DETAILED ACTION
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Applicant’s Request for Continued Examination, Amendment and Arguments/Remarks received on 22 December 2025 have been entered. Claims 27-32 and 35-54 were previously pending in the application. No claims have been newly cancelled nor newly added by Applicant. Claims 27-32 and 35-54 are currently pending in the application. Claims 27, 43, 48, 49, and 54 are independent claims.
The election and rejoinder of Groups I, II, and III drawn to a method for obtaining a composition for tissue regeneration, a composition obtained by a method for obtaining a composition for tissue regeneration, a pharmaceutical composition comprising the composition obtained by a method for obtaining a composition for tissue regeneration, and a method for treating a disease, remains in effect in the instant application.
The following election of species remains in effect in the instant application:
Tissue-specific cells: tenocytes;
Disease: tendinosis.
Claim 54 remains withdrawn from consideration as being directed to a nonelected invention, there being no allowable generic or linking claim.
Claims 27-32 and 35-53 are currently pending and under examination in the instant application. An action on the merits follows.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA . The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Priority
The present application is a 35 U.S.C. 371 national stage filing of International Application No. PCT/EP2020/059365, filed 02 April 2020, which claims priority to EPO 19382247.5, filed 03 April 2019. Filing of a certified copy of the EPO 19382247.5, filed 03 April 2019, is acknowledged.
Thus, the earliest possible priority for the instant application is 03 April 2019.
Claim Objections
The objection to amended claim 32 for reciting “macrophges”, is maintained in view of the amendment to claim 32 wherein claim 32 still recites “macrophges” in line 2.
Claim Rejections - 35 USC § 112(b)
The rejection of amended and previously presented claims 35-36, 39-40, and 43-53 under 35 U.S.C. 112(b) as failing to particularly point out and distinctly claim the subject matter which the inventor(s) regards as the invention for multiple issues of indefiniteness:
Claims 35-36 and 39-40 reciting wherein the monocytes or tissue-specific cells are autologous or allogenic;
Claim 43 reciting “a composition obtained by a method for obtaining a composition for tissue regeneration”, Claims 44-47 reciting recite “the composition according to claim 43”, Claim 48 reciting “the composition of claim 43”, and Claims 51-53 reciting “the composition”;
Claims 46 and 47 reciting “of the total amount of components in the sample”; and
Claim 51 reciting the limitation "the composition administered to the subject is different from a donor subject of the monocytes or the tissue-specific cells”;
is maintained in modified form in view of Applicant’s amendments to the claims.
Regarding a), amended claims 35-36 now recite, “autologous, derived from the same subject intended for treatment” and “allogenic, derived from a donor subject different from the subject intended for treatment”, respectively. Previously presented claims 39-40 have not been amended. Claims 35-36 depend on claim 27 and claims 39-40 depend on claim 38, which then depends on claim 27. Claim 38 has not been amended.
Recitation of “the same subject intended for treatment” and “the subject intended for treatment” in claims 35 and 36, respectively, lack antecedent basis. Claim 27 has been amended to recite, “providing monocytes from a sample obtained from a donor”, but does not recite any subject(s). Additionally, it is unclear whether the “a donor subject” in claim 36 is meant to be the same donor as recited in claim 27. Claim 27 also recites “for tissue regeneration”, but does not indicate any treatment(s).
The claim amendments also do not provide sufficient clarity to the claim in that the indefiniteness of “autologous” and “allogenic” is not rooted in a definition of the terms per se, but in the relative nature of the terms and a lack of clarity indicating relative to what or whom the cells are autologous or allogenic. None of claims 35, 36, or 37 recite any use for the cells beyond being used to produce components of the desired composition. The composition itself cannot be allogenic nor autologous to the source of the monocytes used for producing the composition. The claims additionally do not recite any use for the composition comprising the secreted factors beyond an intended use of “for tissue regeneration”, which is not necessarily required to be a treatment use, but could be a tissue regeneration in vitro, for example.
As such, the metes and bounds of the claims still cannot be determined.
Regarding b), Applicant has amended claim 43 to recite, “A composition comprising a collected acellular supernatant by a method for obtaining a composition for tissue regeneration, the method consisting of”, which has clarified that the composition of claim 43 is the “collected acellular supernatant”. However, it is unclear how the method recited in amended claim 43 is producing “a collected acellular supernatant” in that there are no method steps which require the removal of cells from the supernatant nor that recite obtaining a collected acellular supernatant.
The amendment to claim 43 has also introduced new issues of indefiniteness.
“A composition comprising a collected acellular supernatant by a method for obtaining a composition for tissue regeneration” is indefinite because it is grammatically unclear in what way a composition can comprise a collected acellular supernatant “by” a method for obtaining.
Additionally, “the collected supernatant of the co-culture” recited in lines 10-11 still lacks antecedent basis and it is unclear whether “the collected supernatant of the co-culture” is meant to be the “collected acellular supernatant” newly recited in claim 43 line 1.
Further, it is unclear whether the step of “collecting the supernatant” is meant to require a decellularization step to remove any cell so that “the collected supernatant of the co-culture” is an acellular supernatant.
