Not applicable.
Current State of the Market/Application:
There is a huge market in diagnostics for the use of a peroxidase in both chromogenic, as well as chemiluminescent detection. For example, in most of the tissue diagnostic tests, HRP (horse radish peroxidase) conjugates are utilized, either with an antibody, primary or secondary, or with something like avidin/streptavidin, which can detect a biotinylated preferential locator. This includes many of the automated IHC systems on the market. This could also include approaches like ISH (in situ hybridization), which is labeled with hapten and then detected with a peroxidase antibody conjugate
However, in storage or in use, the HRP is not a particularly stable peroxidase, hence having a peroxidase that performs similarly to HRP and is significantly more stable would be a significant advantage to the diagnostics industry, especially in automated tissue diagnostics, as well as in point of care (POC) and lateral flow immunoassay systems, where stability is essential.
Disclosed is an analytical composition of a peroxidase discrete polyethylene glycol (PEG) conjugate, which conjugate is capable of providing a detectable condition in the presence of peroxidase and hydrogen peroxide. The analytical composition of claim 1, conjugated to avidin/streptavidin. The analytical composition can be conjugated to a biologically active group, which may be one or more of an antibody or an antibody fragment. The antibody or antibody fragment is one or more of a single chain antibody, a divalent antibody, a tetrabody, a triabody, a diabody, a minibody, a camelid derived antibody, or a shark derived antibody.
The analytical composition also can be conjugated with a targeting agent, which may be one or more of a nanoparticle, MMP (matrix metalloprotease) inhibitor substrate, an RGD peptide, engineered scaffold, liposome, a PLGA, silica, or a metal.
The PEG in the analytical composition may be represented as, PEGx, where x ranges between 2 to about 72 and can range between about 8 and about 24.
For a fuller understanding of the nature and advantages of the present method and process, reference should be had to the following detailed description taken in connection with the accompanying drawings, in which:
The drawings will be described in greater detail below.
Disclosed currently is the thermal stability of the soybean peroxidase with streptavidin or a thermally stable antibody using a discrete PEG based conjugation system to create a discrete PEG base conjugate.
Using and conjugating with discrete PEG (preferred), we see a linear response over a concentration range that is not seen using the conventional conjugation chemistry in the HRP conjugates. This is assumed to be due to the use of the discrete PEG conjugation chemistry and creating a very hydrophilic conjugate that makes the SA optimally available to the biotin and eliminates the non-specific binding that causes the non-linearity at high concentrations.
The term “stability” as used herein is related to the activity of the SBP being maintained over a longer period of time and at a higher temperature than other peroxidases, especially the common HRP. More preferably “stability” refers to the activity of the enzyme remaining essentially constant over a period of time at room temperature or above. It also relates to reducing or eliminating the ambient or heat-induced aggregation that often is seen with biologically active compounds.
The terms “discrete PEG based conjugate” or “discrete PEG based conjugation” is used herein to relate to conjugation methods that use the discrete PEG spacers as the linker unit, or more simply as a wavy line, .
“Wavy line”, “”. The wavy line, , is a linear chain containing a discrete polyethylene glycol (dPEG) residue optionally substituted with N, S, Si, Se, or P, and optionally having branching side chains. Such wavy line may contain aryl groups, alkyl groups, amino acids, and the like. The end components of have independently chemically reactable or reactive moieties at each end. These are incorporated such that each end can be reacted independently during its incorporation to any branched discrete PEG construct or intermediates in the process of building the same. When the ends of the wavy line are chemically reactive groups, they can be reactive on their own, or can be masked groups, e.g., an azide as an amine, or protected reactable groups that must be converted to chemically reactive groups. The chemical construction of these compositions can have multiple wavy lines, being the same or different. When they are different, the end groups, “A” must not react at the same time, and can be biorthogonal, or other combinations of masked or protected reactable groups known in the art. (Ref.: E. M. Sletten and C. R. Bertozzi, “Biorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality,” Angew. Chem. Int. Ed., 48, 6974-6998(2009); G. Hermanson, Bioconjugate Techniques, 2nd Edition, Academic Press, 2008; T. Greene and Wutz). The use is the same as that disclosed in our U.S. Pat. No. 7,888,536. Some of the more preferred options are shown in Tables 1 and 2. The chemically reactable or chemically reactive moieties as end groups on the wavy line also can be converted to biologically active groups. Generally this will be a final step or series of steps in the building of the compositions in this disclosure.
