DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status
Applicant’s submission filed 06/09/2026 has been received and entered. No claims are amended. Claims 11-17, 21-22 and 27 remain withdrawn as being directed to non-elected inventions. Accordingly, claims 1 and 3-9 are pending and under current examination.
This is a second non-final office action, and the prosecution has been re-open.
Status of Prior Rejections/Response to Arguments
The rejection to claims 1 and 8 under 35 U.S.C. 103 over Aricha et al., as evidenced by Sun et al. is withdrawn:
The rejection to claims 1 and 3-8 under 35 U.S.C. 103 over Aricha et al., as evidenced by Sun et al., further in view of Wang et al. and Samanta et al., as evidenced by Lee et al. is withdrawn:
The rejection to claims 1, 8 and 9 under 35 U.S.C. 103 over Aricha et al., as evidenced by Sun et al., further in view of Marshak et al. is withdrawn:
In the submission filed 06/09/2026, Applicant points out that Sun et al. teach using room temperature incubation to associate curcumin, a polyphenol, with exosomes, wherein the curcumin is not a protein, but rather a hydrophobic polyphenolic compound with a molecular weight of 368.39 Da (Remarks, p6). Applicant’s argument is persuasive, the Sun et al. reference has been withdrawn as a prior art. The rejection to the claims is therefore withdrawn.
Response to Arguments:
In the submission filed 06/09/2026, Applicant has traversed the rejection, asserting that Aricha teaches the method of producing and purifying exosomes from a differentiated state of MSC (Remarks, p5). In response, the Examiner submits that as noted in the office action mailed 04/16/2026, Aricha et al. teach in Example 2, modified MSC or MSC-NTF derived exosome for use as nanocarriers for siRNA, miRNA, or proteins (see parag 00405): by encapsulating molecules within their membranes, exosomes can protect proteins or RNAs from degradation (parag 00405). Herein the MSC derived exosome encapsulating proteins within the membranes reads on “ EVs associated with protein”. Aricha et al. also emphasize that it is expected that MSC derived exosomes could be similarly isolated using the methods described herein and MSC or genetically modified MSC (parag 00358). The teachings above indicate that both modified and unmodified MSC derived exosomes can encapsulate proteins within the membranes to form “EVs associated with protein”.
Applicant has traversed the rejection, asserts that Aricha uses saponin to cause permeabilization, freeze-thaw cycles, sonication, extrusion, and room temperature incubation for loading protein into an exosome; Aricha does not provide any example of how to use room temperature incubation to load protein into exosomes (Remarks, p5). In response, the Examiner submits that working examples are not required for a prior art reference to proof of efficacy. See MPEP § 2121. Moreover, a new reference Haney et al. (J Control Release. 2015 Jun 10;207:18-30) is set forth as the evidence to show that catalase (a protein) can be incorporated into exosome by incubation at room temperature with or without saponin (p19, right column). Given that Aricha et al. and Haney et al. teach incubation or mixing exosomes and protein in room temperature is capable of forming extracellular vesicle associated with protein, and TFF procedure provides the purified exosomes and temperature (e.g., room temperature) as needed, one of ordinary skill in the art would have substituted Aricha et al.’s step of loading protein directly to exosomes by incubation, and conduct the mixing and incubation during TFF by supplementing the exogenous proteins to TFF device or a vessel connect to the TFF device (i.e., container of collecting the purified exosomes) during the TFF procedure depends on their research interest or preference (i.e., quickly obtain protein associated EVs without extra step of exosome handling and transferring). This simple substitution of one known element (incubating or mixing the exogenous protein and exosomes during TFF) for another known element (Aricha et al.’s method of loading protein directly to exosomes, i.e., by incubation) is likely to be obvious when predictable results are achieved.
Applicant has further traversed the rejection, asserts that the use of tangential flow filtration (TFF) within Aricha is to remove any small molecules that are not included or associated with an exosome, which is counter to instant claim 1 (Remarks, p6). The argument is not found persuasive. Aricha et al. teach during TFF, the small molecules, including free proteins not included within or associated with the membrane are removed (see parag 00376), but the protein associated with exosomes will not be removed, which is benefit for the accumulating of the protein associated exosomes. Moreover, instant claims do not require the TFF operation (i.e., continuously circulating) for the association of EV and the one or more exogenous proteins, but rather only limits that the association of EV and the one or more exogenous proteins occurs in the TFF device, or a vessel outside of (but connect to the) TFF device during the TFF step (which produces purified exosomes).
New grounds of Rejections
Priority
The instant application is a national stage entry of PCT application PCT/EP2021/068991, filed 01/03/2023 under 35 USC 371. Acknowledgment is made of applicant's claim for foreign priority based on an application EP20184901.5 filed in Europe on 07/09/2020.
