METHODS OF PRODUCING MICROBUBBLE DRUG CONJUGATES, VIRAL GENE THERAPY MICROBUBBLE CONJUGATES AND TARGETED MICROBUBBLES
20250177557 ยท 2025-06-05
Assignee
Inventors
Cpc classification
A61K47/6913
HUMAN NECESSITIES
A61K47/6849
HUMAN NECESSITIES
International classification
A61K47/69
HUMAN NECESSITIES
A61K47/68
HUMAN NECESSITIES
A61K48/00
HUMAN NECESSITIES
Abstract
The present invention relates to methods of producing microbubble drug conjugates, viral gene therapy microbubble drug conjugates, and disease-targeting microbubbles, for clinical and preclinical ultrasound-mediated therapeutic and diagnostic applications. It includes methods to produce viral vector gene therapy microbubble drug conjugates with antibody linkers conjugated to lipid shelled microbubbles that both bind to and neutralize viral vectors such that the viral gene therapy can transduce and effect permanent genetic changes only after ultrasound is used to disassociate the viral gene therapy from microbubbles at diseased regions of the body.
Claims
1. Methods to produce lipid-shelled microbubble drug conjugates (MDCs) for ultrasound mediated treatments and diagnosis whereby the drug or disease-targeting biologic is conjugated to functionalized lipid vesicles by means such as, but not limited to, conjugation to amine-PEG, carboxyl-PEG, or azide-PEG, followed by microbubble formulation using methods disclosed in Microbubble Medical Devices U.S, patent U.S. Pat. No. 8,679,051 and Medical Microbubble Generation U.S. patent U.S. Pat. No. 8,257,338, inventors James Keenan et al.
2. Methods to produce viral gene therapy microbubble drug conjugates (MDC) s whereby: a) Antibody linkers with binding specificity to particular viral vectors (adeno associated viral of different serotypes [AAV2, AAV9, etc.], adenovirus, lentiviral, viral immunotherapy, herpes simplex, retroviral, etc.) are developed using methods such as, but not limited to, mouse rapid prime immunization to generate mouse hybridomas secreting monoclonal antibodies specific for the particular virus b) The antibody linkers are covalently conjugated to functionalized lipid vesicles by means such as, but not limited to, conjugation to amine-PEG, carboxyl-PEG, or azide-PEG. c) Lipid-shelled microbubbles are generated with the antibody linkers conjugated to the shell surface d) Viral gene therapy is added to the microbubble solution, mixed, and incubated to form viral gene therapy MDCs
3. Methods to optimize the binding strength of the antibody linkers of the viral gene therapy MDCs of claim 2 for in vivo ultrasound treatments by ensuring sufficient binding strength to maintain viral gene therapy and microbubble conjugation during in vivo circulation while also permitting localized delivery to diseased regions using therapeutic ultrasound to sonicate said regions to controllably disassociate (shed) the viral gene therapy from the MDCs: a) Produce antibody linkers with varying binding strengths to a particular virus b) Generate viral gene therapy MDCs with varying linker binding strengths c) Sonicate the MDCs with therapeutically relevant ultrasound exposures in a flow chamber over a cell bed to quantify transduction and confirm that ultrasound disruption and/or resonating of the MDCs releases the viral gene therapy on demand while maintaining its capacity to transduce
4. Viral gene therapy MDCs of claim 2 whereby the antibody linker with binding specificity to a viral vector also neutralizes the viral vector and prevents transduction while the gene therapy is conjugated to the microbubble. MDCs circulating in the bloodstream will be unable to transduce and so prevent systemic adverse effects to healthy tissue. Focused or non-focused ultrasound may then be applied in vivo to MDCs in circulation to dissociate and release the viral gene therapy at diseased regions for transduction and treatment. Viral gene therapy MDCs not used in treatment will deflate as the gas leaks out and is expelled through the lungs. Viral gene therapy will remain conjugated to the deflated MDC lipid shells which will primarily clear in the liver by Kuppfer cells but will be unable to transduce and effect permanent genetic changes to healthy liver tissue.
5. Microbubble drug conjugates of claim 1 or 2 where the lipids selected to form the MDC-shells promote extended in vivo persistence (half-life in circulation) in order to maximize dose delivered to diseased regions as well as to maximize ultrasound/microbubble therapeutic effects such as tumor vascular disruption and/or temporary blood brain barrier opening for non-invasive, targeted drug delivery to the brain.
6. Microbubble drug conjugates of claim 1 or 2 whereby biologic therapeutics (antibodies, proteins, ligands, bispecific T cell engaging antibodies, etc.) are covalently attached to the microbubble shell by means such as, but not limited to, conjugation to amine-PEG, carboxyl-PEG, or azide-PEG functionalized lipid vesicles.
7. Microbubble drug conjugates featuring small molecule drugs whereby the drug is reacted with lipid powders to form powdered lipids linked to small molecules, water added, and the solution purified using a high performance liquid chromatography (HPLC) column, followed by microbubble drug conjugate generation of claim 1.
8. Microbubble conjugates of claim 1 or 2 whereby disease targeting biologics, for example anti VEGFR2 or anti-ICAM antibodies, are covalently attached to the microbubble shell by means such as, but not limited to, conjugation to amine-PEG, carboxyl-PEG, or azide-PEG functionalized lipid vesicles and the disease targeting microbubbles used to improve ultrasound diagnostic sensitivity. For example, tumor-targeting, echogenic, gas-filled microbubbles with contrast agent properties will bind to microtumors undetectable by conventional ultrasound in order to provide early-stage ultrasound diagnosis of disease.
9. Microbubble drug conjugates of claim 1 whereby microbubbles incorporating azide-PEG vesicles or other suitable click chemistry means are generated and biologic drugs are added to the microbubble solution for incubation and conjugation to the microbubbles.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0076] These and other features of the invention will now be described by way of example, with reference to the appended drawings, wherein:
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DESCRIPTION OF PREFERRED EMBODIMENTS
[0090] The present invention relates to methods of producing microbubble drug conjugates (MDCs), viral gene therapy MDCs, and targeted microbubbles. More specifically, the present invention relates to methods of producing MDCs and targeted microbubbles for clinical applications.
[0091] Biologics with therapeutic or selective targeting capabilities are to be added in a covalent manner to lipid vesicles, prior to microbubble formation. In this way, the addition of biologics does not impact the integrity of the gas contained within the microbubble. To do this, pre-attachment of biologics to, for example, amine-PEG, carboxyl-PEG, or azide-PEG functionalized lipid vesicles prior to conversion to microbubbles is a viable solution to develop clinically useful MDCs or targeted microbubbles ready for injection. This sequence for producing these will allow for MDCs and targeted microbubbles to be produced in the clinic without the requirement of chemistry knowledge.