Furthermore, the wavy line , which in the art also is termed a linker or spacer or spacer arm, means a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches a “preferential locator”, like an antibody, or to a diagnostic or therapeutic group, like a drug moiety. Exemplary linker abbreviations include: MC=6-maleimidocaproyl, MPS=maleimidopropanoyl, val-cit=valine-citrulline, dipeptide site in protease-cleavable linker, ala-phe=alanine-phenylalanine, dipeptide site in protease-cleavable linker, PAB=paminobenzyloxycarbonyl, SPP=N-Succinimidyl 4-(2-pyridylthio) pentanoate, SMCC=N-Succinimidyl 4-(Nmaleimidomethyl) cyclohexane-I carboxylate, SIAB=NSuccinimidyl (4-iodo-acetyl) aminobenzoate, and these and others known in the art can be and preferred to be used in the disclose composition containing a linear discrete PEG, as well as those containing discrete PEG constructs described and defined below.
The wavy line also is defined such that it contributes important properties to be incorporated into or as part of the composition, as part of controlling and including the length and size of the discrete PEG. These also have practical considerations as they variably control the accessibility for reaction and also the dynamics and size on the final construct, as well as other design functions desirable to the application, e.g., cleavable/releasable, multifunctional. And the optimal lengths of the wavy line are preferred in this disclosure, where for discrete PEGx, x is preferred from 2 to 72, more preferred from 8-24. The inherent properties of the discrete PEG as a type of PEG are well known in the art.
The wavy line is defined to optionally incorporate a bond or chemical construct known in the art that will result in a cleavable bond or construct. Also see Tables 1 and 2 below for the preferred chemistries to use in this disclosure as part of the definition for the wavy line, .
“Detectable” conjugate is the term used for any signal that can be produced by the peroxidase that can be detected by methods available in the art. These are most often chromogenic or chemiluminescent (ECL, Supersignal), but others can be used where appropriate.
The term “preferential locator” as used herein often can be used largely interchangeably with ligand or “targeting group” and can be either a “diagnostic group” or a “therapeutic group”or the like. Broadly, preferential locators are molecularly targeted agent defined as drugs that target growth factor receptors and signal transduction pathways. NPOA molecule is used for targeting molecular entities, cells, tissues or organs in a biological system. With respect to neoplastic tissue (cancer cells), a “preferential locator” (or “locator”) specifically binds a marker produced by or associated with, for example, neoplastic tissue, antibodies and somatostatin congeners being representative such locators. Broader, however, a “locator” includes a substance that preferentially concentrates at the tumor sites by binding with a marker (the cancer cell or a product of the cancer cell, for example) produced by or associated with neoplastic tissue or neoplasms. Appropriate locators today primarily include antibodies (whole and monoclonal), antibody fragments, chimeric versions of whole antibodies and antibody fragments, and humanized versions thereof. It will be appreciated, however, that single chain antibodies (SCAs, such as disclosed in U.S. Pat. No. 4,946,778, incorporated herein by reference) and like substances have been developed and may similarly prove similarly efficacious. For example, genetic engineering has been used to generate a variety of modified antibody molecules with distinctive properties. These include various antibody fragments and various antibody formats. An antibody fragment is intended to mean any portion of a complete antibody molecule. These include terminal deletions and protease digestion-derived molecules, as well as immunoglobulin molecules with internal deletions, such as deletions in the IgG constant region that alter Fc mediated antibody effector functions. Thus, an IgG heavy chain with a deletion of the Fc CH2 domain is an example of an antibody fragment. It is also useful to engineer antibody molecules to provide various antibody formats. In addition to single chain antibodies, useful antibody formats include divalent antibodies, tetrabodies, triabodies, diabodies, minibodies, camelid derived antibodies, shark derived antibodies, and other antibody formats. Aptamers form yet a further class of preferential locators. All of these antibody-derived molecules are example of preferential locators.
Various suitable antibodies (including fragments, single chains, domain deletions, humanized, etc.) include, for example, B72.3, CC49, V59, and 3E8 (see U.S. Pat. No. 8,119,132), all directed against adenocarcinomas.
In addition to antibodies, biochemistry and genetic engineering have been used to produce protein molecules that mimic the function of antibodies. Avimers are an example of such molecules. See, generally, Jeong, et al., “Avimers hold their own”, Nature Biotechnology Vol. 23 No. 12 (December 2005). Avimers are useful because they have low immunogenicity in vivo and can be engineered to preferentially locate to a wide range of target molecules, such as cell specific cell surface molecules. Although such substances may not be subsumed within the traditional definition of “antibody”, avimer molecules that selectively concentrate at the sites of neoplastic tissue are intended to be included within the definition of preferential locator. Thus, the terms “locator” was chosen, to include present-day antibodies and equivalents thereof, such as avimers, as well as other engineered proteins and substances, either already demonstrated or yet to be discovered, which mimic the specific binding properties of antibodies in the inventive method disclosed therein. (Refs.: “Engineered protein scaffolds as next-generation antibody therapeutics,” Michaela Gebauer and Arne Skerra, Current Opinion in Chemical Biology, 2009, 13, 245-255; “Adnectins: engineered target-binding protein therapeutics,” D Lipovsek, Protein Engineering, Design & Selection, 2010, 1-7.)
