The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
The following is a quotation of 35 U.S.C. 112(f):
(f) Element in Claim for a Combination. – An element in a claim for a combination may be expressed as a means or step for performing a specified function without the recital of structure, material, or acts in support thereof, and such claim shall be construed to cover the corresponding structure, material, or acts described in the specification and equivalents thereof.
The claims in this application are given their broadest reasonable interpretation using the plain meaning of the claim language in light of the specification as it would be understood by one of ordinary skill in the art. The broadest reasonable interpretation of a claim element (also commonly referred to as a claim limitation) is limited by the description in the specification when 35 U.S.C. 112(f) is invoked.
As explained in MPEP § 2181, subsection I, claim limitations that meet the following three-prong test will be interpreted under 35 U.S.C. 112(f):
(A) the claim limitation uses the term “means” or “step” or a term used as a substitute for “means” that is a generic placeholder (also called a nonce term or a non-structural term having no specific structural meaning) for performing the claimed function;
(B) the term “means” or “step” or the generic placeholder is modified by functional language, typically, but not always linked by the transition word “for” (e.g., “means for”) or another linking word or phrase, such as “configured to” or “so that”; and
(C) the term “means” or “step” or the generic placeholder is not modified by sufficient structure, material, or acts for performing the claimed function.
Use of the word “means” (or “step”) in a claim with functional language creates a rebuttable presumption that the claim limitation is to be treated in accordance with 35 U.S.C. 112(f). The presumption that the claim limitation is interpreted under 35 U.S.C. 112(f), is rebutted when the claim limitation recites sufficient structure, material, or acts to entirely perform the recited function.
Absence of the word “means” (or “step”) in a claim creates a rebuttable presumption that the claim limitation is not to be treated in accordance with 35 U.S.C. 112(f). The presumption that the claim limitation is not interpreted under 35 U.S.C. 112(f), is rebutted when the claim limitation recites function without reciting sufficient structure, material or acts to entirely perform the recited function.
Claim limitations in this application that use the word “means” (or “step”) are being interpreted under 35 U.S.C. 112(f), except as otherwise indicated in an Office action. Conversely, claim limitations in this application that do not use the word “means” (or “step”) are not being interpreted under 35 U.S.C. 112(f), except as otherwise indicated in an Office action.
This application includes one or more claim limitations that do not use the word “means,” but are nonetheless being interpreted under 35 U.S.C. 112(f), because the claim limitation(s) uses a generic placeholder that is coupled with functional language without reciting sufficient structure to perform the recited function and the generic placeholder is not preceded by a structural modifier. Such claim limitation(s) is/are: “the computing platform including a module configured to detect a signal . . . and identify the candidate compound as having an impact” in claim 17.
Because this/these claim limitation(s) is/are being interpreted under 35 U.S.C. 112(f), it/they is/are being interpreted to cover the corresponding structure described in the specification as performing the claimed function, and equivalents thereof.
If applicant does not intend to have this/these limitation(s) interpreted under 35 U.S.C. 112(f), applicant may: (1) amend the claim limitation(s) to avoid it/them being interpreted under 35 U.S.C. 112(f) (e.g., by reciting sufficient structure to perform the claimed function); or (2) present a sufficient showing that the claim limitation(s) recite(s) sufficient structure to perform the claimed function so as to avoid it/them being interpreted under 35 U.S.C. 112(f).
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
Claims 1-33 are rejected under 35 U.S.C. 112(a), as failing to comply with the enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention. Instant claim 1 requires contacting a population of vesicles in which one or more of the vesicles has a single detectable phase and one or more of the vesicles has a membrane lipid raft phase and a membrane non-raft phase domain with a candidate compound, detecting a signal from the population of vesicles and identifying the candidate compound as having an impact on a characteristic of a lipid raft phase domain, a characteristic of a non-raft phase domain and/or a characteristic of one or more membrane proteins based on the detected signal.
In order to determine compliance with the enablement requirement of 35 U.S.C. 112(a), the Federal Circuit developed a framework of factors in In re Wands, 858 F.2d 731, 737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988), referred to as the Wands factors to assess whether any necessary experimentation required by the specification is "reasonable" or is "undue." These factors include, but are not limited to:
(A) The breadth of the claims;
(B) The nature of the invention;
(C) The state of the prior art;
(D) The level of one of ordinary skill;
(E) The level of predictability in the art;
(F) The amount of direction provided by the inventor;
(G) The existence of working examples; and
(H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure.
With respect to the breadth of the claims Winds factor, claim 1 does not define the state of the population of vesicles, the type of detected signal or what if any characteristic of the detected signal is used to identify whether the compound has had an impact. Thus at least claim 1 covers all possible combinations of vesicle populations, detected signals and changes in the detected signals to determine if a candidate compound has had an impact. For example the population of vesicles would include a suspension of vesicles, vesicles in recessed micro- and/or nanowells as taught in the newly cited Wittenberg paper (ACS Nano 2011) or in a microfluidic channel as taught by the newly cited Daniel patent publication (US 2013/0095512). When it comes to the different possible domains, figure 2 of the newly cited Wesolowski shows that there are at least five different types of domains that can be observed in different lipid mixtures. Figure 3 of the Wesolowski paper shows that a vesicle can have multiple separate domains. With respect to the detected signal, figure 1 of the newly cited Katz paper (Pharmaceutical Research 2006) shows a colorimetric change resulting from an interaction between a compound and phospholipid vesicles. Additionally the Kahya (Biochemistry2005), Husen (Biophysical Journal 2012), Hammond (Proceeding of the National Academy of Science 2005), Sych (Bioinformatics 2019) and Jiang (Langmuir 2016) papers give fluorescent signals resulting from phase separation in vesicles. The instant claims also cover detecting non-optical signals.
