tissue, insect Sf9 cells (such as baculoviruses infected SF9 cells) or mammalian cells (somatic or embryonic cells, Human Embryonic Kidney (HEK) cells, Chinese hamster ovary cells, HeLa cells, human 293 cells and monkey COS-7 cells). Host cells suitable in the present invention also include a mammalian cell, a bacterial cell, a yeast cell, an insect cell, and a plant cell.
The nucleic acid molecule can be incorporated or inserted into the host cell by known methods. Examples of suitable methods of transfecting or transforming cells include calcium phosphate precipitation, electroporation, microinjection, infection, lipofection and direct uptake. “Transformation” or “transfection” as used herein refers to the acquisition of new or altered genetic features by incorporation of additional nucleic acids, e.g., DNA. “Expression” of the genetic information of a host cell is a term of art which refers to the directed transcription of DNA to generate RNA which is translated into a polypeptide. Methods for preparing such recombinant host cells and incorporating nucleic acids are described in more detail in Sambrook et al., “Molecular Cloning: A Laboratory Manual,” Second Edition (1989) and Ausubel, et al. “Current Protocols in Molecular Biology,” (1992), for example.
The host cell is then maintained under suitable conditions for expression and recovery of the anti-LL-37 antibody of the present invention. Generally, the cells are maintained in a suitable buffer and/or growth medium or nutrient source for growth of the cells and expression of the gene product(s). The growth media are not critical to the invention, are generally known in the art and include sources of carbon, nitrogen and sulfur. Examples include Luria broth, Superbroth, Dulbecco's Modified Eagles Media (DMEM), RPMI-1640, M199 and Grace's insect media. The growth media can contain a buffer, the selection of which is not critical to the invention. The pH of the buffered Media can be selected and is generally one tolerated by or optimal for growth for the host cell.
The host cell is maintained under a suitable temperature and atmosphere. Alternatively, the host cell is aerobic and the host cell is maintained under atmospheric conditions or other suitable conditions for growth. The temperature should also be selected so that the host cell tolerates the process and can be for example, between about 13-40 degrees Celsius.
EXEMPLIFICATION Example 1 Method of Making an AntibodyThe following methods were used to significantly increase the antigenecity of most of peptides by a chemical modification.
A chemical called “methylglyoxal” can react to arginine (R), lysine (K) or cysteine (C) present in a peptide and increases the peptide's absorption by antigen presenting cells which express the specific receptor (RAGE; the receptor for advanced glycation endproducts) for methylglyoxal-modified peptide 29.7% of total 37 amino acids are R or K.
The rule for this technology is that arginine (R), lysine (K) or cysteine (C) should be present in the peptide. Fortunately, LL-37 is abundant in both arginine (R) and lysine (K).
Using this method, at least three MAbs were generated and have bind at high affinity to different epitopes present in LL-37. Since such chemical modification has never published, none of commercial bio-labs or research scientists can develop high affinity antibodies to LL-37.
Methylglyoxal (MG; 40% aqueous solution) was purchased from Sigma.
Synthetic LL-37 (1 mg/ml; generated by a commercial service of Biomatik corporation) was incubated with MG (1 mm) in phosphate buffered saline (PBS), pH 7.4, at 37° C. for 7 days, with adjustment of the pH to 7.4 with sodium hydroxide solution (5 M) as required. To remove unbound free MG, the resultant LL-37 solution incubated with MG was dialyzed against PBS using a dialysis membrane (1,000 MW cut) for 3 days at 4 C. After filtration of MG-modified LL-37 through 0.2 um pore size disc filter, the MG-modified LL-37 was stored in a −20C freezer.
The MG-modified LL-37 or intact non-treated LL-37 was immunized to BALB/c mice (8 week old male) in the Freund's Complete and Incomplete Adjuvants (FCA and FIA, respectively, from Difco). In order to make the emulsion of adjuvant, 1 vol of LL-37 (1 mg/ml) in PBS was mixed with 1 vol of either FCA or FIA, followed by ultra-sonication of the mixture on ice for 10-20 seconds that completes the formation of emulsion. Primary immunization of mice with LL-37 as well as secondary immunization was carried out by s.c. injection of FCA emulsion and FIA emulsion (100 ug peptide/200 ul emulsion/mouse, respectively) at the two week interval. Two weeks after the secondary immunization, booster immunization was conducted by the i.v. injection of peptide in PBS (100 ug/peptide/100 ul PBS/mouse). Blood sera were sampled from the mice after the 3-5 days from the booster immunization, and IgG antibody response to LL-37 was monitored using an ELISA. For the development of monoclonal antibody, animals were sacrificed 4 days after the booster immunization, and spleen cells isolated from the sacrificed mice was fused with NS-0 mouse myeloma cells using polyethylene glycol (PEG).