[0092] The choice of linker can consist of any functionalized lipid suitable for conjugation to biologics, including amines, carboxy, biotin, maleimide, hydrazide, or azide or other click chemistry methods. These lipid vesicles can encompass any type of unilamellar or multilamellar vesicle, liposome, micelle, bicelle and including those derived from living cells such as exosomes or microvesicles. If required, the lipid vesicles are then purified of unbound biologics before being converted to microbubbles by incorporating gas into the vesicles with various standard mixing or alternative microbubble formation procedures. The lipid vesicle solution can be supplied to clinicians in a vial, cartridge, etc., which also contains perfluorocarbon gas. This vial can then be used on demand and to produce gas-filled microbubble drug conjugates or targeted microbubbles ready for injection when it is processed using various known procedures for microbubble formation.
[0093] This strategy can allow for an easy production of MDCs and targeted microbubbles ready for clinical use
[0094] To do this will require the following steps using, for example, amine-PEG (see
[0101] The use of chemical attachment of biologics to the lipid vesicles stage rather than at the individual lipid stage or at the post microbubble formation stage allows for microbubble drug conjugates (MDCs) to be produced on demand in the clinic. In the clinic, an individual can convert biologic drug-lipid vesicles contained within a vial or cartridge to MDCs without impacting microbubble formation or the biologics' bioactiviy. This strategy would allow MDCs to be produced on demand in the clinic in one step. Vials containing biologics, lipid vesicles, and gas can be safely stored in the fridge until needed without lyophilization and reconstitution. When MDCs are required, a cartridge is mixed by various known methods for microbubble formation without compromising sterility and MDCs or targeted microbubbles are generated automatically and are ready for injection into patients. The ease of generation of MDCs or targeted-microbubbles in this manner, without the requirement of chemistry knowledge post microbubble formation makes clinical use of this technology feasible.
[0102] The present invention will be further illustrated in the following examples. However, it is to be understood that these examples are for illustrative purposes only and should not be used to limit the scope of the present invention in any manner.
TABLE-US-00001 TABLE 1 List of Examples Example Embodiment 1 Production of lipid vesicles 2 Microbubble production and characterization 3 Cy5.5 labeling of lipid vesicle solution 4 Conjugation of biologics to lipid vesicles 5 Microbubble formation from labeled lipid vesicles 6 Conjugated microbubble characterization 7 Binding of conjugated microbubbles 8 Production of Microbubble Drug Conjugates or Targeted Microbubbles 9 Bioactivity Confirmation of Microbubble Drug Conjugates 10 Microbubble Drug Conjugate and Focused Ultrasound Blood Brain Barrier Opening for Non-invasive, Targeted Drug Delivery to the Brain. 11 Production of Viral Vector Gene Therapy Antibody Linkers 12 Production and Characterization of Viral Gene Therapy Microbubble Drug Conjugates with Antibody Linkers 13 Viral Gene Therapy Microbubble Drug Conjugate Non- invasive, Targeted Blood Brain Barrier Delivery 14 Production and Treatment Methods for Viral Gene Therapy MDCs With Neutralizing Antibody Linkers 15 Production of Small Molecule Drug and Microbubble Conjugates 16 Production of Disease-targeting Microbubbles for Diagnosis with in vitro and in vivo characterization
Example 1: Production of Lipid Vesicles
[0103] Lipid vesicles were formed that incorporate amine-PEG-lipids or in various molar ratios. These vesicles will be used in subsequent examples; where biologic molecules are conjugated to the lipid vesicles prior to microbubble formation.
[0104] Lipid vesicles were formed using methods well-known in the art. Briefly, a lipid mixture (i.e. amino functionalized PEG-DSPE lipid, phosphotidylcholine lipids or other lipids suitable to generate lipid vesicles such as DSPC, DBPC, others) was dissolved in chloroform to achieve desired lipid composition and ratio. The lipids were then vortexed. The chloroform was evaporated overnight under reduced pressure and elevated temperature (>45 C.) using a Vacufuge to ensure complete removal of the chloroform. A thin lipid film was then rehydrated with PBS, pH 7.4 at 60 C. using a hot water bath to create a lipid solution comprising lipid vesicles with an aqueous center.
[0105] A lipid mixture of DBPC (1,2-Distearoyl-sn-glycero-3-phosphocholine) plus DSPE-PEG (2000) was formed to increase the in vivo persistence of the MDCs. Extended in vivo persistence improves ultrasound and microbubble therapies such as non-invasive, targeted, temporary opening of the blood brain barrier for drug delivery as well as tumor vascular disruption to boost immunotherapy and chemotherapy efficacy. Extended in vivo persistence also permits increased drug concentration locally delivered to diseased regions
[0106] A lipid mixture of DBPC and DSPE-PEG-Azide (2000) was developed to permit microbubble drug conjugates using click chemistry (also described in Example 3). The utility of using click chemistry versus covalent chemical conjugation is to minimize drug losses during the conjugation process.
Example 2: Microbubble Production and Characterization
[0107] The lipid vesicles of Example 1 were converted to microbubbles. The microbubble solutions were then characterized to determine the total microbubble count and their size distribution.
[0108] The lipid vesicles are converted to microbubbles by either piezoelectric transducer 5 agitation of sealed liposome solution and gas, electrolysis and ultrasound generation, or use of a microbubble generation apparatus through reciprocally driving the drug-liposome solution and gas through a bubble generation module (Reference U.S. Pat. No. 8,257,338),
[0109] Microbubble characterization results are shown in Tables 2 and 3. Results indicate that the addition of 1, 2, or 3 amine-PEG-DSPE linker lipid into the lipid vesicles did not impact the gas microbubble formation, size, or concentration of generated microbubbles.