Claim Interpretation
Instant claim 1 recites “extracellular vesicles (EVs) associated with proteins”, the Specification provides the definition that the term "associated with EV(s)" in relation to substances means that said substance is either a) attached or bound to the surface layer of the EVs (by any type of binding, such as covalent or non-covalent binding) preferably by means of a non-covalent bond; and/or b) attached or bound within the surface layer of said EVs; and/or c) is internalized within said EVs(Specification, p5, L26-30). Claims are interpreted in light of this definition.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1 and 8 stand rejected under 35 U.S.C. 103 as being unpatentable over Aricha et al. (WO 2019/198077 A1, published in 2019), as evidenced by Haney et al. (J Control Release. 2015 Jun 10;207:18-30).
Aricha et al. teach highly-characterized isolated exosomes, methods to produce such exosomes, and methods for the use of such exosomes in treating diseases such as neurodegenerative diseases (Abstract).
Regarding claim 1, Aricha et al. teach an isolated exosome population derived from mesenchymal stem cells secreting-neurotrophic factors (MSC-NTF cells), said isolated exosomes comprising an increased quantity of at least one cargo protein comprising a neurotrophic factor (NTF), compared with the quantity of said at least one cargo protein in an isolated exosome population derived from control MSCs. production, purification, and characterization of MSC-NTF Exosomes (parag 0011). Exosomes are nano-sized, secreted cell vesicles (extracellular vesicles, parag 00127). Aricha et al. teach Example 1, preparation of differentiated human BM-MSC (MSC-NTF) and exosome production and purification (parag 00358-00379). It is noted that Aricha et al. describe the method of produce and purify the exosomes from a differentiated state of MSC (MSC-NTF) in this example, however, Aricha et al. also emphasize that it is expected that MSC derived exosomes could be similarly isolated using the methods described herein and MSC or genetically modified MSC (parag 00358). Aricha et al. teach culture medium collected, and cell debris and large vesicles were removed by filtration through a 0.22-μm filter. The exosome containing filtrate was collected under sterile conditions and subjected to tangential flow filtration (TFF, see parag 00366). Aricha et al. further teach the isolation of the exosomes by TFF (see parag 00375-00376): the exosome-containing sample (filtrate) was continuously pumped through the fiber system and recirculated. Small molecules, including free proteins not included within or associated with the membrane vesicles, were driven through the membrane pores, subsequently eluted as permeate, and eventually discarded. Molecules too large to pass through the pores, such as exosomes (or larger microvesicles), were kept in circulation as retentate (parag 00376). This teaching of the purification of exosomes reads on “a process (for the manufacturing of extracellular vesicles (EVs) associated with proteins, derived from MSCs, said process) comprises the steps of: purifying EVs from a cell medium comprising MSCs, wherein said purifying occurs via at least one filtration step of said medium; followed by a concentration step of filtrate of said at least one filtration step, wherein said EVs are concentrated by means of tangential flow filtration (TFF) in a TFF device” in instant claim.
Aricha et al. do not specifically teach “during said TFF step the EVs are associated with one or more exogenous proteins inside of said TFF device, or in a vessel fluidly connected to said TFF device to which said EVs are transferred from said TFF” and “said proteins are supplemented to said TFF device or said vessel during the concentrating of said EVs”.
However, regarding the limitation “during said TFF step the EVs are associated with one or more exogenous proteins inside of said TFF device, or in a vessel fluidly connected to said TFF device to which said EVs are transferred from said TFF”, Aricha et al. teach in Example 2, modification of MSC or MSC-NTF exosome cargo (see parag 00405): to prepare modified MSC or MSC-NTF derived exosomes for use as nanocarriers for siRNA, miRNA, or proteins, thereby producing a targeted population of MSC-NT derived exosomes. By encapsulating molecules within their membranes, exosomes can protect proteins or RNAs from degradation. Moreover, while serving as nano-carriers for a specific target, MSC or MSC-NTF derived exosomes still maintain their original therapeutic potential (parag 00405). Herein the MSC derived exosome encapsulating proteins within the membranes reads on “ extracellular vesicles (EVs) associated with protein” as recited in instant claims. Proteins or miRNA can be loaded into exosomes either directly or through genetic engineering of the donor MSC cells (see parag 00415), or protein loading directly into exosomes including incubation at room temperature, permeabilization with saponin, freeze-thaw cycles, sonication, or extrusion (parag00416). Herein the protein loading directly into exosomes including incubation at room temperature is evidenced by Haney et al..