The newly cited Kenworthy paper (Journal of Cell Biology 2004) looked at dynamics of putative raft-associated proteins at the cell surface and found that raft proteins diffused freely over large distances pointing to their diffusion not being part of discrete, stable raft domains. This finding/teaching points to a problem with any vesicle population and detecting signals from such a population: the raft domains and the proteins potentially part of them change over time without any outside influence. Thus, for one of ordinary skill in the art to have an expectation that the invention is enabled without undue experimentation, there needs to be an expectation that the detected signal can distinguish between interactions that have an impact and signals that are a result of the natural changes that are occurring in the vesicles. Figure 1 of Kenworthy shows the four models they used: stable, immobile rafts; stable, mobile rafts; dynamic partitioning and no rafts. The fact that raft associated proteins were found to diffuse with no particular association/relationship to the rafts points to a disconnect between the characteristics of one or more proteins and the detected signal. One of ordinary skill in the art would recognize that in a vesicle suspension, and in a flowing system, the vesicles are capable of rotating so that even though a raft may be immobile, its ability to be detected over time would change. Especially using a detection process such as described by one or more of Kahya, Sych and Jiang would potentially result in modulations of the detected signal that have nothing to do with an interaction between the candidate compound and the vesicle domains. The fact that the rafts appear to be capable of movement within the vesicle membrane and that proteins can move into and out of the raft or through non-raft sections of the vesicle membrane without outside influence further complicates this aspect of enablement from a detection signal actually being representative of a candidate compound having some form of impact on the characteristics of a raft domain, a non-raft domain and/or a protein in the vesicle. Examiner also notes that vesicle domains such as shown in figure 2A and 2D of the Wesolowski paper would present their own challenges to the Kahya, Sych and Jiang signal detection methods if the vesicles and/or domains are capable of change and/or movement in the absence of an interaction with a candidate compound.
While there is a disclosure of a method of detecting the fluorescent signal that appears to be similar to and/or derived from the teachings of Kahya, Sych and Jiang, there is no disclosure that examiner was able to find that shows that the detection method is capable of distinguishing between changes that result from natural processes and the changes resulting from a candidate compound having an impact a characteristic of a raft domain, a non-raft domain or a membrane protein.
With respect to the state of the prior art Wands factor, references of record clearly teach that different vesicles are a tool to visualize phase separation and lipid rafts in model systems (see Wesolowski or Husen), a tool for probing effects of drugs and other conditions on membrane stability (see the newly cited Gerstle paper (Methods in Enzymology 2018)) or lipid vesicles as membrane models for assessment of compounds (see the newly cited Zepik paper (Critical Reviews in Toxicology 2008). The prior art has also contacted various vesicles with at least drugs and anesthetics to determine/monitor the effect they have on the membrane through detecting changes of the vesicle membrane. This art shows that different compounds (anesthetics or drugs) can have different effects on similar vesicles. In particular the newly cited Ramakrishnan paper (Langmuir 2018) shows that software is either available and/or developable by those of ordinary skill in the art for automated image analysis.
While the prior art has the various teachings outlined above, the scope of those teachings fail to show that the invention of claim 1 was fully enabled.
With respect to claim 17, the computing platform limitation is required to identify a candidate compound as having an impact on a characteristic of a lipid raft phase domain, a characteristic of a non-raft phase domain and/or a characteristic of one or more membrane proteins between the lipid raft phase and the non-raft phase domains based on a detected signal. This computing platform is required to do at least the method of claim 1 at the scope of claim 1. Thus it is also not enabled for the reasons given above relative to the method of claim 1.
With respect to claim 33, the one or more non-transitory computer readable media having executable instructions stored thereon is required to identify a candidate compound as having an impact on a characteristic of a lipid raft phase domain, a characteristic of a non-raft phase domain and/or a characteristic of one or more membrane proteins between the lipid raft phase and the non-raft phase domains based on a detected signal. This executable instructions are required to do at least the method of claim 1 at the scope of claim 1. Thus it is also not enabled for the reasons given above relative to the method of claim 1.
While the dependent claims narrow the scope of claims 1 or 17, they fail to limit the scope to a point that the detected signal is capable of distinguishing between normal processes occurring in a vesicle population and changes in a characteristic of a lipid raft phase domain, a characteristic of a non-raft phase domain and/or a characteristic of one or more membrane proteins between the lipid raft phase and the non-raft phase domains resulting from contact of the vesicle population with a candidate compound.