TABLE-US-00002 TABLE 2 Summary comparison of total microbubble counts/ml of various lipid microbubbles containing 1X, 2X or 3X PEG amine linker. PEG Total Microbubble amine Microbubble Solution Dilution Counts/ml 1X 0.8 ml MB solution, 1.2 ml PBS, 5.34 10.sup.8 pH 7.4 2X 0.8 ml MB solution, 1.2 ml PBS, 2.60 10.sup.8 - run 1 pH 7.4 2.89 10.sup.8 - run 2 3X 0.8 ml MB solution, 1.2 ml PBS, 4.41 10.sup.8 - run 1 pH 7.4 5.44 10.sup.8 - run 2 3.96 10.sup.8 - run 3
TABLE-US-00003 TABLE 3 Coulter counter results of microbubble size distribution and concentration in the sample with 1X, 2X, 3X PEG amine linker. Size 1X 2X - run 1 2X - run 2 3X - run 1 3X - run 2 3X - run 3 M [C]/mL [C]/mL [C]/mL [C]/mL [C]/mL [C]/mL 0.75-1.00 1.953 10.sup.8 2.836 10.sup.8 1.640 10.sup.8 2.836 10.sup.8 2.650 10.sup.8 2.428 10.sup.8 1.01-2.00 2.421 10.sup.8 1.163 10.sup.8 7.354 10.sup.7 1.163 10.sup.8 2.074 10.sup.8 1.077 10.sup.8 2.01-3.00 6.719 10.sup.7 3.364 10.sup.7 1.874 10.sup.7 3.364 10.sup.7 5.184 10.sup.7 3.677 10.sup.7 3.01-4.00 8.407 10.sup.6 6.482 10.sup.6 3.057 10.sup.6 6.482 10.sup.6 1.618 10.sup.7 7.912 10.sup.6 4.01-5.00 3.500 10.sup.5 5.499 10.sup.5 1.692 10.sup.5 5.499 10.sup.5 3.189 10.sup.6 6.747 10.sup.5 5.01-6.00 1.869 10.sup.4 2.475 10.sup.4 1.818 10.sup.4 2.475 10.sup.4 3.732 10.sup.5 6.111 10.sup.4 6.01-7.00 5.555 10.sup.3 7.575 10.sup.3 3.535 10.sup.3 7.575 10.sup.3 6.262 10.sup.4 8.080 10.sup.3 7.01-15.00 6.575 10.sup.3 3.030 10.sup.3 1.010 10.sup.3 3.030 10.sup.3 1.263 10.sup.4 6.565 10.sup.3 15.01-30.00 3.030 10.sup.3 0.000 1.010 10.sup.3 0.000 5.050 10.sup.2 0.000
[0110] Novel refinements were done to the microbubble generation to increase counts in order to improve in vivo performance for anticipated clinical use.
Example 3: Cy5.5 Labeling of Lipid Vesicle Solution
[0111] The lipid vesicles prepared as described in Example 1 were functionalized with Cy5.5 prior to microbubble formation.
[0112] Cy5.5 dye was conjugated to the lipid vesicles (Example 1). 200 l of carbonate buffer (10% v/v) was added to 2 ml of lipid vesicle solution, followed by 1 l (1 Cy5.5), 2 l (2 Cy5.5), or 3 l (3 Cy5.5) of NHSester Cy5.5 (10 g/l). The solution was mixed and the reaction allowed to proceed for 2 hr at room temperature. Free Cy5.5 was separated from the lipid vesicle-conjugated Cy5.5 using an Amicon 10 kDa cutoff concentrator. The Cy5.5-lipid vesicle solution volume was reduced to 1 ml.
[0113] A further example to label MD.Cs with a Cy5.5 is though click chemistry by incorporating a functionalized azide-PEG lipid to the lipid vesicles (Example 1) and then adding, mixing, and incubating the Cy5.5. dye. An Amicon concentrator may be used to remove unconjugated dye.
Example 4: Conjugation of Biologics to Lipid Vesicles
[0114] A common method for associating protein molecules (antibodies, ligands, proteins, Bispecific T cell engaging antibodies, antibodies with binding specificity to viral vector gene therapies, others) to microbubbles is by covalent linkage (also referred to as chemical conjugation). Chemical conjugation techniques are best to use for in vivo studies as they are stable in the blood and have proven successful when linking biologics to lipids. A variety of standard chemical conjugation reactions have been utilized in the literature, including attaching a peptide or protein via carbodiimide chemistry (Palmowski et al, 2008) or stable thioether bond chemistry (Anderson et al, 2010). Polymer spacers of various length, such as PEG, may be used to maintain distance between the protein and the microbubble shell (Kim et al, 2000).
[0115] Lipid vesicles comprised of DSPC plus DSPE-PEG (2000) amino linker were formulated as per Example 1.
[0116] Proteins were covalently attached directly to the functionalized, stable lipid vesicles in aqueous buffers prior to (perfluorocarbon) gas microbubble formation. The antibody's c-terminal carboxy group were chemically conjugated to the amine groups incorporated on the lipid vesicles. In this method, the antibody was attached to amine-PEG functionalized lipid vesicles prior to conversion to microbubbles; The antibody-lipid vesicles were then purified of unbound antibodies before being converted to microbubbles by incorporating gas into the vesicles.
[0117] Antibodies were dialyzed into MES (2-ethanesulfonic acid) buffer, followed by the addition of 1.1 mg sulfo-NHS (N-hydroxysulfosuccinimide) (final concentration 5 mM) and 0.4 mg EDC (final concentration 2 mM). The mixture was allowed to react for 15 min, after which it was purified with an Amicon 10 kDa cutoff using MES buffer; the solution volume was reduced to 100-200 l. The free antibody was removed using an Amicon 100 kDa cutoff concentrator.
[0118] The inclusion of the biologics to the amine-PEG-DPSE lipid vesicles did not impact gas microbubble formation, the size of generated microbubbles, or their concentration or the bioactivity of the biologics (see example 9)
[0119] A further example to conjugate biologic drugs to MDCs is though click chemistry by incorporating a functionalized azide-PEG lipid to the lipid vesicles (Example 1) and then adding, mixing, and incubating, for example, an anti CTLA-4 antibody. Focused ultrasound can then be used with immunotherapy MDCs circulating through the bloodstream to disrupt the MDCs to increase drug concentration at tumors and to disrupt the tumor vasculature to further increase immunotherapy drug efficacy.
[0120] In a targeted diagnostic microbubble variant, a single domain antibody targeting IGFBP7 (VHH 4.43, as described in WO2010043037A1) was conjugated to the lipid vesicles of Example 1 prior to microbubble formation.
[0121] Briefly, 1 mg VHH 4.43 (3 mg/ml) was dialized into MES buffer, followed by the addition of 1.1 mg sulfo-NHS (final concentration 5 mM) and 0.4 mg EDC (final concentration 2 mM). The mixture was allowed to react for 15 min, after which it was purified with an Amicon 10 kDa cutoff using MES buffer; the solution volume was reduced to 100-200 l. The NHS-ester modified protein (100 l) was mixed with carbonate (100 l, 10%) and Cy5.5-labeled lipid solution (2-10 ml); the reaction was allowed to proceed for 2 hr. The free antibody was removed using an Amicon 100 kDa cutoff concentrator.
[0122] This method can be used to prepare both labelled and unlabeled antibody vesicles and to prepare labelled vesicles with a variety of dyes such as, for example, rhodamine.
Example 5. Conjugated Microbubble Formation from Labeled Lipid Vesicles
[0123] The lipid vesicles conjugated to Cy5.5 (Example 3) or VHH 4.43 (Example 4) were converted to microbubbles, which were then characterized.