Haney et al. teach the development of a new exosomal-based formulation of catalase
(exoCAT) that was obtained by drug loading into naïve exosomes ex vivo (p19, left column). Haney et al. teach the approaches for catalase incorporation into exosome including the incubation at RT with or without saponin (Method I) (p19, right column) catalase solution in PBS (0.5 mg/mL) was added to
250 μL of exosomes to the final concentration of 0.1mg/mL total protein, and incubated at RT for 18 h. In case of a saponin treatment, a mixture of catalase and exosomes was supplemented with 0.2% saponin and placed on shaker for 20 min at room temperature (RT) (p19, left column).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to utilize Aricha et al.’s method of preparing MSCs derived extracellular vesicles associated with protein(s), which is comprising a step of tangential flow filtration (TFF), and add one or more exogenous proteins inside of TFF device or in a vessel connected to the TFF device to form extracellular vesicles associated with protein(s), based on the principle that protein loading directly into exosomes by incubation at room temperature as taught by Aricha et al. and Haney et al.. The only difference between instant claim and Aricha et al.’s method of preparing MSCs derived extracellular vesicles associated with protein(s) is instant claim loads protein(s) directly to extracellular vesicles in the TFF device or in a vessel connect to the TFF device. Given that Aricha et al. and Haney et al. teach incubation exosomes and protein in room temperature is capable of forming extracellular vesicle associated with protein, and TFF procedure provides the purified exosomes and temperature (e.g., room temperature) as needed, one of ordinary skill in the art would have substituted Aricha et al.’s loading protein directly to exosomes by incubation, and incubate or mix the protein and exosomes in TFF device or in a vessel connected to the TFF device depends on their research preference (i.e., quickly obtain protein associated EVs without extra exosome handling step). This simple substitution of one known element (incubating or mixing the exogenous protein and exosomes in TFF device) for another known element (Aricha et al.’s method of loading protein directly to exosomes, i.e., by incubation) is likely to be obvious when predictable results are achieved. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (see MPEP § 2143, B.).
Regarding claim 8, Aricha et al. teach either 100, 300, 500-kDa MWCO membranes were used tangential flow filtration (TFF) (paraf 00376).
Claims 1 and 3-8 stand rejected under 35 U.S.C. 103 as being unpatentable over Aricha et al. (WO 2019/198077 A1, published in 2019), as evidenced by Haney et al. (J Control Release. 2015 Jun 10;207:18-30), further in view of Wang et al. (Molecules. 2017 Dec 19;22(12):2256) and Samanta et al. ( Acta Pharmacologica Sinica (2018) 39: 501–513), as evidenced by Lee et al. (Front Neurosci. 2019 Oct 18;13:1067).
The teaching of Aricha et al. is set forth above.
Regarding claims 3, 4, 6 and 7, Aricha et al. do not teach supplement calcium to the TFF device, the supplemented calcium would mix with proteins in the TFF device, and the proteins are annexins, thioredoxins or lactadherin, and more specifically, these proteins are at least one of Annexin V, Trx or Mfge8. However, this was disclosed by Wang et al. and Samanta et al..
Wang et al. teach the deletion of domain IV will affect the functions, especially the central PS-binding function, of anxA5 through the in vitro activity assay of purified proteins (p2).
Samanta et al. review studies that have been reported regarding how exosomes serve as a new
molecular target for various diseases, and discuss the use of exosomes in therapy (p501, right column).
Regarding claims 3, 4, 6 and 7, Wang et al. teach annexin A5 (anxA5 or Annexin V) has high affinity in binding phosphatidylserine (PS) exposed on apoptotic cells. Additionally, anxA5 possesses various functions and applications, such as cell membrane repair, anticoagulation, in vivo therapy, and mediating targeted therapy, the above functions and applications of anxA5 are closely related to its PS-binding ability, either directly or indirectly (see p1-2, Introduction). Wang et al. also teach the interaction between anxA5 and PS is calcium-dependent (p4, parag 2). Samanta et al. teach extracellular vesicles (EVs) such as microvesicles are characterized by the presence of phosphatidylserine (PS) in the outer leaflet of the membrane and a size ranging between 100 and 1000 nm in diameter (p503, left column). This teaching indicates Annexin V can be mixed with calcium and increasing the binding to extracellular vesicles which contain phosphatidylserine.