As such, the metes and bounds of the claims still cannot be determined.
Regarding c), amended claim 46 now recites, “wherein platelets, red blood cells or leukocytes are present in an amount less than 0.1% by cell count relative to a total number of components in the collected supernatant.” Note that by claiming that “platelets, red blood cells or leukocytes are present in an amount less than 0.1% by cell count relative to a total number of components in the collected supernatant”, Applicant is indicating that “the collected supernatant” of claim 43 is not acellular, and as such there is not an equivalency between “the collected supernatant of the co-culture” and “a collected acellular supernatant” recited in the preamble of claim 43. The amendment to claim 46 has clarified in what way the platelets, red blood cells, and leukocytes are to be quantitated. However, the claim is still indefinite because it is unclear what constitutes a “component” in the collected supernatant.
Previously presented claim 47 has not been amended. Therefore, Applicant has not overcome any issues of indefiniteness associated with claim 47. As such, the metes and bounds of the claims still cannot be determined.
Regarding d), Previously presented claim 51 has not been amended. As such, the metes and bounds of the claim still cannot be determined.
Applicant has requested reconsideration and withdrawal of the rejection in view of the amendments to the claims, but has provided no arguments traversing the rejection. Therefore, Applicant’s amendments and arguments do not overcome a finding of indefiniteness 35 U.S.C. 112(b) for amended and previously presented claims 35-36, 39-40, and 43-53.
Amended claim 32 is newly rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Amended claim 32 now recites, “wherein the differentiating the purified monocytes into M2-macrophges comprising culturing the monocytes in serum free medium in presence of macrophage colony-stimulating factor (M-CSF) carried out for 3-5 days”, which is indefinite because the verb “is” has been removed from the sentence. As such, the metes and bounds of the claim cannot be determined.
Claim Rejections - 35 USC § 103
The rejection of amended and previously presented claims 27-32 and 35-48 under 35 U.S.C. 103 as being unpatentable over Stolk et al. [2017, Scientific Reports, 7, 9801, 1-14]; in view of Schoenenberger et al. [2018, Acta Biomaterialia, 71, 306-317]; da Silva et al. [2016, Atherosclerosis, 248, 170-178]; Sardana et al. [2007, Clinical Chemistry, 53(3), 429-437]; Lehmann et al. [2017, Journal of Visualized Experiments, 129, e56440, 1-10]; de Santa et al. [2019, Antioxidants & Redox Signaling, 30(12), 1553-1598, ePub 9 October 2018]; and Committee for Proprietary Medicinal Products & Committee for Veterinary Medicinal Products [2002, Note for the Guidance on Quality of Water for Pharmaceutical Use, The European Agency for the Evaluation of Medicinal Products, London, 1-6], is maintained. Applicant's amendments to the claims and arguments have been fully considered but have not been found persuasive in overcoming the rejection for reasons of record as discussed in detail below.
Applicant amended claim 27 to recite, “providing M2-macrophages by purifying monocytes from a sample obtained from a donor” in lines 3-4 and “collecting acellular supernatant of the co-culture, thereby obtaining a composition comprising soluble factors secreted by the co-cultured M2 macrophages and tissue specific cells” in lines 9-10.
Stolk was cited for teaching a method of obtaining a composition comprising purifying monocytes from a sample, differentiating the purified monocytes into macrophages by culturing the monocytes in the presence of macrophage colony-stimulating factor (M-CSF), co-culturing the macrophages with tenocytes directly or in a Transwell™ system, collecting the supernatant of the co-culture, and freezing/cryopreserving the supernatant of the co-culture for later use [page 10 ¶ 4- page 11 ¶ 1, page 11 ¶ 6-7 ]. The teaching by Stolk of collecting the supernatant of the co-culture and freezing/cryopreserving the supernatant of the co-culture for later use is a teaching for obtaining a composition comprising soluble factors secreted by the co-cultured M2 macrophages and tissue specific cells. Additionally, Stolk teaches that the monocytes were purified from buffy coats from healthy volunteers/donors [page 10 ¶ 4].
Applicant’s amendments to claims 35-36, 43, and 46 are directed to clarifying issues of indefiniteness but have not altered the scope of the claims in a way to overcome the obviousness rejection under 35 U.S.C. 103 of record.