[0124] Briefly, the labelled lipid vesicles solution was mixed with gas (i.e. perfluorocarbon or air) and surfactant (i.e. tween80, stearic acid.Math. or an alternative detergent) in a tube or cartridge. Vigorous mixing, pressure, and/or sonication was applied to convert liposomal solution to larger gas-filled microbubbles.
[0125] Alternatively, a bubble-generating apparatus may be used in an automated process to provide more reproducible microbubbles/results as described in the following patents assigned to Artenga Inc.: [0126] Microbubble Medical Devices U.S, patent U.S. Pat. No. 8,679,051 issued Mar. 25, 2014. [0127] Medical Microbubble Generation U.S. patent U.S. Pat. No. 8,257,338 issued Sep. 4, 2012
Example 6. Conjugated Microbubble Characterization
[0128] The conjugated microbubbles prepared in Example 5 were characterized using methods described in Example 2.
[0129] Table 3 shows absorbance measurements at 280 nm, 555 nm, and 678 nm. These results confirmed successful conjugation of antibody (280 nm), Cy5.5 (678 nm), Cy5.5-labeled antibody, and/or rhodamine-labeled antibody (555 nm) to the lipid vesicles in solution and maintenance of the link after gas microbubble formation.
TABLE-US-00004 TABLE 3 Summary of absorbance measurements for antibody and dye incorporation in lipid vesicles and microbubbles (MB). Construct A.sub.280 A.sub.555 A.sub.678 1x PEG lipid vesicle 0.172 0.036 0.004 2x PEG lipid vesicle 0.255 0.009 0.009 3x PEG lipid vesicle 0.359 0.006 0.002 1x PEG Ab lipid vesicle 2.879 0.019 0.002 1x PEG Rhod Ab lipid vesicle 3.468 2.510 0.066 1x PEG 1x Cy5.5 lipid vesicle 0.802 0.181 0.511 1x PEG 2x Cy5.5 lipid vesicle 0.820 0.106 1.960 1x PEG 3x Cy5.5 lipid vesicle 0.884 0.150 2.963 3x PEG Rhod Ab lipid vesicle and 1x Cy5.5 1.207 0.488 1.603 3x PEG Rhod Ab lipid vesicle and 1x Cy5.5 1.21 0.608 1.501 1x PEG MB 0.241 0.226 0.077 1x PEG Ab MB 0.987 0.053 0.025 1x PEG Rhod Ab MB 1.425 0.909 0.038 1x PEG MB 2x Cy5.5 0.419 0.048 0.710 1x PEG MB 3x Cy5.5 0.420 0.080 1.120 3x PEG lipid vesicle 0.536 0.001 0.020 3x PEG Cy5.5 protein lipid vesicle 1.719 0.009 0.481 (no purification) 3x PEG Cy5.5 protein lipid vesicle 1.079 0.004 0.357 (with purification)
[0130] Results for Cy5.5-conjugated microbubbles (Table 4) show that chemical attachment of 1, 2 or 3 fluorescent Cy5.5 onto the external shell of amine-PEG-DSPE-containing lipid vesicles prior to gas microbubble formation did not impact gas microbubble formation, size or concentration of generated microbubbles.
TABLE-US-00005 TABLE 4 Coulter counter results of microbubble size/concentration distribution in Cy5.5-conjugated microbubbles with 3X PEG amine linker. Total microbubble counts/ml: 3.546 10.sup.8 (1x Cy5.5); 2.892 10.sup.8 (2x Cy5.5); 1.690 10.sup.8 (3x Cy5.5). Size 1X 2X 3X M [C]/mL [C]/mL [C]/mL 0.75-1.00 2.120 10.sup.8 1.806 10.sup.8 9.234E+07 1.01-2.00 9.550 10.sup.7 7.150 10.sup.7 5.725 10.sup.7 2.01-3.00 3.770 10.sup.7 3.051 10.sup.7 1.745 10.sup.7 3.01-4.00 9.126 10.sup.6 5.943 10.sup.6 1.613 10.sup.6 4.01-5.00 1.106 10.sup.5 5.414 10.sup.5 2.889 10.sup.5 5.01-6.00 9.797 10.sup.4 4.899 10.sup.4 6.818 10.sup.4 6.01-7.00 1.212 10.sup.4 6.565 10.sup.3 2.576 10.sup.4 7.01-15.00 5.555 10.sup.3 4.040 10.sup.3 1.162 10.sup.4 15.01-30.00 0.000 0.000 0.000
[0131] Tables 5 and 6 show the size distribution for various microbubble preparations. Chemical conjugation of Rhodamine-labeled or unlabeled antibody, in addition to chemical conjugation of Cy5.5, onto the external shell of amine-PEG-DSPE-containing lipid vesicles prior to gas microbubble formation did not impact gas microbubble formation, size, or concentration of generated microbubbles.
TABLE-US-00006 TABLE 5 Coulter counter results of microbubble size/concentration distribution in 1x Cy5.5-conjugated microbubbles with 3X PEG amine linker (3.546 10.sup.8/ml); 1x Cy5.5- and Rhod-Ab-conjugated microbubbles with 3X PEG amine linker (4.814 10.sup.8/ml); and 1x Cy5.5- and Ab-conjugated microbubbles with 3X PEG amine linker (2.769 10.sup.8/ml). Size 3x PEG 3x PEG 1x Cy5.5 3x PEG 1x Cy5.5 M 1x Cy5.5 MB Rhod Ab MB Ab MB 0.75-1.00 2.120 10.sup.8 2.966 10.sup.8 1.623 10.sup.8 1.01-2.00 9.550 10.sup.7 1.423 10.sup.8 7.917 10.sup.7 2.01-3.00 3.770 10.sup.7 3.476 10.sup.7 2.888 10.sup.7 3.01-4.00 9.126 10.sup.6 7.298 10.sup.6 5.938 10.sup.6 4.01-5.00 1.106 10.sup.5 4.338 10.sup.5 5.449 10.sup.5 5.01-6.00 9.797 10.sup.4 3.838 10.sup.4 7.979 10.sup.4 6.01-7.00 1.212 10.sup.4 1.010 10.sup.4 9.090 10.sup.3 7.01-15.00 5.555 10.sup.3 6.060 10.sup.3 8.585 10.sup.3 15.01-30.00 0.000 0.000 0.000
TABLE-US-00007 TABLE 6 Coulter counter results of microbubble size/concentration distribution in microbubbles with 3X PEG amine linker (4.128 10.sup.8/ml); and 1x Cy5.5-Ab-conjugated microbubbles with 3X PEG amine linker with (3.645 10.sup.8/ml) or without (1.082 10.sup.9/ml) purification of protein from lipid vesicles. 3x PEG 1x Cy5.5 3x PEG 1x Cy5.5 Size Ab MB Ab MB M 3x PEG MB (no purification) (purification) 0.75-1.00 2.626 10.sup.8 8.643 10.sup.8 2.487 10.sup.8 1.01-2.00 1.008 10.sup.8 1.809 10.sup.8 8.669 10.sup.7 2.01-3.00 4.095 10.sup.7 3.089 10.sup.7 2.439 10.sup.7 3.01-4.00 7.872 10.sup.6 5.584 10.sup.6 4.301 10.sup.6 4.01-5.00 5.378 10.sup.5 2.909 10.sup.5 3.934 10.sup.5 5.01-6.00 3.485 10.sup.4 2.576 10.sup.4 5.202 10.sup.4 6.01-7.00 5.555 10.sup.3 2.525 10.sup.3 1.061 10.sup.4 7.01-15.00 5.050 10.sup.2 1.515 10.sup.3 3.535 10.sup.3 15.01-30.00 0.000 1.515 10.sup.3 0.000
Example 7. Conjugated Microbubble Payload Delivery Using Ultrasound
[0132] Using surface plasmon resonance (SPR).