For other disease types or states, other compounds will serve as preferential locators.
The term “preferential locator” also can include terms like “targeting group” and “targeting agent” and are intended to mean a moiety that is (1) able to direct the entity to which it is attached (e.g., therapeutic agent or marker) to a target cell, for example to a specific type of tumor cell or (2) is preferentially activated at a target tissue, for example a tumor. The targeting group or targeting agent can be a small molecule, which is intended to include both non-peptides and peptides. The targeting group also can be a macromolecule, which includes saccharides, lectins, receptors, ligands for receptors, proteins such as BSA, antibodies, and so forth. (Refs.: (a) “Peptides and Peptide Hormones for Molecular Imaging and Disease Diagnosis,” Xiaoyuan Chen, et al., Chemical Reviews, 2010, 110, 3087-3111; (b) “Integrin Targeted Therapeutics,” N. Neamati, et al., Theranostics, 2011, 1, 154-188; (c) “Integrin Targeting for Tumor Optical Imaging,” Yunpeng Ye, et al., Theranostics, 2011, 1, 102-126.)
A is a biologically active group with diagnostic significance, e.g., a peptide, PNA, aptamer, antibody fragments, whole antibodies. “A” as a “Biologically active group” is a biologically active group that is either able to target (preferential locator) a particular compound that is matched to A with a specific non-covalent affinity, e.g., or one that can interact with a target in specific and complementary ways, e.g., enzyme inhibitor peptide (A) to an enzyme released at a disease sight. Any of these biologically active groups inhibitor can be delivered with a radiolabel or a toxic drug that would kill the target, or can deliver a detectable probe as a diagnostic agent, or both.
“A” as a biologically active group is introduced into the branched discrete PEG construct by the many chemistries known in the art, e.g., references: E. M. Sletten and C. R. Bertozzi, “Biorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality,” Angew. Chem. Int. Ed., 48, 6974-6998(2009); G. Hermanson, Bioconjugate Techniques, 2nd Edition, Academic Press, 2008. In addition the option for incorporating a cleavable chemistry into the linkage formed also is a preferred option. This could include, but not limited to, a cleavable peptide, a disulfide, or a hydrazone.
As used herein, “A” can be a targeting agent, or carrier with targeting agent (e.g., a nanoparticle that has the targeting agents attached to the particle with various linear and branched discrete PEG constructs), the targeting agent matched to a particular target. A can be, e.g., an MMP (matrix metalloprotease) inhibitor substrate, an RGD peptide, antibody, antibody fragment, engineered scaffold, liposome, a PLGA, silica or a metal nanoparticle, such as gold or silver, all well known in the art or targeting for diagnostics and therapeutics.
When there is more than one “A” as a “biologically active group”, the term used is a multivalent group. The “A” independently can be the same or different depending on the intent and need of the particular application of “A”. E.g., Two different “A's” give a bispecific interaction, or where “A” is the same, a single interaction can be enhanced, but in both cases there can be a very large advantage over having just one “A” and the design of the can control that synergy of having more than one “A.”
Shown in
Conjugate varied between 200-0.2 ng/ml or 20-0.02 ng/well. SA qSP sustained its activity after 75° C., 60 min and showed identical levels of activity to SA qSP stored at 4° C. The SA qSP conjugate shows higher sensitivity at lower concentrations than its leading competitor.
The results demonstrate the thermal stability of the qSP conjugate as well as its higher sensitivity at lower concentrations in comparison to commercially available Streptavidin conjugates.
Protocols for Conjugation of the HRP conjugate. The SBP conjugation is done identically.
This disclosure describes the process for quantifying the number of aminooxy groups that are present on IgGs and Streptavidin after these proteins have been modified with phthalamidooxy-dPEG®12-NHS ester (product number 11135). The assay is based on the reaction between 4-nitrobenzaldehyde and aminooxy-dPEG®12-t-butyl ester which leads to the formation of an oxime adduct with maximum absorbance at 350 nm.
SCOPE: We have developed this assay for and have tested it with proteins that have aminooxy groups connected to proteins through a dPEG®12 chain.
PPE must be worn at all times. All chemical reaction steps must be performed in a fume hood. Dimethylacetamide (DMAC) is a colorless, water miscible, liquid which is commonly used as a polar solvent in organic chemistry. It is a potential hazard to human health and should be handled in a fume hood at all times.