Applicant argues that:
none of the cited references teaches or suggests producing a tissue-specific acellular product via co-culture of M2-macrophages with differentiated tissue-specific cells in serum-free medium where the secreted factor composition is modulated by cross-talk and subsequently isolated as the final therapeutic composition, in that Stolk and Schoenenberger merely identify differential cytokine secretion patterns in experimental models aimed at mechanistic insights and not clinical compositions without disclosing, suggesting, or motivating one skilled in the art to collect and preserve the supernatant itself as a stand alone product for regenerative therapy;
the claimed process yields a reproducible, cell-free composition with stable growth factor profiles after cryopreservation or lyophilization, retaining full regenerative bioactivity, such that they maintained activity after >12 months of storage is a feature not predicted from the transient secretome activity observed in Stolk and Veronesi and represents unexpected results not rendered obvious by any combination of the cited references, see page 13, lines 12-17 of the specification as filed;
Inventor’s Declaration under 37 CFR 1.132 clarifies that the M2-macrophages of the present invention are provided by purifying monocytes from a sample obtained from a donor, and collecting acellular supernatant of the co-culture, thereby obtaining a composition comprising soluble factors secreted by the co-cultured M2-macrophages and tissue specific cells such that there are no additional impurities or degradation products expected in the secretome; and
the invention achieves immune tolerance through removal of cell material (<0.001% cellular residue, which can be considered to be 100% cell-free product) and provides an off the shelf therapeutic composition derived from controlled in vitro co-cultures resulting in a composition with nonobvious structural and functional characteristics, i.e., long-term stability of the cryopreserved secretome, retention of biological activity post-thaw, tissue-specific regenerative effects in vivo, and reproducibility across batches to solve long-standing problems in regenerative medicine, and that none of Stolk, Schoenenberger, Veronesi, or Lipman teaches or suggests producing an acellular, immune safe, ready to use allogenic formulation stored and reconstituted for direct tissue targeted use in that the prior art co-cultures primarily involve transient, contact dependent systems providing non specific macrophage conditioned media in contrast to the defined, programmable secretome of the instant invention.
However, this is not agreed.
In response to Applicant’s arguments against the references individually, it is noted that the test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981). One cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). Further, the Examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In addition, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971).
Specifically, regarding 1), note that claims 27-32 and 35-48 as written do not require that the composition be a final therapeutic composition nor a stand along product for regenerative therapy. Additionally, Stolk was cited for teaching a method of obtaining a composition comprising purifying monocytes from a sample, differentiating the purified monocytes into macrophages by culturing the monocytes in the presence of macrophage colony-stimulating factor (M-CSF), co-culturing the macrophages with tenocytes in at a ratio of 5 macrophages to 1 tenocyte for 3 days directly or in a Transwell™ system, collecting the supernatant of the co-culture, and freezing/cryopreserving the supernatant of the co-culture for later use [page 10 ¶ 4- page 11 ¶ 1, page 11 ¶ 6-7 ]. Additionally, Stolk teaches that during co-culture of the macrophages with tenocytes, the macrophages are polarized into a mixed M1/M2 phenotype [page 9 ¶ 2-3]. Additionally, Da Silva was cited for teaching a method of purifying monocytes from a sample and differentiating the purified monocytes into M2 macrophages by culturing the monocytes in serum free medium in the presence of macrophage colony-stimulating factor (M-CSF) [column 3 ¶ 5- column 4 ¶ 2]. Da Silva was further cited for teaching that the use of serum-free medium avoids the potentially confounding and batch-dependent effects of serum components, such as polarizing factors and endogenous serum lipoproteins [column 7 ¶ 5]. Further, Sardana was cited for teaching hat the use of protein- and peptide-free chemically defined serum-free media simplifies analysis of conditioned media compared to the use of serum-containing media which would contaminate the conditioned media [column 10 ¶ 4-column 11 ¶ 1].
Therefore, Stolk, Da Silva, and Sardana teach producing a tissue-specific acellular product via a co-culture of M2-macrophages with differentiated tissue-specific cells in serum-free medium where the secreted factor composition is modulated by cross-talk and subsequently isolated.
Regarding Applicant’s argument 2), note that while dependent claim 37 requires that the method further comprise cryopreservation or lyophilization of the collected supernatant, the claims do not require that the cell-free composition retain stable growth factor profiles or full regenerative bioactivity after cryopreservation or lyophilization nor maintain activity after >12 months of storage.
Additionally, the combined teachings of Stolk, Da Silva, and Sardana motivate the ordinarily skilled artisan to arrive at the instantly claimed invention, such that the combination would be expected to have the same structure, and therefore the same functions as the claimed invention.
Further, Applicant’s assertion that maintaining activity after >12 months of storage is an unexpected result of the instant Application amounts to arguments of counsel. The arguments of counsel cannot take the place of evidence in the record. In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965); In re Geisler, 116 F.3d 1465, 43 USPQ2d 1362 (Fed. Cir. 1997) ("An assertion of what seems to follow from common experience is just attorney argument and not the kind of factual evidence that is required to rebut a prima facie case of obviousness."). See MPEP § 716.01(c) for examples of attorney statements which are not evidence and which must be supported by an appropriate affidavit or declaration. Examples of attorney statements which are not evidence and which must be supported by an appropriate affidavit or declaration include statements regarding unexpected results, commercial success, solution of a long-felt need, inoperability of the prior art, invention before the date of the reference, and allegations that the author(s) of the prior art derived the disclosed subject matter from the applicant. MPEP 716.01(c). Attorney argument is not evidence unless it is an admission, in which case, an examiner may use the admission in making a rejection. See MPEP § 2129 and § 2144.03 for a discussion of admissions as prior art.