Claims 1-33 are rejected under 35 U.S.C. 112(b), as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention. In claim 1, the preamble identifies the method as an “automated method” for identifying a compound. However, the steps that follow are all capable of being performed manually. Thus it is not clear if the steps are all required to be performed in an automated manner or if one or more of the steps can be performed in a manual manner. Furthermore it is not clear if the detected signal is required to have any form or if it simply could be the increase or decrease in the intensity of a property such as an absorption at a particular optical frequency of the population of vesicles. Finally, it is not clear if the effect on the characteristic that is identified is anything in particular such as a change in porosity a shift in vesicle components, the loss or inclusion of a function or something else. For examination purposes, examiner will not place any particular limitation on the detected signal, the number of automated steps or the characteristic being impacted. With respect to claims 2-3, it is not clear if the claimed changes are in response to or correlated with specific detected signals or if there are multiple detected signals that can be used to determine the induced change. For examination purposes, examiner will not place any specific relationship requirement between a detected signal and the induced characteristic change. With respect to claim 4, “the vesicle” does not have proper antecedent basis in the population of “vesicles” language of claim 1. Is applicant requiring only one vesicle of the population of vesicles to be a Giant Plasma-Membrane Derived Vesicle (GPMV) or is applicant attempting to require all of the population of vesicles to be GPMV? With respect to claim 7, it is not clear how taking one or more images of one or more vesicles in the population of vesicles constitutes detecting a signal in the population of vesicles. Is there a reason that the images are taken or additional processing of the images that is required to actually detect a signal? With respect to claim 8, it is not clear what selection criteria would result in the exclusion of one or more vesicles. With respect to claim 9, “the fluorescent labels and/or fluorescently labeled membrane proteins” language does not have antecedent basis in claims 8 or 1. With respect to claim 12, “the imaging” language does not have antecedent basis in claim 1. With respect to claim 13, it is not clear how one can detect a change of either phase separation or movement of one or more membrane proteins without the optional comparison of the signal after contacting the population of vesicles with the candidate compound with the signal prior to contacting the population of vesicles with the candidate compound. With respect to claim 16, it appears that the claim requires the population of vesicles to be present in multiple reaction wells however claim 1 does not have such a requirement. With respect to claim 17, the computing platform appears to be required to perform the detecting and identifying steps of claim 1 so that the issues related to these steps of claim 1 are also issues with respect to the computing platform of claim 17. Additionally, it appears that claim 17 is claiming a plate comprising a plurality of reaction wells, but not the population of vesicles. Since this appears to be the case, examiner will not require the presence of vesicles in the wells of the plate and treat the description of vesicles as non-limiting for examination purposes. However, if applicant intended for the vesicle language to have patentable moment, it is not clear if each well is required to have both one or more vesicles with only a single detectable membrane phase and one or more vesicles having a membrane lipid raft phase domain and a membrane non-raft phase domain that are separated or if the types of vesicles in each reaction well are not limited as long as the population of vesicles has both types of vesicles. Claims 18-19 appear to correspond to the limitations of claims 2-3 above so that they suffer from the problems described above for claims 2-3. Claim 20 corresponds to the limitation of claim 4 so that it suffers from the issues described with respect to claim 4 above. Claim 23 corresponds to the limitation of claim 7 above so that it suffers from the issues described above for claim 7. Claim 24 corresponds to the limitation of claim 8 above so that it suffers from the issue described with respect to claim 8 above. Claim 25 corresponds to the limitation of claim 9 so that it suffers from the same issue described with respect to claim 9 above. Claim 28 corresponds to the limitation of claim 12 so that it suffers from the issue described with respect to claim 12 above. Claim 29 corresponds to claim 13 above so that it suffers from the same issue described with respect to claim 13 above. Claim 33 requires one or more non-transitory computer readable media having executable instructions stored thereon to perform the steps of claim 1 including the detecting and identifying steps of claim 1 so that the issues related to these steps of claim 1 are also issues with respect to the one or more non-transitory computer readable media having executable instructions stored thereon of claim 33. Additionally, “the signal” in the first paragraph of claim 33 does not have antecedent basis.
The following is examiner’s statement of how certain claim limitations are being interpreted for examination purposes. With respect to vesicle production, page 57, lines 4-21 of the instant specification is the only place that examiner can find that teaches a preparation of the vesicles (GPMV). That method involves treating HeLa cells in a manner that GPMV are produced and separated from the cells. Since that method is the only production method described and the examples and/or figures show/teach that both types of vesicles are present, examiner is treating the described production method as inherently producing a vesicle population that meets the requirements of claim 1. There does not appear to be any part of the process that could be used to produce either type of vesicle exclusively. For that reason, examiner is treating any vesicle production method as producing a population of a random mixture of vesicles for examination purposes. Thus, for examination purposes, examiner is treating any vesicle production method (i.e. from cells as described in the instant specification or from a mixture of lipids) as producing a population of vesicles in which a portion of the population of vesicles has only a single detectable membrane phase and a portion of the population of vesicles has a phase separated membrane lipid raft phase domain and a membrane non-raft phase domain.