[0133] Microbubble drug conjugate surrogates were prepared as described in Example 5 with a fluorescent dye payload and injected in tumor-bearing rodent models and ultrasound exposures applied locally to the tumors. Optical imaging (GE Healthcare explore Optix) was used to demonstrate enhanced perfusion and retention in vivo of microbubble payload in tumor bearing rodent models. In a clinical application this would permit enhanced perfusion and retention of drug from microbubble drug conjugates. In a clinical application using approved focused ultrasound systems capable of precision targeting of diseased regions of the body (including but not limited to tumors) this would permit high dose, localized drug delivery with enhanced perfusion and delivery.
[0134] The embodiments and examples described herein are illustrative and are not meant to limit the scope of the invention as claimed. Variations of the foregoing embodiments, including alternatives, modifications and equivalents, are intended by the inventors to be encompassed by the claims. Furthermore, the discussed combination of features might not be necessary for the inventive solution.
Example 8: Production of Microbubble Drug Conjugates and Targeted Microbubbles
[0135] The lipid vesicle production of Example 1, conjugation of biologics to lipid vesicles and purifying the solution without the need of an organic solvent of Example 4, and microbubble generation of Example 2, were done using high lipid concentrations (30 mg lipids per 10 ml solution for example) whereby the resulting solution produced therapeutically relevant quantities of microbubble drug conjugates.
[0136] Different lipid-drug linking modalities may be incorporated in the liposome solution such as covalent (amine, carboxyl, others), electrostatic (cationic, anionic), click chemistry (azide, cyclooctyne, others), or biotin-avidin conjugation of the liposomes to the drug.
[0137] The present invention is a method to produce an MDC solution whereby a biologic drug's bioactivity is maintained post conjugation as shown in Example 9. The simple, one step process described avoids the cost, complexity, and potential sterility compromise of producing and then mixing separate MDC and DLL solutions prior to infusion. [0138] MDCs can be used with FUS for non-invasive, targeted, and temporary BBB opening of diseased brain regions for active drug transport and greater drug perfusion as shown in Example 10 [0139] Ultrasound manipulation of circulating microbubbles can increase immunotherapy efficacy for cancer patients without the added toxicity typically associated with combining other drugs or treatments through: [0140] Promoting the release of tumor antigens, modulating the tumor microenvironment, and increasing cytotoxic T-cells (Lui et al (J Trans Medicine, 2012) and Hunt et al (J Ultrasound Med, 2015)) [0141] Boosting checkpoint inhibitor efficacy (tumor eradication and an active immune response) by slowing or stopping blood flow within a tumor (David E. Goertz, et al Enhancing Checkpoint Inhibitor Therapy with Ultrasound Stimulated Microbubbles Ultrasound in Med.&Biol., Vol.00,No.00,pp. 1_14, 2018) A preferred embodiment of the invention to produce and administer MDC solutions is described below:
[0142] A checkpoint inhibitor antibody (anti PD-1, anti PDL-1, or anti CTLA-4) that is an ineffective monotherapy once cancer spreads to the brain is conjugated to lipid vesicles as described in Examples 1 and 4, using high lipid concentrations and microbubble generation of Example 2, thereby producing a solution of therapeutically relevant quantities of microbubble drug conjugates.
[0143] The MDC solution is administered intravenously and circulates throughout the bloodstream. Focused ultrasound (FUS) is used to target brain tumors and sonication rapidly compresses and expands the MDCs at the frequency of the FUS. The resulting mechanical action temporarily opens the tight endothelial junctions that form the blood brain barrier (BBB) and promotes release of antibodies from the MDCs for active drug transport into tumors with enhanced perfusion.
[0144] The FUS acoustic pressure is then increased and ultrasound exposures modified in order to induce MDC expansions and contractions sufficient to slow or stop blood flow within the tumors. This mechanical action has a synergistic effect to promote tumor reduction (as shown in
[0145] The BBB starts closing immediately after the few minutes of FUS sonications cease but remains open for a few hours sufficient for small molecule drug delivery.
[0146] MDCs that are not disrupted by FUS exposures circulate and clear in the standard way of ultrasound contrast agent microbubbles used for diagnosis: the PFC gas leaks from the MDCs and is expelled through the lungs and the collapsed lipid microbubble shells with conjugated antibodies is cleared by the liver's Kupffer cells. This liver clearance will prevent system wide dosing and potential endocrine immune-related adverse effects.
[0147] The therapy described in this embodiment can also be used on other indications such as primary triple negative breast cancer, ovarian, liver, pancreatic cancers, and others. MDC solutions can be produced with a variety of biologics including antibody fragments, targeted therapy, bispecific T cell engaging antibodies (BiTEs), and others.
Example 9: Bioactivity Confirmation of Microbubble Drug Conjugates
[0148] Cerebral Dopamine Neurotrophic factor (CDNF). protein (a Parkinson's Disease neuroprotective drug) was used to produce CDNF: MDCs and the retention of the neuroprotective activity of CDNF-MDCs confirmed in vitro.
[0149] Neuroprotection was tested in the well-established neuronal model of human SHSY-5Y cells (Lopes, F. M.,. et al. (2010) Brain Res. 1337, 85-94) treated with parkinsonian neurotoxins. Human SHSY-5Y cells were grown in 24 well dishes and differentiated into neuron-like cells by treatment with 10 M retinoic acid (RA) for 7 days (
[0150] The neurotoxic effects of MPP+ (active metabolite of MPTP) and thapsigargin (thap, ER stress inducer) were first established by treating the cells with 0.1-1.0 mM MPP+ or 0.1-1.0 M thap for 16 h. Cell viability was quantified by the CFDA assay as described previously (Sandhu, J. K., et al. (2009) Neurobiol. Dis. 33, 405-414.).