Using these Anti-LL-37 MAbs, the Following was Done:
1) Develop ELISA to quantify the amount of LL-37 (none of the commercially available antibody can be applicable for ELISA).
2) Detect both pro-form (18 kD) and active form (4 kD) of LL-37 by Western blot (All commercially available antibodies react to only pro-form (18 kD) LL-37 or called CAP18)
Two hybridoma cell lines that produce IgG antibody (IgG1 isotype) that react specifically to LL-37 were established. The two hybridoma cell lines were termed as AL37-2 and AL37-7, respectively. Total RNA from each hybridoma cell line was extracted using a RNA-Bee solution (phenol and guanidine thiocyanate, TEL-TEST inc.) and chloroform. The amount of isolated total RNAs were quantified by spectrometry at 260 and 280 nm. Five micrograms of each total RNA sample was used to perform a first-strand cDNA synthesis using a reverse transcriptase (SuperScript First-Strand Synthesis System, Invitrogen). The generated cDNA was then used as a template DNA for the subsequent PCR using an Expand high-fidelity PCR system (Roche) with sense primer; 5′-CCTGGGGCTTCAGTGAAG-3′ and antisense primer; 5′-GTGTCTTGCACAGTAATA-3′. Such primer set was designed to specifically bind to the frame work sequence present in the hyper variable region of mouse IgG1 heavy chain. The resulting PCR products were separated by electrophoresis on a 1.5% agarose gel and purified by gel extraction using Nucleospin (Clontech). The PCR products were subjected to DNA sequence using a commercial service (Genwiz, Inc.). The sequences are shown in
Induction of LL-37 and hBD2 mRNA Expression in Human Gingival Epithelial Cells:
Both LL-37 and hBD2 mRNA expression in OBA9 cells are induced after a 4-hour exposure to green tea polyphenol extract, Teaflan 90S (EGCG 50%, ECG 13%), and purified EGCG (95% pure, SIGMA) at concentrations of 0.1, 1.0 and 10 ug/ml. EGCG increases the production of LL-37 and hBD2 at the mRNA level in the human gingival epithelial cell line OBA9 (
Using ELISA systems to detect LL-37 (Forsyth in-house) and hBD2 (Peprotech), 33 natural compounds (20 ug/ml) are examined for their ability to induce LL-37 and hBD2 from OBA9 cells. Although the sensitivity of the hBD2 ELISA is higher than that of LL-37 ELISA (10 pg/ml vs 1 ng/ml, respectively), the induction of LL-37 is more prominent than hBD2 (
The Trihydroxybenzoate Derivatives of the Invention and rosemarinic acid, courmarin, catechin gallate, epicatechin gallate, epigallocatechin, and tannic acid are tested to induce LL-37 from OBA9 cells in the absence of EGCG. Rosemarinic acid and courmarin, which lack 3,4,5-trihydroxy benzoate, showed little or no induction of LL-37 expression by OBA9 cells. Therefore, gallic acid appears to be more potent than 3,4,-dihydroxy benzoate in the induction of LL-37 expression by cultured gingival epithelial cells.
Culture Supernatant of EGCG-Treated Human Gingival Epithelial Cells:The culture supernatant isolate from OBA9 is treated with 1) EGCG, 2) catechin, 3) 2,4-dihydroxy benzoic acid, and 4) 3,4,5-tri-hydroxy benzoic acid (20 ug/ml, respectively) and examined for their bactericidal effects on Actinobacillus actinomycetemcomitans Y4 (Aa Y4). The culture supernatant of EGCG- and 3,4,5-tri-hydroxy benzoic acid-treated OBA9 cells show more bactericidal effects than control non-treated OBA9 supernatant or supernatant of OBA9 treated with catechin or with 2,4-dihydroxy benzoic acid. Therefore, 3,4,5-tri-hydroxy benzoic acid and EGCG which contains gallic acid both appear to have higher antimicrobial effects than the compounds which do not have the structural isomers of gallic acid.