The following is a quotation of pre-AIA 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under pre-AIA 35 U.S.C. 103(a) are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1, 16-17 and 32-33 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Katz (Pharmaceutical Research 2006) in view of Doranz (US 2007/0225227) or Califano (US 2009/0269772). With regards to claim 1, Katz teaches a method for identifying a compound that impacts a characteristic of a lipid raft phase domain, a characteristic of a non-raft phase domain, and/or a characteristic of one or more membrane proteins (see at least the abstract; develop a rapid colorimetric assay for evaluating membrane interactions and penetration through lipid barriers and to create a platform, amenable to high-throughput screening formats, for predicting the extent of penetration of pharmaceutical compounds through lipid layers), the method comprising: contacting a population of vesicles (see at least the abstract; vesicles of phospholipids and the chromatic lipid-mimetic polymer polydiacetylene. The polymer undergoes visible, concentration-dependent blue-red transformations induced through interactions of the vesicles with the molecules examined and at least the UV-Vis Measurement section on page 581)), wherein one or more vesicles in the population of vesicles optionally comprises one or more membrane proteins (the presence of proteins is optional), with a candidate compound (see table 1 for a listing of candidate compounds and the sensed colorimetric data), wherein in a portion of the population of vesicles there is only a single detectable membrane phase and in a portion of the population of vesicles a membrane lipid raft phase domain and a membrane non-raft phase domain are phase separated (figure 1 shows a representation of a vesicle with separated domains and since the vesicle preparation on page 581 produces a random set of vesicles at least some of the vesicles would have been expected to be single phase vesicles); detecting a signal from the population of vesicles (see at least figure 1 on page 581, the UV-Vis measurement section on page 581 figure 2 on page 582 and its associated discussion); and identifying the candidate compound as having an impact on a characteristic of a lipid raft phase domain, a characteristic of a non-raft phase domain, and/or a characteristic of one or more membrane proteins based on the signal (see at least figure 1 and its associated discussion in combination with at least figure 4 and its associated discussion). The paragraph bridging the columns of page 587 teaches that figure 5 visually summarizes the practical utilization of the colorimetric assay for rapid screening of membrane interactions of pharmacological molecules. Each tested compound would be examined in three concentrations: single micromolar range (row A in figure 5), millimolar range (row B), and molar range (row C). The produced blue-red color coding would be predictive as to the type of membrane interaction properties of the examined compound. For example, a surface-attached molecule (column I in figure 5) would induce a purple (or red) color at 50 2M (A), and maximal red colors at the higher concentrations employed (B, C). The corresponding color code for a bilayer-penetrating compound (column II in figure 5) would be blue/purple/red (figure 5, middle column), whereas a substance belonging to the third group (nonmembrane active compounds) would produce blue/blue/blue (or blue/blue/purple), respectively, at the three concentrations examined (figure 5, column III). The final paragraph on page 587 teaches that the phospholipid/PDA assay exhibits important practical advantages for application as a generic tool for drug screening. The vesicle solutions can be placed and stored for long periods in conventional 96-well (or 384-well) plates. The colorimetric transitions are induced within a very short time (seconds) after mixing the reagents. The new technique is robust and easy to apply, and data for large compound libraries can be obtained in a few minutes. The colorimetric assay can thus become a highly useful tool for predicting membrane interactions and bilayer permeation at early stages of drug development and profiling. While the vesicle solutions are placed in plates with a plurality of reaction wells for contacting the population of vesicles with a candidate compound and the results are measured at two wavelengths, Katz does not specifically teach an automated process.
In the patent publication, Doranz teaches the use of lipoparticles and testing ion channel function and modulators of ion channels therewith. Paragraph [0112] teaches that a variety of detection formats can be used to detect changes in membrane potential dyes. Microplate-based detectors such as the Wallac Victor2V and Molecular Devices FlexStationII and FLIPR-Tetra can be used. These detection devices are capable of detecting samples in 96, 384, and/or 1536-well microplates. In some cases, the detection devices permit ratiometric detection of two wavelengths. In order to capture signals generated quickly after addition, an automated injector is preferred to initiate the assay (e.g. the addition of high-K buffer to samples). In other cases, end point readings can be used to compare fluorescence before and after stimulation of the ion channels. Additional detection devices could also be used, including microfluidic devices, a 96-well plate, a 384-well plate, a 1536-well plate, a glass slide, a plastic slide, an optical fiber, a flow cytometer, a microscope, a fluorometer, a spectrometer, or a CCD camera. Alternative formats for ion channel detection within lipoparticles also include confocal-based detection (e.g. Evotec's FCS Opera system that detects nanometer-scale events within femtoliter volumes) and FMAT (Applied Biosystems). Other methods of detection can also be used, such as, for example, confocal microscopy, fluorescent microscopy, and the like. Relative to detection with microplates, paragraph [0239] teaches that experimental conditions for monitoring ion channel activity in lipoparticles in microplates using a fluorescence microplate reader were established. Lipoparticles pre-treated with valinomycin (1 µM, 5 minutes, room temperature) were suspended in Hepes (pH 7.0) containing FMP dye (amounts of lipoparticles and FMP were optimized as described with respect to figures 18 A and B). Aliquots (50 µl) were dispensed across a 96-well microplate. A Wallac Victor 2V microplate reader was programmed to automatically inject 5 µl of 500 mM K2SO4 (45 mM K2SO4 final), simultaneously to each well, and to measure fluorescence at 2 sec intervals starting 10 sec before the addition of K+. Paragraph [0240] teaches that to establish experimental reproducibility under these conditions, twelve replicates each of valinomycin-treated and non-treated null-lipoparticles (no ion channel) were mixed with FMP dye, exposed to K+, and fluorescent signals recorded in the Wallac Victor2V. Valinomycin-treated lipoparticles produced a signal of 1.347 ± 0.036, while untreated lipoparticles produced a signal of 1.023 ± 0.009 (mean ± standard deviation) (see figure 18). These results yielded a statistical Z'-factor of 0.577, considered an excellent score for high-throughput screening purposes. The Z'-factor is an often-used, unit-less statistical measure of the signal-to-noise and reproducibility of an assay. A Z'-factor greater than 0.5 is highly desired in high-throughput screening assays. Collectively, the results demonstrate the utility of lipoparticles for high throughput-screening of potential ion channel modulators in miniaturized format, and suggest they are likely to be useful for high-throughput drug screening of potential ion channel modulators. Similar experiments were performed using valinomycin-treated lipoparticles in 384-well plates in a Molecular Devices FlexStationII and a Molecular Devices FLIPR-tetra (see figure 19). These data establish the usefulness of lipoparticles on a high-throughput device for drug screening, especially drug screening for novel modulators of ion channels. Example 23, paragraph [0249], describes the discovery of inhibitors of closed ion channels through a process similar to that described in paragraph [0239]. Example 24, paragraph [0250], describes the discovery of inhibitors of open ion channels through a process similar to that described in paragraph [0239]. Example 26, paragraph [0252], describes the discovery of drugs that block hERG activity through a process similar to that described in paragraph [0239]. Paragraph [0260] teaches that the device being used had the ability to monitor fluorescence by recording the intensity every 2 seconds and calculate the change in fluorescence by subtracting the mean fluorescence of the first 30 seconds of recording (baseline) from the mean fluorescence of the final 30 seconds of recording (maximum).