[0151] Recombinant human GDNF at 50 ng/ml was used as a positive control. Cells pre-incubated for 2 hours with 50 ng/ml rhGDNF before exposure and during the treatment with either 1.0 mM MPP+ or 1.0 M thapsigargin were protected from cell death (
[0152] The neuroprotective effects of CDNF-MDCs was tested using a similar protocol (as described above for rhGDNF) by treating cells with 1.0 mM MPP+ or 1.0 UM thap for 16 h in the absence or presence of 25-250 ng/ml CDNF-MDCs. The pre-treatment with 25-250 ng/ml CDNF-MDCs completely protected the cells from the neurotoxic effects of MPP+ (
[0153] These results demonstrate that CDNF retains its biological activity after conjugation to microbubbles. The neurotoxic effects of MPP+ or thapsigargin were abolished in cells treated in the presence of CDNF-MDCs. Maximum neuroprotection was reached at concentrations ranging from 25-250 ng/ml.
Example 10: Microbubble Drug Conjugate and Focused Ultrasound Blood Brain Barrier Opening for Non-Invasive, Targeted Drug Delivery to the Brain
[0154] Focused ultrasound (FUS) can be used to target deep seated brain regions and compress and expand microbubbles in circulation. This temporarily opens the tight vascular endothelial cell junctions of the blood brain barrier (BBB) to permit active drug transport. Microbubbles are essential to open the BBB with FUS at lower, safe acoustic pressures.
[0155] AAV2-GFP MDCs were injected into healthy rodents and FUS sonications used to open the BBB in the two brain regions affected by Parkinson's disease, the striatum (ST) and substantia nigra (SN). MR contrast agent gadolinium was co-injected and MR imaging performed to confirm BBB opening in the two regions.
[0156] CDNF protein neurotrophic drug was conjugated to MDCs and delivered to the SN and ST in this manner. Only one hemisphere was sonicated so the animals acted as their own control. See
Example 12: Production of Microbubble Drug Conjugate Viral Vector Gene Therapy Linkers
[0157] The Mouse Rapid Prime immunization approach was used to generate mouse hybridomas secreting IgG monoclonal antibodies specific for adeno-associated virus 2 (AAV2).
[0158] Immunizations were performed with whole virus on female BALB/c mice with a total of 2*1011 recombinant AAV2-GFP per mouse over the immunization period for cell fusion and hybridoma cell line generation. Lymphocytes collected from immunized mice were harvested and counted and fused with murine SP2/0 myeloma cells in the presence of poly-ethylene glycol. The fusion product of up to 10.sup.8 lymphocytes were cultured using a single step cloning method (HAT selection) and remaining fusion product frozen and stored in liquid nitrogen as backup.
[0159] Indirect ELISAs were used to screen for hybridomas that bind to AAV2 and custom downstream assays, including affinity testing and viral neutralization assays, were used to test for antibodies with a range of affinities and properties.
[0160] Clones were transferred to cell wells and grown in HT containing medium. Primary screening was performed by indirect ELISA on whole live AAV2-GFP probing with secondary antibody for both IgG and IgM isotypes. Cultures were retested separately on AAV2-GFP and recombinant AAV5-GFP antigens, the later as a specificity control. Positive hybridomas were isotyped for IgF, IgM, and IgA.
[0161] Twenty different mouse anti AAV2 protein purified monoclonal antibodies were produced by hybridoma. Ten were assessed. for binding affinity. to AAV2 by calculating the ratio of virus/trapping Elisa with three fold titrations as shown in Table 7 below.
TABLE-US-00008 TABLE 7 Anti-AAV2 Antibody Binding Affinity Title: Titration of anti-AAV2 Mouse Hybridomas on AAV2 antigen by Indirect ELISA and by Antibody Trapping ELISA Date: Nov. 24 2017 SOP IPASOP-G-15b Top 10 Reference: Operator: K. M. McIntosh 1E6 2B2 3H3 3H4 4C10 Data D. R. Kroeger 4D1 5A8 5H11 6G6 9D4 reviewed by: Antigen: AAV2, Goat anti- mouse(g/m(H + L) Source of current culture - Primary mid log growth Antibody: ELISA Conditions: Corning Costar 96 well ELISA plates coated with: AAV2 at 10{circumflex over ()}8 viral genome units per well in Carbonate Coating Buffer (pH 9.6) at 100 l/well ON at 4 C. Blocked with 3% Skim Milk Powder in PBS (pH 7.6) at 100 l/well for 1 hour at RT with shaking Primary antibody: TC sup neat at 100 uL/well, incubate for 1 hour at 37 C. with shaking Secondary Antibody 1:5000 Goat anti-mouse IgG-HRP Homemade mix at 100 uL/well in PBS-Tween at 37 C. w/shaking All washing steps performed for 30 mins with PBS-Tween TMB Substrate added at 50 uL/well, developed in the dark and stopped with equal volume 1M HCl Development time: 9 mins Plate read at 450 nm Antibody Trap Conditions: Primary antibody: Hybridoma TC sup neat at 100 L/well for 1 hour at Room Temperature with shaking Corning Costar 96 well ELISA plates coated with: 1:10000 Goat anti-Mouse IgG/IgM(H + L) in Carbonate Coating Buffer (pH 9.6) at 100 L/well in PBS (pH 7.4) O/N at 4 C. No Blocking Primary antibody: Hybridoma TC sup neat at 100 L/well for 1 hour at Room Temperature with shaking Secondary Antibody 1:5000 Goat anti-mouse IgG-HRP Homemade mix at 100 uL/well in PBS-Tween at RT w/shaking All washing steps performed for 30 mins with PBS-Tween TMB Substrate added at 50 L/well, developed in the dark and stopped with equal volume 1M HCl Development time: 3 mins Plate read at 450 nm 0D > 0.20 AAV21 1E6 2B2 2E9 2F4 2H5 3C3 Antibody Titration 1 2 3 4 5 6 Neat A 0.49 0.414 0.411 0.386 0.324 0.316 1:3 B 0.465 0.4 0.346 0.328 0.299 0.323 1:9 C 0.48 0.387 0.231 0.206 0.313 0.279 1:27 D 0.468 0.42 0.157 0.145 0.32 0.183 1:81 E 0.26 0.345 0.105 0.099 0.241 0.11 1:243 F 0.128 0.226 0.087 0.082 0.155 0.083 1:729 G 0.082 0.137 0.064 0.07 0.101 0.062 1:2187 H 0.125 0.115 0.072 0.07 0.092 0.07 0D > 0.20 AAV21 3H3 3H4 4C10 4D1 PBS Controls Antibody Titration 7 8 9 10 11 12 Neat A 0.505 0.501 0.377 0.496 0.068 0.084 IgG Non Secretor 1:3 B 0.498 0.493 0.379 0.466 0.056 0.097 IgG1 Secretor 1:9 C 0.519 0.496 0.325 0.469 0.055 0.075 IgG2a Secretor 1:27 D 0.542 0.533 0.269 0.46 0.055 0.072 IgM Secretor 1:81 E 0.559 0.531 0.157 0.231 0.056 0.242 1/500 Serum 1:243 F 0.509 0.5 0.096 0.106 0.051 0.065 1/500 PreImmune Serum 1:729 G 0.382 0.383 0.066 0.075 0.053 0.072 1:2187 H 0.204 0.208 0.077 0.076 0.074 0.095
[0162] Selected clones were then subcloned by plating parental clones into single cell colonies and antibody purified from culture supernatants using protein G columns. Purified antibody is stored in a carrier-free neutral low endotoxin buffer containing no azide.