Note that Applicant cites a passage in the specification (page 13, lines 12-17) as evidence of the unexpected results. However, the referenced paragraph in the specification merely states:
“The cryopreserved or lyophilized composition may be stored between -80 and -86 oC until its use. Storage from -40 oC has been experimentally proved to ensure stability and biological activity of the GF and/or cytokines present in the composition and their activity in vivo restoring an injured tissue as well. The composition cryopreserved may be maintained at least 1 year between -20 and -196 oC. Preferably no more than 3 years. In a preferred embodiment, the supernatant is dosed in single-doses.”
The paragraph cited says that storage from -40 oC has been experimentally proven to ensure stability and biological activity of the GF and/or cytokines present in the composition and their activity in vivo restoring an injured tissue as well, but does not reference any data to support the assertion. Additionally, the cited paragraph does not indicate that cryopreservation beyond one year has been tested, merely that it could be done.
Looking to the disclosure for any additional support, Example 1 teaches preparation of the composition, wherein the collected supernatants were centrifuged and stored at -80oC for further analysis, without any reference to a storage duration [page 31 lines 6-9]. Example 2 discloses the analysis of the compositions comprising cytokines and growth factors, but does not indicate how long the samples had been frozen nor a comparison of fresh samples, freshly frozen samples, and long-term frozen samples to indicate stability and preservation of biological activity of the GF and/or cytokines present in the composition or their activity for restoring an injured tissue in vivo [page 21 lines 20-33, Figures 1-3]. Example 2 further states, “In fact, concentrating formulations either by centrifugation, lyophilization, drying, vacuum, purification columns or by any other method will retain the described modification patterns or tissue-specific composition signatures”, which addresses an issue of concentration but not storage and does not provide any data to support the assertion. Example 3 presents clinical assays using compositions of the invention which had been frozen at -35oC, cryopreserved at -80oC, and thawed prior to their clinical use, but likewise does not indicate how long the samples were stored prior to use [page 23 lines 1-3]. Examples 4-11 present the clinical experiments and data without indicating any storage times for the compositions.
The in vivo clinical data presented in the Drawings in Figures 4-6 and 8-12 present measurements of different symptoms each presented as “% Disease” over time broken down by two grades of disease severity (II or III), wherein the “control” is merely the starting condition prior to treatment. Figure 7 shows the result of an echography done on the day previous to the treatment (-1), the day 4 after the treatment (+4) and the day 8 after the treatment (+8) of a single patient. No untreated nor standard of care treatment data over time is provided. Therefore, there is no actual indication of efficacy of the samples applied because there is no data presenting either the natural, untreated healing process over time nor a standard of care healing process over time to which the treated data can be compared.
Therefore, the disclosure does not provide data in support of Applicant’s assertion that the claimed process yields a reproducible, cell-free composition with stable growth factor profiles after cryopreservation or lyophilization, retaining full regenerative bioactivity, such that the maintained activity after >12 months of storage is a feature not predicted from the transient secretome activity observed in Stolk and Veronesi and represents unexpected results not rendered obvious by any combination of the cited references.
Applicant has provided a Declaration under 37 CFR 1.132 by inventor Jara Sanz, which clarifies that the M2-macrophages of the present invention are provided by purifying monocytes from a sample obtained from a donor, and collecting acellular supernatant of the co-culture, thereby obtaining a composition comprising soluble factors secreted by the co-cultured M2-macrophages and tissue specific cells [page 3 bullet 4], but does not provide evidence of the composition following long-term storage. The Declaration does not indicate storage times and does not provide comparison control data for the clinical experiments.
Therefore, Applicant’s arguments amount to arguments of counsel in that they are not supported by any evidence of record.
It is also noted that any evidence of unexpected results must be commensurate in scope with the claimed invention, and that a greater, or greater than additive, effect is not necessarily sufficient to overcome a prima facie case of obviousness because such an effect can either be expected or unexpected MPEP 716.02 (a) and (d). Whether the unexpected results are the result of unexpectedly improved results or a property not taught by the prior art, the "objective evidence of nonobviousness must be commensurate in scope with the claims which the evidence is offered to support." In other words, the showing of unexpected results must be reviewed to see if the results occur over the entire claimed range. In re Clemens, 622 F.2d 1029, 1036, 206 USPQ 289, 296 (CCPA 1980). Specifically, to the extent that Applicant may rely on the clinical data presented in the disclosure to support the efficacy of the composition in treating various diseases, note again that the data presented does not provide evidence of any improved efficacy in that no control samples are provided for comparison, nor do the examples specifically provide evidence of efficacy following long-term storage. Additionally, the Declaration provided does not provide any evidence of long-term storage stability nor efficacy. It is also noted that the scope of the claims as written encompass the production and use of a composition without any storage, purification, nor concentration.
The scopes of the composition claims encompass culturing M2-macrophages with any tissue specific cells to generate collected supernatant. The scope of the treatment claims additionally encompasses treatment of any disease by administering any composition according to claim 43 to any subject in need. Therefore, although Applicant has provided examples of 8 tissue-specific cell types for production of compositions of the invention and has administered 4 of those compositions to patients suffering from 8 different diseases/conditions, such compositions and administrations are not representative of all tissue-specific cell types nor all diseases. Therefore, Applicant’s data is not commensurate in scope with the claimed invention.