In the patent publication Califano teaches systems, methods, and apparatus for searching for a combination of compounds of therapeutic interest. Cell-based assays are performed, each cell-based assay exposing a different sample of cells to a different compound in a plurality of compounds. From the cell-based assays, a subset of the tested compounds is selected. For each respective compound in the subset, a molecular abundance profile from cells exposed to the respective compound is measured. Targets of transcription factors and post-translational modulators of transcription factor activity are inferred from the molecular abundance profile data using information theoretic measures. This data is used to construct an interaction network. Variances in edges in the interaction network are used to determine the drug activity profile of compounds in the subset of compounds. The drug activity profiles are used to form a filter set of compound combinations from the subset of compounds. Paragraph [0067] teaches that to assess the end-point phenotype in high-throughput fashion, fully automated fluorescent or luminescent readout is performed in some embodiments using standard robotically integrated plate-readers. In some embodiments, the fluorescent readout is proportional or otherwise indicative of the number of cells in a culture that are undergoing apoptosis or that are viable. In some embodiments, after readout, the top 2,000 compounds, the top 1,000 compounds, the top 500 compounds or some other user specified upper threshold number of compounds with the highest activity (e.g., greatest ability to reduce viability in malignant cells) are selected for further analysis. In some embodiments, after readout, the top 2,000 compounds, the top 1,000 compounds, the top 500 compounds or some other user specified lower threshold number of compounds with the highest activity are selected for further analysis. Step 202 achieves about a 10.sup.3 fold search space reduction (e.g. from one million compounds to one thousand compounds) in some embodiments. Paragraphs [0200]-[0201] teach that the invention can be implemented as a computer program product that comprises a computer program mechanism embedded in a computer-readable storage medium. Further, any of the methods disclosed herein can be implemented in one or more computers or other forms of apparatus. Examples of apparatus include but are not limited to, a computer, and a spectroscopic measuring device (e.g., a microarray reader or microarray scanner). Further still, any of the methods disclosed herein can be implemented in one or more computer program products. Some embodiments disclosed herein provide a computer program product that encodes any or all of the methods disclosed herein. Such methods can be stored on a CD-ROM, DVD, magnetic disk storage product, or any other computer-readable data or program storage product. Such methods can also be embedded in permanent storage, such as ROM, one or more programmable chips, or one or more application specific integrated circuits (ASICs). Such permanent storage can be localized in a server, 802.11 access point, 802.11 wireless bridge/station, repeater, router, mobile phone, or other electronic devices. Paragraph [0204] teaches that in some embodiments, cell growth is measured in cell-based assays. For example, in some embodiments cell growth is measured by a homogeneous, vital dye method in which one of several choices of dye is added to cells in a 96, 384 or 1536 well plate (or other form of plate), incubated for increasing hours, and read directly in a plate reader. The dye is enzymatically changed in healthy cells so that development of color or fluorescence is measured using a different wavelength than the unaltered dye.
With respect to claim 1, it would have been obvious to one of ordinary skill in the art to provide the Katz method as an automated method using the teachings of Doranz or Califano because Katz is directed to high-throughput assays and Doranz or Califano show the benefits of processing lipid vesicles in a high-throughput automated manner. With respect to claim 16, figure 5 of Katz shows that the method includes analyzing multiple vesicles in the population of vesicles across multiple reaction wells so that modification of Katz by Doranz or Califano would show the obviousness of performing it in an automated fashion for the same reasons as given above for claim 1.
With respect to claim 17, Katz teaches the use of plate comprising a plurality of reaction wells for contacting a population of vesicles with a candidate compound. Katz does not teach a computing platform including a processor and memory, the computing platform including a module configured to detect a signal from the population of vesicles using a microscope, camera or a sensor communicatively coupled to the computing platform; and to identify the candidate compound as having an impact on a characteristic of a lipid raft phase domain, a characteristic of a non-raft phase domain, and/or a characteristic of one or more membrane proteins based on the signal.
With respect to claim 17, it would have been obvious to one of ordinary skill in the art to automate the Katz method through adding the automated structure of Doranz or Califano to process the plate with a computing platform including a processor and memory, the computing platform including a module configured to detect a signal from the population of vesicles using a microscope, camera or a sensor communicatively coupled to the computing platform as taught by Doranz or Califano; and to identify the candidate compound as having an impact on a characteristic of a lipid raft phase domain, a characteristic of a non-raft phase domain, and/or a characteristic of one or more membrane proteins based on the signal in accordance with the teachings of Katz because Katz is directed to high-throughput assays and Doranz or Califano show the benefits of processing lipid vesicles with a computing platform capable of a high-throughput automated manner. With respect to claim 32, figure 5 of Katz shows that the method includes analyzing multiple vesicles in the population of vesicles across multiple reaction wells so that modification of Katz by teachings of Doranz or Califano would show the obviousness of performing it in an automated fashion for the same reasons as given above for claim 17.