[0163] The same MDC antibody linker development process can be used to produce MDC linkers for other adeno-associated virus (AAV) vector serotypes (AAV1, AAV9 etc.) with controllably varying binding affinities. It can be used to develop MDC antibody linkers for other viral vectors (adenovirus, oncolytic viruses, retrovirus; herpesvirus, etc.) and used to develop binding/neutralizing antibody linkers (BNAbs) as shown in Example 15.
Example 13: Production and Characterization of Viral Gene Therapy Microbubble Drug Conjugates
[0164] The following is a preferred example of a means to conjugate viral based gene therapy to microbubbles for targeted, non-invasive delivery to the brain and other organs. A variety of gene therapies and indications, including oncology, neurodegenerative disorders, and others may be treated using this method.
[0165] Six antibodies with varying binding affinity to AAV2 were selected for MDC configuration. The lipid vesicle production of Example 1 and conjugation of antibodies to lipid vesicles and purification of Example 4 were done. Antibody loading was quantified as shown in Table 8.
TABLE-US-00009 TABLE 8 Antibody Loading of Lipid Vesicle Solution Anti-AAV2 Mab bio-conjugation to LPN1X microbubble solution. 2018-05-06 Antibody Protein in 2 ml solution (mg) 3H3 0.474 9D4 0.263 4C10 0.428 6G6 0.223 2E9 0.406 3F8 0.532
[0166] Microbubble generation of Example 2 was done for eachantibody. The microbubble solution was centrifuged, isolated, and resuspended with 2 ml phosphate buffered saline (PBS) to remove excess shell lipids. 100 l at (610.sup.12 virus molecules/mL) of AAV2-GFP (green fluorescent protein gene therapy surrogate) was added to the solution and the solution inverted and incubated for 15 minutes to promote binding.
[0167] The AAV2-SIRT3 microbubbles were centrifuged, isolated, and resuspended with PBS three times to remove remaining 99.9% of the buffer liquid and unbound AAV2-SIRT3 to produce AAV2-GFP MDCs.
[0168] In vitro experiments (see FIG. 10 experimental apparatus) were done with AAV2-GFP MDCs to confirm gene therapy bioactivity and that focused ultrasound (FUS) can be used to induce dissociation of AAV2 from MDCs in a controllable manner: [0169] We demonstrated using cell-based flow cytometry that focused ultrasound exposures relevant to clinically scalable BBB opening and drug delivery can promote the release of AAV gene therapy from MDCs to permit localized transfection (p=0.049 and p=0.0586 AAV MDC only control vs. AAV MDC and FUS)see Normalized 6G6 and 3F8 and 6G6 MDC results below. [0170] AAV2 GFP bioactivity was maintained after conjugation to microbubbles loaded with antibody linkers and FUS exposures. Four different linkers (4C10, 2E9, 9D9, and 6G6)*with a range of AAV2 binding affinities were used with varying FUS pressures (300 KPa, 600 KPa, and 900 KPa).
[0171] Our findings (
[0172] We compared the percent of GFP transfected cells with MDCs versus MDCs and ultrasound for different AAV2 antibody linkers. We see a statistically significant increase in transfection with the ultrasound groups indicating that FUS can be used to release viral gene therapy from MDCs at diseased regions in vivo. Three antibody linkers (6G6, 3F8, & 4C10) were tested and the 4C10 linker selected for the in vivo experiments had the highest transfection as well as highest yield (data not shown).
TABLE-US-00010 NORMALIZED 6G6 AND 3F8 MDC'S Testing of 6G6 Cartridge (2019) Trial # 1 Trial # 1 (Scheme (Scheme Trial # 1 Trial # 1 3 values) 5 values) (Scheme 3 (Scheme 5 AAV2-GFP AAV2-GFP normalized) normalized) MDCs MDCs & FUS (3.81) (3.81) 4.18 3.83 1.10 1.00 3.47 5.22 0.91 1.37 4.06 5.33 1.07 1.40 3.52 6.85 0.92 1.80 Testing of 3F8 Cartridge (2019) Trial # 2 Trial # 2 (Scheme (Scheme Trial # 2 Trial # 2 3 values) 5 values) (Scheme 3 (Scheme 5 AAV2 = GFP AAV2 GFP normalized) normalized) MDCs MDCs & FUS (1.92) (1.92) 1.48 2.11 0.77 1.10 2.06 2.67 1.07 1.39 2.29 2.70 1.19 1.41 1.84 1.85 0.96 0.97 2.55 1.33 2.34 1.22 2.39 1.25 2.20 1.15
[0173] The embodiments and examples described herein are illustrative and are not meant. to limit the scope of the invention as claimed. Variations of the foregoing embodiments, including alternatives, modifications and equivalents, are intended by the inventors to be encompassed by the claims. Furthermore, the discussed combination of features might not be necessary for the inventive solution.
Example 13: Viral Gene Therapy Microbubble Drug Conjugate Non-invasive, Targeted Blood Brain Barrier Delivery
[0174] Sirtuin 3 (SIRT3) gene therapy has been shown to rescue neurons in alpha synuclein rodent models of Parkinson's disease (Nash, J. E., et al, Neurobiol. Dis 106 (2018) 133-146) so AAV2-SIRT3 microbubble drug conjugates (MDCs) were developed as a potential clinical treatment for Parkinson's disease as follows:
[0175] The lipid vesicle production of Example 1, the production of 4C10 antibody linker with binding specificity to AAV2 of Example 12, the conjugation of antibodies to lipid vesicles and purification of Example 4, and microbubble generation of Example 2, and AAV2-SIRT3 conjugation of Example 13 was done to produce AAV2-SIRT3-MDCs.