In Vivo Induction of LL-37 mRNA and Protein Expression by Human Cheek Epithelial Cells:
Cheek epithelium is utilized for monitoring LL-37 mRNA and protein expression to determine if Trihydroxybenzoate Derivatives of the Invention can induce LL-37 in vivo. Total RNA is extracted from cheek epithelial cells after application of mouthwash with EGCG (40 ug/ml), and LL-37 mRNA expression by the cheek epithelial cells is determined by RT-PCR. mRNA for LL-37 is up-regulated after only 5 minutes from application of the mouthwash with EGCG, whereas the internal control β-actin is expressed constitutively. The isolated cheek cells are also subjected to Western blot analysis for protein expression of LL-37 by cheek epithelium using anti-LL-37 monoclonal antibody. The expression of both the proform (18 kD, also termed as hCAP18) and secreted form (4.5 kD) of LL-37 (12) in cheek epithelial cells is induced by Trihydroxybenzoate Derivatives of the Invention (e.g., EGCG (95% SIGMA), or by EGCG (90% Teavigo)). The immunohistochemical analysis for LL-37 expression stained with anti-LL-37 monoclonal antibody also demonstrate the increased expression of LL-37 in the cheek epithelial cells after ex vivo stimulation.
Tri-Hydroxy Benzoic Acid:In order to investigate the structure-function relationship underlying EGCG-mediated induction of LL-37 from human gingival epithelial cell line OBA9, different isomers of benzoic acids are examined for their effects on the LL-37 expression by OBA9 cells. Each isomer of benzoic acids is coupled to NHS-activated Sepharose gel (Pierce) using a cross-linker, 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC, Pierce). The isomers of benzoic acids include 1) 2,6-di-hydroxy benzoic acid, 2) 2,3,4-tri-hydroxy benzoic acids, 3) 3,4,5-tri-hydroxy benzoic acids, and 4) 2,4,6-tri-hydroxy benzoic acids. Only 2,3,4-tri-hydroxy benzoic acids and 3,4,5-tri-hydroxy benzoic acids, but not 2,6-di-hydroxy benzoic acids or 2,4,6-tri-hydroxy benzoic acids, show the induction of LL-37 expression by OBA9 cells.
The teachings of related Application No. (not yet assigned, attorney Docket no: 0024.0012-002), filed Apr. 16, 2010, entitled “Antibacterial Compositions” by Toshihisa Kawai, et al. are incorporated herein by reference in this entirety.
The relevant teachings of all the references, patents and/or patent applications cited herein are incorporated herein by reference in their entirety.
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.
Claims
1. A method of preparing an immunizing antigen to be used in making an antibody to a native antigen, the method comprises the steps of:
- a. selecting a native antigen having an amino acid sequence with a high content of arginine, lysine, cysteine or a combination thereof; and
- b. contacting the antigen with methylglyoxal in an amount sufficient to chemically modify the antigen to thereby obtain a chemically modified antigen; wherein the chemically modified antigen is prepared for use in making an antibody specific to the native antigen.
2. The method of claim 1, further comprising selecting an amino acid sequence having a content of arginine, lysine, cysteine or a combination thereof in a range between about 1% and about 30%.
3. The method of claim 1, further comprising contacting the antigen with an amount of methylglyoxal in a range between about 1 nM and about 1 mM.
4. The method of claim 1, further comprising immunizing an animal with the chemically modified antigen.
5. A method of making an antibody to a native antigen, the method comprises the steps of:
- a. selecting a native antigen having an amino acid sequence with a high content of arginine, lysine, cysteine or a combination thereof;
- b. contacting the antigen with methylglyoxal in an amount sufficient to chemically modify the antigen to thereby obtain a chemically modified antigen; and
- c. injecting the chemically modified antigen into an animal in an amount sufficient to illicit an immune response by the animal, wherein the immune response includes the production of one or more antibody secreting cells that is specific to the native antigen;
- wherein the antibody to the native antigen is made.
6. The method of claim 5, further comprising contacting the antibody secreting cell with a myeloma cell under conditions suitable for fusion thereof to thereby obtain a hybridoma that secretes an antibody specific to the native antigen.
7. The method of claim 6, further comprising selecting for hybridomas made during fusion.
8. The method of claim 7, further comprising isolating the hybridoma that secretes an antibody specific to the native antigen.
9. An antibody made from the methods of claim 5.
10. An isolated polypeptide molecule comprises an amino acid sequence selected from the group consisting of:
- a. an amino acid sequence encoded by a nucleic acid molecule having a sequence of SEQ ID NO: 1, 3, or combination thereof;
- b. an amino acid sequence encoded by a complement of SEQ ID NO: 1, 3, or combination thereof;
- c. an amino acid sequence encoded by a nucleic acid molecule that hybridizes to SEQ ID NO: 1, 3, or combination thereof; and
- d. an amino acid sequence set forth in SEQ ID NO: 2, 4, or combination thereof.