Regarding Applicant’s argument 3), Inventor’s Declaration under 37 CFR 1.132 clarifies that the M2-macrophages of the present invention are provided by purifying monocytes from a sample obtained from a donor, and collecting acellular supernatant of the co-culture, thereby obtaining a composition comprising soluble factors secreted by the co-cultured M2-macrophages and tissue specific cells such that there are no additional impurities or degradation products expected in the secretome. The Declaration as filed adds details to the processing of the composition after collection to support the claim that the composition is acellular, in that the examples in the specification only teach that the collected supernatants were subjected to low speed centrifugation and subsequently frozen at -80 oC [page 21 lines 6-9] or that the compositions were frozen at -35oC, cryopreserved at -80oC, and thawed before use [page 23 lines 1-3]. The specification as filed provides support for removing cell debris by filtering with a 0.22 um membrane to purify the sample [page 12 lines 20-23], but does not specifically teach that the compositions used in the examples were filtered, nor that all cells were removed from the compositions.
Claim 27 does not require that the composition be acellular. Additionally, claim 43 states in the preamble that the composition is acellular, but does not provide any limitation within the claim which would require the composition to be acellular or which would necessarily result in a composition which is acellular. Claims 46-47 limit the residual number of platelets, red blood cells, or leukocytes present in the collected supernatant to an amount less than 0.1% by cell count (claim 46) or in the collected sample to less than 0.001% of the amount of components in the collected sample. By further limiting the amount of cells within the collected supernatant or the sample collected, particularly to a level as high as 0.1%, the claims themselves teach that the composition according to claim 43 encompasses compositions comprising an amount of platelets, red blood cells, and leukocytes which is greater than 0.1% in addition to any other cells which may be present.
Therefore, given the teachings of Stolk, Schoenenberger, Da Silva, and Sardana, which teach all the limitations of independent claims 27 and 43, the teachings of Stolk to freeze the collected supernatants at -80 oC, the statement in the Declaration that freezing the supernatant at -80 oC eliminates any remaining cellular material [page 4 bullet 6], the lack of clarity in the claims regarding the collection of the supernatant in generating an acellular composition, and the lack of requirements in the claims for steps which would render the collected supernatant acellular, the cited references are still considered to teach the compositions and methods of the instant invention as claimed.
Regarding Applicant’s argument 4), that the invention achieves immune tolerance through removal of cell material, note that the claims as written do not require the removal of cell material. Additionally, Applicant has provided no evidence of any immune tolerance. As discussed above, Applicant additionally has provided no evidence of long-term stability of the cryopreserved secretome, no evidence of retention of biological activity post-thaw for either short term or long-term storage, no evidence of tissue-specific regenerative effect in vivo, and no evidence of reproducibility across batches. Additionally, Applicant has not provided any evidence that the issues attempting to be solved are indeed long-standing problems in regenerative medicine.
Applicant asserts that the cited references do not teach an allogenic formulation. Note that claims 35-36 and 39-40 recite wherein the monocytes or tissue-specific cells are autologous or allogenic, but they do not provide any reference cells or subject for which they are autologous or relative to. As discussed above for the indefiniteness rejection under 35 U.S.C. 112(b), references in claims 35-36 to “the subject” and “for treatment” are indefinite in that claim 27 does not recite any subject or any treatment. Therefore, these claims have been interpreted such that the limitations “autologous” and “allogenic” do not add any further structural limitations to the claimed invention. Therefore, by teaching the co-culture of M2-macrophages and tenocytes according to claim 27, Stolk, Schoenenberger, da Silva, and Sardana teach the limitations of claims 35-36 and 39-40. Additionally, Applicant is arguing specifically for allogenic formulation although the claims as written encompass autologous, allogenic, xenogenic, or any other relationship among cellular source and intended recipient.
Therefore, Applicant’s amendments and arguments do not overcome a finding of obviousness over Stolk, Schoenenberger, da Silva, Sardana, Lehmann, de Santa, and Committee for Proprietary Medicinal Products & Committee for Veterinary Medicinal Products, and the rejection under 35 U.S.C. 103 is maintained.
The rejection of amended and previously presented claims 49-53 under 35 U.S.C. 103 as being unpatentable over Stolk et al. [2017, Scientific Reports, 7, 9801, 1-14]; in view of Schoenenberger et al. [2018, Acta Biomaterialia, 71, 306-317]; da Silva et al. [2016, Atherosclerosis, 248, 170-178]; Sardana et al. [2007, Clinical Chemistry, 53(3), 429-437]; Lehmann et al. [2017, Journal of Visualized Experiments, 129, e56440, 1-10]; de Santa et al. [2019, Antioxidants & Redox Signaling, 30(12), 1553-1598, ePub 9 October 2018]; and Committee for Proprietary Medicinal Products & Committee for Veterinary Medicinal Products [2002, Note for the Guidance on Quality of Water for Pharmaceutical Use, The European Agency for the Evaluation of Medicinal Products, London, 1-6]; as applied to claims 27-32 and 35-48 above; and further in view of Veronesi et al. [2017, Journal of Cellular Physiology, 233, 4423-4442]; and Lipman et al.[ 2018, Drug Design, Development & Therapy, 12, 591-603]; is maintained. Applicant's amendments to the claims and arguments have been fully considered but have not been found persuasive in overcoming the rejection for reasons of record as discussed in detail below.