With respect to claim 33, automation of claim 1 by the teachings of Doranz or Califano would have required one or more non-transitory computer readable media having stored thereon executable instructions that when executed by one or more processors of one or more computers control the one or more computers to perform steps for automatedly identifying the candidate compound as having an impact on a characteristic of a lipid raft phase domain, a characteristic of a non-raft phase domain, and/or a characteristic of one or more membrane proteins based on a signal detected from the population of vesicles. Thus claim 33 is obvious over Katz in view of Doranz or Califano for the same reasons as given above for claim 1.
Claims 2-15 and 18-31 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Katz in view of Doranz or Califano as applied to claims 1 and 17 above, and further in view of Levental (Proceedings of the National Academy of Sciences 2011) and Sych (Bioinformatics 2019). With respect to claims 2-15 and 18-31 Katz does not teach that the vesicles are Giant Plasma-Membrane Derived Vesicle (GPMV) or that the detection is based on a fluorescent image of the vesicles using labels for the membrane lipid raft phase domain. the membrane non-raft phase domain and/or the membrane proteins.
In the paper Levental teaches that biological membranes are compartmentalized for functional diversity by a variety of specific protein–protein, protein–lipid, and lipid–lipid interactions. A subset of these are the preferential interactions between sterols, sphingolipids, and saturated aliphatic lipid tails responsible for liquid–liquid domain coexistence in eukaryotic membranes, which give rise to dynamic, nanoscopic assemblies whose coalescence is regulated by specific biochemical cues. Microscopic phase separation recently observed in isolated plasma membranes (giant plasma membrane vesicles and plasma membrane spheres) (i) confirms the capacity of compositionally complex membranes to phase separate, (ii) reflects the nanoscopic organization of live cell membranes, and (iii) provides a versatile platform for the investigation of the compositions and properties of the phases. They show that the properties of coexisting phases in giant plasma membrane vesicles are dependent on isolation conditions—namely, the chemicals used to induce membrane blebbing. They observed strong correlations between the relative compositions and orders of the coexisting phases, and their resulting miscibility. Chemically unperturbed plasma membranes reflect these properties and validate the observations in chemically induced vesicles. Most importantly, they observed domains with a continuum of varying stabilities, orders, and compositions induced by relatively small differences in isolation conditions. These results show that, based on the principle of preferential association of raft lipids, domains of various properties can be produced in a membrane environment whose complexity is reflective of biological membranes. The cell culture and labeling section on page 11415 teaches that RBL cells and A431 cells were cultured under similar conditions in respective mediums. Prior to GPMV/PMS isolation, cell membranes were stained with 5 μg∕mL of FAST-DiO, a lipidic dye that strongly partitions to disordered phases due to double bonds in its fatty anchors. The GPMV and PMS isolation, labeling and treatment section on page 11415 teaches that GPMVs were isolated and imaged under temperature-controlled conditions as previously described. For postisolation treatments, isolation chemicals were removed by dialysis and GPMVs were treated for 1 h at 37 °C. PMS were also isolated as previously described. Relative order magnitudes were measured by two-photon microscopy of C-Laurdan in phase-separated GPMVs at 5 °C as previously described. The labeling of cell surface proteins section on page 11415 teaches that surface-exposed proteins were biotinylated using a membrane-impermeable, amine-reactive biotin reagent, as previously described by incubating cells on ice for 30 min in the presence of Sulfo-NHS-biotin (1 mg∕mL), followed by GPMV isolation and staining with a monomeric Fab’ fragment of goat antibiotin coupled to Texas red (10 μg∕mL). Images of the Texas red signal were quantified as previously described to derive Kp,raft, the relative concentration of surface protein in the raft phase. Figure 2 on page 11413 shows that postisolation treatment reproduces PFA/DTT phenotype only when both PFA and DTT are present. (A–F) Representative images (all taken at 10 °C) of GPMVs isolated with the conditions shown on the left of the images, then dialyzed to remove isolation chemicals, then treated with the chemicals indicated above the arrows. Phase separation at this temperature is observed only when PFA and DTT are present, either as the isolation condition or the postisolation treatment. (G) Percentage of phase-separated GPMVs as a function of temperature for different isolation and treatment conditions. Each point represents 50–100 vesicles; curves are sigmoidal fits to data. Results are representative of three independent experiments. Figure 3 shows phase separation in PMS. (A) PMS isolated by cell swelling without additional chemicals show clear liquid–liquid phase separation below 5 °C (staining with 1 μg∕mL FAST-DiO, disordered phase marker). (B) Treatment of PMS with PFA + DTT reproduces the PFA + DTT phenotype in GPMVs, whereas treatment with PFA or DTT alone does not have a significant effect on phase behavior. Figure 5 shows that the order difference between coexisting phases is dependent on [DTT]. (A) Exemplary GP images of coexisting domains in GPMVs (all imaged at 5 °C). GP is a relative indicator of the membrane order (higher GP equals more ordered membranes). (B and C) Although the inclusion of PFA did not have a major effect on order (comparing NEM versus 25 mM PFA + 0.2 mM DTT), increasing [DTT] reduced the overall order and increased the relative order difference between the coexisting phases. Average ± SD from 10 vesicles per condition and representative of three experiments. Figure 6 shows that the relative protein concentration in coexisting phases is dependent on [DTT]. (A) Relative protein concentration in coexisting phases of GPMVs can be quantified by fluorescent imaging of antibiotin Fab’ after nonspecific protein labeling with Sulfo-NHS-biotin. (B) Kp,raft (ratio of intensity in the raft versus