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Aricha et al.’s method of preparing MSCs derived extracellular vesicles associated with protein(s), and mix the protein anxA5 and calcium together with the extracellular vesicles, for the reason that calcium can increased the anxA5 binding to the phosphatidylserine in extracellular vesicles, as taught by Wang et al. and Samanta et al.. The only difference between instant claim and Aricha et al.’s method of preparing MSCs derived extracellular vesicles associated with protein(s) is instant claim mix protein (i.e., Annexin V) and calcium together with extracellular vesicles. Given that Wang et al. teach anxA5 binding to phosphatidylserine is calcium-dependent (p4, parag 2), and Samanta et al. teach extracellular vesicles contain phosphatidylserine, one of ordinary skill in the art would have substitute Aricha et al.’s method of preparing MSCs derived extracellular vesicles associated with protein(s) (i.e., by incubation), and mix or incubate the anxA5 and calcium together with the extracellular vesicles depends on their research interest or preference. This simple substitution of one known element (mix or incubate the anxA5 and calcium together with the extracellular vesicles to form extracellular vesicles associated anxA5) for another known element (Aricha et al.’s method of preparing MSCs derived extracellular vesicles associated with protein) is likely to be obvious when predictable results are achieved. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (see MPEP § 2143, B.).
Regarding claim 5, following the discussion above, Aricha et al. teach for high-purity exosomes, TFF was followed by size exclusion chromatography (SEC) (parag 00377). The procedure of size exclusion chromatography is evidenced by Lee et al.. Lee et al. teach that purification of EVs by size-exclusion liquid chromatography protocol (LC) comprises that the filtered (culture medium) CM was concentrated using the Amicon 100k-Da molecular weight cut-off (MWCO) filters at 3,500 g for 15 min. The concentrate retentate was then loaded onto a Sephacryl S-400 16/60 LC column and run with PBS at 0.5 ml/min. Fixed-volume 2 ml fractions of the eluted solutions were then collected with a fraction collector (p3, right column). Lee et al.’s teaching of the size-exclusion chromatography procedure indicates that EVs associated with proteins formed in TFF device can be washed (when filling in the filter), concentrated, and collected during the size exclusion chromatography procedure, indicates the EVs associated with proteins formed in TFF device are washed, re-concentrated, and collected outside said TFF device (e.g., in size exclusion chromatography system), as recited in instant claim.
Claims 1, 8 and 9 stand rejected under 35 U.S.C. 103 as being unpatentable over Aricha et al. (WO 2019/198077 A1, published in 2019), as evidenced by Haney et al. (J Control Release. 2015 Jun 10;207:18-30), further in view of Marshak et al. (US-5908782-A. patented in 1999).
The teaching of Aricha et al. is set forth above.
Regarding claim 9, Aricha et al. teach using a serum free DMEM medium for culturing MSC (see e.g., parag 0020), do not teach MSCs are cultured and expanded in a cell medium comprising purified human serum albumin, wherein the albumin concentration in said medium is between 1 g/Land 5 g/L. However, it was disclosed by Marshak et al..
Marshak et al. teach a composition and method for maintaining the viability of human mesencnymal precursor cells in a serum-free environment which composition includes (1) a minimum essential medium; (2) serum albumin; (3) an iron Source; (4) insulin or an insulin-like growth factor; and (5) at least one amino acid selected from the group consisting of glutamine, arginine and cysteine, and is free of Serum (Abstract).
Regarding claim 9, Marshak et al. teach Example 1, culture expansion of human MSCs in a serum free medium (Col 7), Iscove’s modified Dulbecco's Medium (IMDM) was supplemented with human serum albumin 5 mg/ml (Col 7, L64-67). 5 mg/ml human serum albumin is 5 g/L human serum albumin. This teaching reads on MSCs are cultured and expanded in a cell medium comprising purified human serum albumin, wherein the albumin concentration in said medium is between 1 g/l and 5 g/1, as recited in instant claim.
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Aricha et al.’s culturing MSC with serum free medium, and use serum free medium supplemented by 5 g/L human serum albumin, as taught by Marshak et al.. The only difference between instant claim and Aricha et al.’s culturing MSC with serum free medium is instant claim use a serum free cell medium comprising purified human serum albumin, wherein the albumin concentration in said medium is between 1 g/L and 5 g/L. Given that Marshak et al. teach using serum free medium supplemented with 5 g/L human serum albumin (Col 7, L64-67), and serum albumin is helpful for maintaining the viability of human mesencnymal precursor cells (see Abstract), one of ordinary skill in the art would have substitute Aricha et al.’s method of culturing MSC with serum free medium, and use Marshak et al.’s serum free medium supplemented with 5 g/L human serum albumin depends on their research preference. This simple substitution of one known element (Marshak et al.’s serum free medium supplemented with 5 g/L human serum albumin) for another known element (Aricha et al.’s culturing MSC with serum free medium) is likely to be obvious when predictable results are achieved. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (see MPEP § 2143, B.).
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to QINHUA GU whose telephone number is (703)756-1176. The examiner can normally be reached M-F: 9:00 - 5:00.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Christopher Babic can be reached at (571)272-8507. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/Q.G./Examiner, Art Unit 1633
/CHRISTOPHER M BABIC/Supervisory Patent Examiner, Art Unit 1633
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