Applicant has not amended claims 49-53. As discussed above, Applicant’s amendments and arguments have not overcome a finding of obviousness over Stolk, Schoenenberger, da Silva, Sardana, Lehman, de Santa, and Committee for Proprietary Medicinal Products & Committee for Veterinary Medicinal Products, as applied to claims 27-32 and 35-48 above.
Applicant argues that:
none of the cited references teaches or suggests producing a tissue-specific acellular product via co-culture of M2-macrophages with differentiated tissue-specific cells in serum-free medium where the secreted factor composition is modulated by cross-talk and subsequently isolated as the final therapeutic composition, in that Stolk and Schoenenberger merely identify differential cytokine secretion patterns in experimental models aimed at mechanistic insights and not clinical compositions without disclosing, suggesting, or motivating one skilled in the art to collect and preserve the supernatant itself as a stand- alone product for regenerative therapy;
the claimed process yields a reproducible, cell-free composition with stable growth factor profiles after cryopreservation or lyophilization, retaining full regenerative bioactivity, such that they maintained activity after >12 months of storage is a feature not predicted from the transient secretome activity observed in Stolk and Veronesi and represents unexpected results not rendered obvious by any combination of the cited references, see page 13, lines 12-17 of the specification as filed;
Inventor’s Declaration under 37 CFR 1.132 clarifies that the M2-macrophages of the present invention are provided by purifying monocytes from a sample obtained from a donor, and collecting acellular supernatant of the co-culture, thereby obtaining a composition comprising soluble factors secreted by the co-cultured M2-macrophages and tissue specific cells such that there are no additional impurities or degradation products expected in the secretome; and
the invention achieves immune tolerance through removal of cell material (<0.001% cellular residue, which can be considered to be 100% cell-free product) and provides an off the shelf therapeutic composition derived from controlled in vitro co-cultures resulting in a composition with nonobvious structural and functional characteristics, i.e., long-term stability of the cryopreserved secretome, retention of biological activity post-thaw, tissue-specific regenerative effects in vivo, and reproducibility across batches to solve long-standing problems in regenerative medicine, and that none of Stolk, Schoenenberger, Veronesi, or Lipman teaches or suggests producing an acellular, immune safe, ready to use allogenic formulation stored and reconstituted for direct tissue targeted use in that the prior art co-cultures primarily involve transient, contact dependent systems providing nonspecific macrophage conditioned media in contrast to the defined, programmable secretome of the instant invention.
However, this is not agreed.
In response to Applicant’s arguments against the references individually, it is noted that the test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981). One cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). Further, the Examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In addition, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971).
Specifically, regarding 1), note that claims 49-53 as written do not require that the composition be a stand along product for regenerative therapy. In fact, claim 49 encompasses any administering of the composition along with any other treatment steps to treat any disease. The intended use “for tissue regeneration” within the preamble of claim 43, which states “A composition comprising a collected acellular supernatant by a method for obtaining a composition for tissue regeneration” is an intended use for a composition produced by the method which is used to produce the composition of claim 43, but does not limit the actual use of the product as recited in claim 49.
Additionally, Stolk was cited for teaching a method of obtaining a composition comprising purifying monocytes from a sample, differentiating the purified monocytes into macrophages by culturing the monocytes in the presence of macrophage colony-stimulating factor (M-CSF), co-culturing the macrophages with tenocytes in at a ratio of 5 macrophages to 1 tenocyte for 3 days directly or in a Transwell™ system, collecting the supernatant of the co-culture, and freezing/cryopreserving the supernatant of the co-culture for later use [page 10 ¶ 4- page 11 ¶ 1, page 11 ¶ 6-7 ]. Additionally, Stolk teaches that during co-culture of the macrophages with tenocytes, the macrophages are polarized into a mixed M1/M2 phenotype [page 9 ¶ 2-3]. Additionally, Da Silva was cited for teaching a method of purifying monocytes from a sample and differentiating the purified monocytes into M2 macrophages by culturing the monocytes in serum free medium in the presence of macrophage colony-stimulating factor (M-CSF) [column 3 ¶ 5- column 4 ¶ 2]. Da Silva was further cited for teaching that the use of serum-free medium avoids the potentially confounding and batch-dependent effects of serum components, such as polarizing factors and endogenous serum lipoproteins [column 7 ¶ 5]. Further, Sardana was cited for teaching hat the use of protein- and peptide-free chemically defined serum-free media simplifies analysis of conditioned media compared to the use of serum-containing media which would contaminate the conditioned media [column 10 ¶ 4-column 11 ¶ 1].
Therefore, Stolk, Da Silva, and Sardana teach producing a tissue-specific acellular product via a co-culture of M2-macrophages with differentiated tissue-specific cells in serum-free medium where the secreted factor composition is modulated by cross-talk and subsequently isolated.