nonraft phase) of extracellularly accessible proteins decreases with [DTT] from a high concentration of raft proteins in NEM GPMVs to barely observable raft protein signal in 2 mM DTT. Points are average ± SD of >15vesicles per condition, representative of three independent experiments. The paragraph bridging the columns of page 11415 teaches that obviously, the perturbations observed by Levental are artifactual consequences of treatment of membranes with exogenous chemical agents. They previously showed that DTT can cause protein removal from the raft phase by depalmitoylation, and this effect may be partially responsible for the perturbations observed in this study. However, the concentration range at which these DTT-dependent effects are observed here (0.2–1 mM) is significantly lower than that necessary to induce depalmitoylation (at least 2 mM DTT). Additionally, pretreatment of cells with the palmitoylation inhibitor 2BP did not have an effect on phase separation (Figure 1C), confirming the limited role of palmitoylation in the observed effects. Rather, the activity that seems to be the biggest driver of the observed effects is the coupling of PE to membrane proteins mediated by PFA and DTT (Figure 7). This activity would be expected to deplete the raft phase of TM proteins because the acyl chains of PE are nearly always unsaturated and therefore a covalent protein–PE complex would be nonraft preferring. A testable hypothesis that follows from this proposal is that protein compositions and properties of raft domains could be regulated by a switchable affinity of membrane proteins for certain membrane phospholipids. Although several specific interactions between integral proteins and membrane lipids have been observed (e.g., for the epidermal growth factor receptor; and others reviewed as reported in certain cited literature), there has been no systematic analysis to determine how widespread this phenomenon is, nor whether it can be reversibly regulated. This field represents a neglected area of membrane biology that is ripe for further investigation.
In the paper Sych teaches a multifunctional tool for quantitative image analysis of giant unilaminar vesicles. Giant Unilamellar Vesicles (GUVs) are widely used synthetic membrane systems that mimic native membranes and cellular processes. Various fluorescence imaging techniques can be employed for their characterization. In order to guarantee a fast and unbiased analysis of imaging data, the development of automated recognition and processing steps is required. They developed a fast and versatile Fiji-based macro for the analysis of digital microscopy images of GUVs. This macro was designed to investigate membrane dye incorporation and protein binding to membranes. Moreover, they propose a fluorescence intensity-based method to quantitatively assess protein binding. The ImageJ distribution package FIJI they used is freely available online and the developed macro file GUV-AP.ijm is also available. the introduction teaches that GUVs represent cell-sized (tens of micrometers in diameter) spherical lipid bilayers. In combination with fluorescence microscopy techniques, GUVs became a frequently used approach to study cellular processes such as membrane permeabilization, adhesion and endocytosis. GUVs with a spatial phase-separation in liquid disordered (Ld) and liquid ordered (Lo) domains are of particular interest to mimic the heterogeneity of the plasma membrane including lipid rafts. Acquired fluorescence microscopy data requires processing of a large number of data sets, especially if a quantitative description is needed. As manual image analysis is biased and highly time consuming, the use of high throughput methods implemented in automated software can significantly speed up the analysis and reduce biases. Current automated software packages for GUV analysis based on 3D micrographs or GUV contour require commercial software licenses, are not compatible with different microscope output formats, or lack effective high throughput analysis capabilities. In this application note, they introduce a powerful FIJI-based macro for fast and versatile analysis of multiple GUVs per image. The developed macro used the ImageJ (FIJI) environment, an open source microscopy image processing software. It has multiple built-in functionalities and most importantly it is compatible with the majority of microscopy data output formats. Briefly, the macro detects circular particles (e.g. GUVs, Supplementary figure S1) in the microscopy image and determines their centers and radii. The algorithm is explained in detail and step by step in its associated Manual. Section 3 discusses the GUV processing, validation and application. When GUVs are detected, various processing approaches can be applied. They demonstrated a selection. They tested the performance of GUV-AP based on laser scanning confocal microscopy images of GUVs. These GUVs were composed of DOPC, sphingomyelin and cholesterol in different ratios and labeled by various fluorescent probes. The materials and methods section of the attached supplementary material teaches that fluorescence intensity measurements of GUVs (Figure 1, Figures S1, S2, S4) were performed on a confocal fluorescence microscope. For fluorescence excitation, 405 nm (F2N12SM), 488 nm (ß-Bodipy FL C5-HPC and Di-4ANEPPDHQ) and 642 nm (StxB-Cy5) lasers were used. The emission of ß-Bodipy FL C5-HPC and F2N12SM, as well as the green part of the emission di-4ANEPPDHQ were recorded using a 525/50 BP filter. The emission of the red part of di-4ANEPPDHQ emission and the emission of StxB-Cy5 were detected using a 700/75 BP filter. Section 3.1 teaches that Figure 1 as well as Supplementary Figures S2 and S3 show that the macro can be applied to decipher the fluorescence properties (intensity, lifetime) of a membrane dye incorporated in the lipid bilayer as well as its preference to distinct lipid phases and its orientation in the lipid bilayer. For instance, the lipophilic membrane probe ß-Bodipy FL C5-HPC (Bodipy) is known to have preferences for the Ld phase (Figure 1A), whereas it is almost completely excluded from the Lo phase. Furthermore, the GUV analysis macro can also be used to display the circular profile that visualizes the variations of fluorescence intensity along the GUV contour as a 2D plot (Figure 1B). The legend of figure 1 further explains that the figure shows GUV processing. (A) StxB-Cy5 (red channel) binds to the Lo domain of a GUV composed of DOPC/chol/SM and the glycosphingolipid Gb3 (40/15/40/5). The Lo domains are not visible since the membrane probe Bodipy (green channel) preferentially localizes to Ld domains. (B) Circular profiles of the membrane marker Bodipy (green curve) and StxB-Cy5 (red curve). The units of StxB-Cy5 circular profile (right Y axis) are quantified as shown in (C). (C) Calculation of the relative binding efficiency. Section 3.2 discusses the quantification of protein binding to GUVs. In order to highlight the performance of the macro for the observation and quantification of protein binding, they imaged phase-separated GUVs containing the neutral glycosphingolipid globotriaosylceramide (Gb3; also known as CD77 or Pk blood group antigen). Gb3 is the receptor for the galactose-binding B-subunit of Shiga toxin (StxB) from Shigella dysenteriae. In phase-separated GUVs (Figure 1A), Cy5-labeled StxB preferentially binds to Lo domains. This binding can be more accurately displayed using the circular profiles of Bodipy (Figure 1B—green) and StxB-Cy5 (Figure 1B—red). To quantitatively evaluate the protein binding, they calculated the relative binding efficiency ε as shown in Figure 1C. ⟨Bound protein⟩ is the mean fluorescence intensity signal from the protein located on the vesicle contour and ⟨Free protein⟩ is the mean fluorescence intensity signal from the free protein in solution, extracted from the area of the image, which surrounds the GUV (Figure 1C). In absence of protein binding, the ⟨Bound protein⟩ value is equal to the ⟨Free protein⟩ value, so that the relative binding efficiency is ≈ 0. If ε is above 0, binding occurs. This binding efficiency value depends on the number of protein molecules bound to the membrane. Moreover, it is possible to link such fluorescence intensity quantifications to the actual number of protein molecules bound to the membrane using proper calibration. The robustness of the method was validated by analyzing StxB binding to GUVs differing in their Gb3 content. As expected, the binding efficiency of StxB to GUVs increased with the amount of Gb3 molecules in the membrane (see Supplementary Figure S4). Figure S2 and its associated legend show/teach membrane partitioning of the membrane probe F2N12SM. A and B) Partitioning of the F2N12SM probe into Ld and Lo phases. A) Phase-separated GUV composed of DOPC/chol/SM (40/15/40) labeled by F2N12SM. The yellow dot and the dashed arrow respectively indicate the starting point and the direction used to extract the circular profile given in (B). This circular profile shows that the F2N12SM intensity is high in Ld phase and negligible in Lo phase. C) and D) show the polarization effect, which results in the variation of the fluorescence intensity of the probe in the bilayer along the GUV contour. Imax is the fluorescence intensity where the molecules of the probe have parallel orientation to the excitation laser polarization. At Imin probes are oriented perpendicular to the laser polarization. This effect can be used to determine the orientation of the probe in the bilayer; C) GUV composed of SM/chol (70/30) labeled by F2N12SM. The fluorescence intensity of F2N12SM varies along the GUV contour, as a result of the orientation of the dye in the membrane. D) Circular profile of the GUV in (C). The conclusion section teaches that they developed a script for the recognition and analysis of circular segments of GUVs in order to study the partition of membrane probes as well as protein binding to membranes. They additionally proposed a quantitative analysis to compare the binding efficiency of proteins to membranes of different compositions and biophysical properties. The script is not limited to the analysis of GUVs, but can also process other spherical membranes like native giant plasma membrane vesicles, or any circular object. The script can also be combined with time-frame analysis to study dynamics of binding processes and, with a tracking algorithm, to monitor GUV motions.
It would have been obvious to one of ordinary skill in the art at the time the application was filed to use other vesicles such as the GPMV of Levental (claims 4 and 20 specifically) or the GUV of Sych (all claims not specifically limited to GPMV) in the Katz method and system because the method and system of Katz are intended for detecting/studying the interaction of various chemicals with vesicle membrane domains in combination with the recognized ability of GPMV and/or GUV to produce domain separations which can be used model membranes and be tested for the interaction of different compounds/proteins with the different domains as shown/taught by Levental and Sych. It further would have been obvious to one of ordinary skill in the art at the time the application was filed to utilize the separately detectable/distinguishable dyes/indicators taught by Levental or Sych (all claims generally with specific reference to claims 8-12 and 24-28) in the Katz method and system because of the dyes’/indicators’ known ability and use to label/combine with membrane phase domains and/or proteins in a manner that will allow detection and analysis of the phase domains and interactions with proteins and/or other compounds that can be localized in and/or affect the amount of the different phase domains present in a vesicle as shown/taught by Levental and Sych. It also would have been obvious to one of ordinary skill in the art at the time the application was filed to incorporate an automated software image analysis routine such as taught by Sych (all claims generally with specific reference to claims 5-7, 13-15, 21-23 and 29-31) into the Katz method and system because the method and system of Katz are intended for detecting/studying the interaction of various chemicals with vesicle membrane domains in a high-throughput manner in combination with the recognized fast and unbiased analysis of imaging data that the Sych method and software enables for high-throughput analysis of vesicle systems.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure. The additionally cited art is related to vesicles being used to identify/measure the effects on membranes cause by interactions with various chemicals and the analysis of microplates with reaction wells.
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/ARLEN SODERQUIST/Primary Examiner, Art Unit 1797
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