11. An isolated nucleic acid molecule having a nucleic acid sequence selected from the group consisting of:
- a. a nucleic acid sequence set forth in SEQ ID NO: 1, 3, or combination thereof;
- b. a nucleic acid sequence that is a complement of SEQ ID NO: 1, 3, or combination thereof;
- c. a nucleic acid sequence that hybridizes to SEQ ID NO:1, 3, or combination thereof; and
- d. a nucleic acid sequence that encodes SEQ ID NO: 2, 4, or combination thereof.
12. The isolated nucleic acid molecule of claim 11, further comprising a nucleic acid sequence that encodes a detectable label.
13. The isolated nucleic acid molecule of claim 12, wherein the detectable label is luciferase.
14. A vector or plasmid that comprises the nucleic acid molecule of claim 11.
15. A host cell transformed with the nucleic acid sequence of claim 11.
16. A composition that comprises the polypeptide sequence of claim 10 and a physiologically acceptable carrier.
17. An antibody specific to LL-37, wherein the antibody comprises a variable heavy chain having an amino acid sequence of claim 10.
18. The antibody specific to LL-37 of claim 16, further comprising a detectable label.
19. A method for measuring the presence, absence or amount of LL-37 in a sample, wherein the method comprises:
- a. contacting the sample with an antibody that binds to LL-37 sufficient to allow formation of a complex between the sample and the antibody, to thereby form an antigen-antibody complex; and
- b. assessing the presence, absence or amount of the antigen-antibody complex.
20. The method of claim 19, further comprising comparing the amount of the antigen-antibody complex to a control.
21. The method of claim 19, wherein said antibody is detectably labeled.
22. The method of claim 19, wherein the method further includes contacting the sample with a second antibody specific to LL-37 or said antigen-antibody complex.
23. The method of claim 19, wherein the antibody that binds to LL-37 has an amino acid sequence set forth in SEQ ID NO: 1,3, or combination thereof.
24. The method of claim 19, wherein the anti-microbial peptide or the antibody is bound to a solid support.
25. A method for assessing a compound for stimulation of LL-37 in a sample, wherein the method comprises:
- a. contacting the sample with a compound to be tested to allow stimulation of LL-37;
- b. contacting the sample with an antibody that binds to LL-37 sufficient to allow formation of a complex between the sample and the antibody, to thereby form an antigen-antibody complex; and
- c. assessing the presence, absence or amount of the antigen-antibody complex; wherein the presence or an increased level of the LL-37, as compared to a control, indicates that the compound stimulates production of the LL-37, and the absence or decreased level of LL-37, as compared to a control, indicates that the compound does not stimulate of the production of the LL-37.
26. A method for assessing a compound for stimulation of LL-37 in a sample, the method comprises:
- a. contacting the sample with the compound to be tested sufficiently to allow stimulation of LL-37;
- b. contacting the sample with at least two oligonucleotide primers in a polymerase chain reaction, wherein at least one of the oligonucleotide primers is specific for the nucleic acid sequence of LL-37, sufficiently to allow amplification of the primers; and
- c. detecting in the sample the amplified nucleic acid sequence;
- wherein the presence the amplified nucleic acid sequence indicates that the compound stimulates production of the LL-37, and the absence of the amplified nucleic acid sequence indicates that the compound does not stimulate of the production of the LL-37.
27. The method of claim 26, wherein at least one of the oligonucleotide primers comprises at least about 10 contiguous bases.
28. A method for assessing a compound for stimulation of LL-37 in a sample, the method comprises:
- a. contacting the sample with the compound to be tested sufficiently to allow stimulation of LL-37;
- b. contacting the sample with one or more oligonucleotide probes specific for an isolated nucleic acid molecule of LL-37 under high stringency conditions, sufficiently to allow hybridization between the sample and the probe; and
- c. detecting the nucleic acid molecule that hybridizes to the oligonucleotide probe in the sample;
- wherein the presence the hybridization indicates that the compound stimulates production of the LL-37, and the absence of hybridization indicates that the compound does not stimulate of the production of the LL-37.
Type: Application
Filed: Apr 16, 2010
Publication Date: Dec 30, 2010
Inventors: Toshihisa Kawai (Brookline, MA), Philip P. Stashenko (Medfield, MA), Yoshitaka Hosokawa (Tokushima), Kazuhisa Ohara (Hiroshima-city)
Application Number: 12/762,212
International Classification: A61K 39/395 (20060101); C07K 14/00 (20060101); A61K 39/00 (20060101); C12P 21/04 (20060101); C07K 16/00 (20060101); C07H 21/04 (20060101); C12N 15/63 (20060101); C12N 5/10 (20060101); A61K 38/16 (20060101); C12Q 1/68 (20060101); G01N 33/53 (20060101); A61P 37/00 (20060101);