Regarding Applicant’s argument 2), note that while dependent claim 52 requires that the composition is cryopreservation or lyophilization, the claims do not require that the cell-free composition retain stable growth factor profiles or full regenerative bioactivity after cryopreservation or lyophilization nor maintain activity after >12 months of storage.
Additionally, the combined teachings of Stolk, Da Silva, and Sardana motivate the ordinarily skilled artisan to arrive at the instantly claimed invention, such that the combination would be expected to have the same structure, and therefore the same functions as the claimed invention.
Further, Applicant’s assertion that maintaining activity after >12 months of storage is an unexpected result of the instant Application amounts to arguments of counsel. The arguments of counsel cannot take the place of evidence in the record. In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965); In re Geisler, 116 F.3d 1465, 43 USPQ2d 1362 (Fed. Cir. 1997) ("An assertion of what seems to follow from common experience is just attorney argument and not the kind of factual evidence that is required to rebut a prima facie case of obviousness."). See MPEP § 716.01(c) for examples of attorney statements which are not evidence and which must be supported by an appropriate affidavit or declaration. Examples of attorney statements which are not evidence and which must be supported by an appropriate affidavit or declaration include statements regarding unexpected results, commercial success, solution of a long-felt need, inoperability of the prior art, invention before the date of the reference, and allegations that the author(s) of the prior art derived the disclosed subject matter from the applicant. MPEP 716.01(c). Attorney argument is not evidence unless it is an admission, in which case, an examiner may use the admission in making a rejection. See MPEP § 2129 and § 2144.03 for a discussion of admissions as prior art.
Note that Applicant cites a passage in the specification (page 13, lines 12-17) as evidence of the unexpected results. However, the referenced paragraph in the specification merely states:
“The cryopreserved or lyophilized composition may be stored between -80 and -86 oC until its use. Storage from -40 oC has been experimentally proved to ensure stability and biological activity of the GF and/or cytokines present in the composition and their activity in vivo restoring an injured tissue as well. The composition cryopreserved may be maintained at least 1 year between -20 and -196 oC. Preferably no more than 3 years. In a preferred embodiment, the supernatant is dosed in single-doses.”
The paragraph cited says that storage from -40 oC has been experimentally proven to ensure stability and biological activity of the GF and/or cytokines present in the composition and their activity in vivo restoring an injured tissue as well, but does not reference any data to support the assertion. Additionally, the cited paragraph does not indicate that cryopreservation beyond one year has been tested, merely that it could be done.
Looking to the disclosure for any additional support, Example 1 teaches preparation of the composition, wherein the collected supernatants were centrifuged and stored at -80oC for further analysis, without any reference to a storage duration [page 31 lines 6-9]. Example 2 discloses the analysis of the compositions comprising cytokines and growth factors, but does not indicate how long the samples had been frozen nor a comparison of fresh samples, freshly frozen samples, and long-term frozen samples to indicate stability and preservation of biological activity of the GF and/or cytokines present in the composition or their activity for restoring an injured tissue in vivo [page 21 lines 20-33, Figures 1-3]. Example 2 further states, “In fact, concentrating formulations either by centrifugation, lyophilization, drying, vacuum, purification columns or by any other method will retain the described modification patterns or tissue-specific composition signatures”, which addresses an issue of concentration but not storage and does not provide any data to support the assertion. Example 3 presents clinical assays using compositions of the invention which had been frozen at -35oC, cryopreserved at -80oC, and thawed prior to their clinical use, but likewise does not indicate how long the samples were stored prior to use [page 23 lines 1-3]. Examples 4-11 present the clinical experiments and data without indicating any storage times for the compositions.
The in vivo clinical data presented in the Drawings in Figures 4-6 and 8-12 present measurements of different symptoms each presented as “% Disease” over time broken down by two grades of disease severity (II or III), wherein the “control” is merely the starting condition prior to treatment. Figure 7 shows the result of an echography done on the day previous to the treatment (-1), the day 4 after the treatment (+4) and the day 8 after the treatment (+8) of a single patient. No untreated nor standard of care treatment data over time is provided. Therefore, there is no actual indication of efficacy of the samples applied because there is no data presenting either the natural, untreated healing process over time nor a standard of care healing process over time to which the treated data can be compared.
Therefore, the disclosure does not provide data in support of Applicant’s assertion that the claimed process yields a reproducible, cell-free composition with stable growth factor profiles after cryopreservation or lyophilization, retaining full regenerative bioactivity, such that the maintained activity after >12 months of storage is a feature not predicted from the transient secretome activity observed in Stolk and Veronesi and represents unexpected results not rendered obvious by any combination of the cited references.
Applicant has provided a Declaration under 37 CFR 1.132 by inventor Jara Sanz, which clarifies that the M2-macrophages of the present invention are provided by purifying monocytes from a sample obtained from a donor, and collecting acellular supernatant of the co-culture, thereby obtaining a composition comprising soluble factors secreted by the co-cultured M2-macrophages and tissue specific cells [page 3 bullet 4], but does not provide evidence of the composition following long-term storage. The Declaration does not indicate storage times and does not provide comparison control data for the clinical experiments.