Store the aminooxy-dPEG®12-TBE at −20° C. until use. This chemical is stable in dry dimethylacetamide for several weeks if stored at −20° C. Bring all coupling reagents to room temperature before use and mix by gentle vortexing to assure homogeneity. New chemical solutions should be prepared every 6 to 9 months.
Preparing unknown samples: dilute samples five fold in NaOAc, (0.1 M, pH 5.0)
Incubate in a water bath (37° to 40° C., 30 minutes).
Read the abs @ 350 nm.
Use Quartz Cuvettes and zero the spectrophotometer with the working reagent.
Plot the data as A350 (on Y-axis) versus μmoles of aminooxy (on X-axis) and interpolate the unknown
This disclosure further describes the process of preparing oxidized Horse Radish Peroxidase (HRP) and its subsequent PEGylation with methoxy-dPEG®12-NHS ester (product number 10262). Horseradish peroxidase is a glycoprotein that is widely used as a readout enzyme for immunocytochemical, and general immunological applications. One of its frequent uses is to create enzyme-antibody conjugates. In the current procedure, monosaccharides are oxidized with sodium meta periodate to produce aldehyde groups. The oxidation reaction then is quenched with sodium sulfite, and the enzyme is purified over a PD-10 column. In the final step the enzyme is reacted with methoxy-dPEG-NHS and repurified. The enzyme is subsequently used later to create HRP-antibody conjugates, streptavidin-HRP conjugates, and biotinylated-HRP conjugates (see SOPs).
This disclosure also applies to the preparation and purification of oxidized and dPEGylated HRP. The enzyme should be purchased in powder form. The procedure also works well with the related enzyme, soybean peroxidase, SBP.
PPE must be worn at all times. Sodium metaperiodate is a strong oxidizing agent that is a potential hazard to unprotected skin. Dimethylacetamide (DMAC) is a colorless, water miscible, liquid which is commonly used as a polar solvent in organic chemistry. It is a potential hazard to human health and should be handled in a fume hood at all times.
In this step the enzyme is oxidized with sodium periodate to introduce aldehyde groups into the oligosaccharide groups. These aldehydes are used later in coupling reactions that use aminooxy or amine groups.
In this step the oxidized enzyme is conjugated to an m-dPEG®12-NHS ester. The NHS ester group reacts simultaneously with the amine groups on the target enzyme. The process yields increased stability and water solubility of the modified enzyme.
This disclosure further describes the process for preparing antibody-enzyme conjugates using phthalamidooxy-dPEG®12-NHS ester (product number 11135). Briefly, this procedure involves incubating a pure antibody, present at a concentration of at least 10 mg/mL, with the phthalamidooxy-dPEG®12-NHS ester present at a 10 to 20 fold molar excess over the antibody. After the incubation period, hydrazine is added to remove the protecting group and expose the aminooxy group (AO). The aminooxy-modified antibody then is purified by chromatography on a PD-10 column and the AO content quantified (further characterization is done by capillary electrophoresis, ELISA, and protein analysis). The modified IgG is mixed with an aniline catalyst and a five-fold excess of oxidized and dPEGylated Horseradish Peroxidase (HRP). The AO groups react with the aldehyde on the HRP to form stable oxime linkages. The catalyst is removed by dialysis in a Slide-A-Lyzer and stored at 4° C.
This described procedure applies only to the preparation of aminooxy-modified antibodies and its conjugation with oxidized, dPEGylated HRP. Other procedures including assay of aminooxy content and preparation of oxidized, dPEGylated HRP are described in other SOPs. The procedure should work for all IgGs of most animal species, however every antibody should be optimized for the best labeling conditions.
PPE must be worn at all times. All chemical reaction steps must be performed in a fume hood. Dimethylacetamide (DMAC) is a colorless, water miscible, liquid which is commonly used as a polar solvent in organic chemistry. It is a potential hazard to human health and should be handled in a fume hood at all times.
Store the phthalamidooxy-dPEG®12-NHS ester at −20° C. until use. This chemical is stable in dry dimethylacetamide for several weeks if stored at −20° C. Bring all coupling reagents to room temperature before use and mix by gentle vortexing to assure homogeneity.
While the device and method have been described with reference to various embodiments, those skilled in the art will understand that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope and essence of the disclosure. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the disclosure without departing from the essential scope thereof. Therefore, it is intended that the disclosure not be limited to the particular embodiments disclosed, but that the disclosure will include all embodiments falling within the scope of the appended claims. In this application all units are in the metric system and all amounts and percentages are by weight, unless otherwise expressly indicated. Also, all citations referred herein are expressly incorporated herein by reference.
This application claims benefit of provisional application Ser. No. 61/723,397, filed Nov. 7, 2012, the disclosure of which is expressly incorporated herein.
Number | Date | Country | |
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61723397 | Nov 2012 | US |