[0176] Tail vein injection of AAV2-SIRT3-MDCs in healthy rodents was done and focused ultrasound used to temporarily open the BBB and disassociate the viral gene therapy for active transport of AAV2-SIRT3 to the brain. MR imaging with gadolinium confirmed bright regions where FUS targeted the brain, indicating BBB permeabilization.
[0177] Post treatments, rodent brain tissue was harvested and cryosectioned for immunofluorescent analysis. Immunofluorescence labeling and imaging confirmed SIRT3 expression in the ipsilateral striatum using the following antibodies: Myc (SIRT3 myc), SIRT3 (endogenous and ectopic SIRT3), and DAPI (nucleus).
[0178] Reference: Dennison Trinh, Joanne Nash, David Goertz, Kullervo Hynynen, Sharshi Bulner, Umar Iqbal & James Keenan (2022) Microbubble drug conjugate and focused ultrasound blood brain barrier delivery of AAV-2 SIRT-3, Drug Delivery, 29:1, 1176-1183, DOI: 10.1080/10717544.2022.2035855.
[0179] The embodiments and examples described herein are illustrative and are not meant to limit the scope of the invention as claimed. Variations of the foregoing embodiments, including alternatives, modifications and equivalents, are intended by the inventor to be encompassed by the claims. Furthermore, the discussed combination of features might not be necessary for the inventive solution.
Example 14: Production of and Treatment Methods for Viral Gene Therapy MDCs With Neutralizing Antibody Linkers
[0180] Microbubbles in circulation clear from the body as the gas leaks from the microbubbles to be expelled by the lungs and the deflated lipid shells are primarily cleared in the liver by resident Kupffer cell macrophages. Viral gene therapy microbubble drug conjugates cleared in this manner will result in gene therapy transduction of healthy liver tissue. Dependant upon the dose delivered and the toxicity of the gene therapy this may induce adverse effects.
[0181] Therefore, there exists the need for viral gene therapy MDCs whereby the gene therapy cannot transduce while conjugated to the microbubble shells. Only after disassociation from the microbubble at diseased regions of the body using focused ultrasound would transduction and permanent genetic changes be possible.
[0182] Viral-neutralizing antibody therapeutics have been developed to treat SARS-COV-2 and Covid-19.
[0183] In a preferred embodiment of Example 12 of the viral gene therapy MDC invention antibodies that preferentially bind to as well as neutralize specific viral vectors are developed by hybridoma as described in Example 11.
[0184] The antibody linker may be selected on the basis of its capacity to neutralize vectors through, for example, binding affinity to the viral cell surface proteins needed for the virus to enter a cell to permit transduction.
[0185] Neutralization assays will be used to select antibodies to evaluate as MDCs and to confirm that MDC configuration neutralizes viral vector transduction capacity using in vitro cell well assays.
[0186] Antibodies with binding specificity and neutralizing capability may be developed for different adeno-associated vector serotypes (AAV1, AAV9 etc.) and other viral vectors used for gene therapy such as adenovirus, oncolytic viruses, retrovirus, or herpesvirus.
[0187] The lipid vesicle production of Example 1, covalent conjugation of viral binding and neutralizing antibodies to lipid vesicles of Example 4, microbubble generation of Example 5, and viral gene therapy conjugation of Example 12 will be done to produce viral gene therapy MDCs whereby the gene therapy in neutralized while conjugated to the microbubbles.
[0188] Antibodies with varying binding affinity strengths will be conjugated to MDCS and assessed using an in vitro flow chamber as described in Example 12 and with in vivo delivery and biodistribution characterization as shown in Example 13. The binding affinity selected will be sufficiently strong to ensure that all but trace quantities of gene therapy remain conjugated to the MDCs in circulation but still permit the dissociation of viral gene therapy from the MDCs using ultrasound exposures at clinically safe ultrasound parameters including acoustic pressure. An example of typical parameters would be 1 MZ frequency, peak negative pressure of 0.15 and 0.30 MPa, pulse length of 10 ms, and pulse repetition frequency of 1 Hz.
[0189] For clinical treatments the viral gene therapy microbubble drug conjugates will be intravenously infused in the blood stream and circulate while focused ultrasound is used to resonate and/or disrupt the microbubbles in order to disassociate the viral gene therapy at diseased regions of the body for localized transduction and treatment. Diseased regions of most organs in the body are amenable to focused ultrasound treatment including the brain with FUS and MDC targeted, non-invasive, and temporary BBB opening as per Example 10.
[0190] The embodiments and examples described herein are illustrative and are not meant to limit the scope of the invention as claimed. Variations of the foregoing embodiments, including alternatives, modifications and equivalents, are intended by the inventor to be encompassed by the claims. Furthermore, the discussed combination of features might not be necessary for the inventive solution.
Example 15: Production of Small Molecule Drug and Microbubble Conjugates
[0191] Microbubble drug conjugates featuring small molecule drugs are produced whereby the drug is first reacted with lipid powders to form powdered lipids linked to small molecules. Lipid vesicles are then produced as described in Example 1 by combining the small molecule drug lipid with a second lipid (DSPC, DBPC, others) followed by emulsification, and the addition of liquid buffer (PBS, others). The solution is then purified using a high performance liquid chromatography (HPLC) column and small molecule microbubble drug conjugates generated as per Example 5.
Example 16: Production of Disease-targeting Microbubbles for Diagnosis with in vitro and in vivo characterization
[0192] Imaging with ultrasound contrast agent (USCA) microbubbles is used clinically to diagnose, for example, liver tumors. USCA imaging typically has a minimum detection limit of, for example, lesions 2 cm in length or greater. Reducing the detection limit in order to detect microtumors would improve outcome for patients and hence there is a need for microbubbles that target to and bind to microtumors and other diseased regions.
[0193] Approved Insulin-like growth factor-binding protein 7 (IGFBP7) is a protein involved in several cancers including Hepatocellular carcinoma (HCC). IGFBP7 targeting antibodies were developed and covalently conjugated to microbubbles as illustrated in
[0194] Preclincally, fluorescently labeled IGFBP7 were injected intravenously into rodent HCC models and histology performed to confirm IGFBP7 expression.
[0195] Ultrasound imaging was used to compare the IV injection of tumor-targeting IGFBP7 antibody microbubbles versus non-targeted microubbles. Ultrasound imaging confirmed significantly greater contrast enhancement with the targeted microbubbles and that the targeted microbubbles binding to the tumors persisted in vivo longer than non-targeted microbubbles in circulation.
[0196]
Information Disclosure Sheet
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