Therefore, Applicant’s arguments amount to arguments of counsel in that they are not supported by any evidence of record.
It is also noted that any evidence of unexpected results must be commensurate in scope with the claimed invention, and that a greater, or greater than additive, effect is not necessarily sufficient to overcome a prima facie case of obviousness because such an effect can either be expected or unexpected MPEP 716.02 (a) and (d). Whether the unexpected results are the result of unexpectedly improved results or a property not taught by the prior art, the "objective evidence of nonobviousness must be commensurate in scope with the claims which the evidence is offered to support." In other words, the showing of unexpected results must be reviewed to see if the results occur over the entire claimed range. In re Clemens, 622 F.2d 1029, 1036, 206 USPQ 289, 296 (CCPA 1980). Specifically, to the extent that Applicant may rely on the clinical data presented in the disclosure to support the efficacy of the composition in treating various diseases, note again that the data presented does not provide evidence of any improved or unexpected efficacy in that no control samples are provided for comparison, nor do the examples specifically provide evidence of efficacy following long-term storage. Additionally, the Declaration does not provide any evidence of long-term storage stability nor any evidence of efficacy. It is also noted that the scope of the claims as written encompasses the production and use of a composition without any storage, purification, nor concentration.
The scopes of the composition claims encompass culturing M2-macrophages with any tissue specific cells to generate collected supernatant. The scope of the treatment claims additionally encompasses treatment of any disease by administering any composition according to claim 43 to any subject in need. Therefore, although Applicant has provided examples of 8 tissue-specific cell types for production of compositions of the invention and has administered 4 of those compositions to patients suffering from 8 different diseases/conditions, such compositions and administrations are not representative of all tissue-specific cell types nor all diseases. Therefore, Applicant’s limited data is not commensurate in scope with the claimed invention.
Regarding Applicant’s argument 3), Inventor’s Declaration under 37 CFR 1.132 clarifies that the M2-macrophages of the present invention are provided by purifying monocytes from a sample obtained from a donor, and collecting acellular supernatant of the co-culture, thereby obtaining a composition comprising soluble factors secreted by the co-cultured M2-macrophages and tissue specific cells such that there are no additional impurities or degradation products expected in the secretome. The Declaration as filed adds details to the processing of the composition after collection to support the claim that the composition is acellular, in that the examples in the specification only teach that the collected supernatants were subjected to low speed centrifugation and subsequently frozen at -80 oC [page 21 lines 6-9] or that the compositions were frozen at -35oC, cryopreserved at -80oC, and thawed before use [page 23 lines 1-3]. The specification as filed provides support for removing cell debris by filtering with a 0.22 um membrane to purify the sample [page 12 lines 20-23], but does not specifically teach that the compositions used in the examples were filtered, nor that all cells were removed from the compositions.
Claim 43 states in the preamble that the composition is acellular, but does not provide any limitation within the claim which would require the composition to be acellular or which would necessarily result in a composition which is acellular. Claims 46-47 limit the residual number of platelets, red blood cells, or leukocytes present in the collected supernatant to an amount less than 0.1% by cell count (claim 46) or in the collected sample to less than 0.001% of the amount of components in the collected sample. By further limiting the amount of cells within the collected supernatant or the sample collected, particularly to a level as high as 0.1%, the claims themselves teach that the composition according to claim 43 encompasses compositions comprising an amount of platelets, red blood cells, and leukocytes which is greater than 0.1% in addition to any other cells which may be present.
Therefore, given the teachings of Stolk, Schoenenberger, Da Silva, and Sardana, which teach all the limitations of independent claim 43, the teachings of Stolk to freeze the collected supernatants at -80 oC, the statement in the Declaration that freezing the supernatant at -80 oC eliminates any remaining cellular material [page 4 bullet 6], the lack of clarity in the claims regarding the collection of the supernatant in generating an acellular composition, and the lack of requirements in the claims for steps which would render the collected supernatant acellular, the cited references are still considered to teach the compositions and methods of the instant invention as claimed.
Regarding Applicant’s argument 4), that the invention achieves immune tolerance through removal of cell material, note that the claims as written do not require the removal of cell material. Additionally, Applicant has provided no evidence of any immune tolerance. As discussed above, Applicant additionally has provided no evidence of long-term stability of the cryopreserved secretome, no evidence of retention of biological activity post-thaw for either short term or long-term storage, no evidence of tissue-specific regenerative effect in vivo, and no evidence of reproducibility across batches. Additionally, Applicant has not provided any evidence that the issues attempting to be solved are indeed long-standing problems in regenerative medicine.
Applicant asserts that the cited references do not teach an allogenic formulation. Note that claim 51 recites “wherein the composition administered to the subject has been obtained from monocytes sourced from a different subject”, but the method of claim 49 does not limit the method to only allogenic administration.
Veronesi was cited for teaching the administration of CM to patients/ subjects by implanting scaffolds soaked in CM, injection of CM systematically, or directly into the lesion site, wherein the CM employed was allogenic CM or xenogenic without showing signs of inflammatory reaction or systemic and local complications [column 17 ¶ 4-5]. Veronesi additionally teaches that a cell-free approach which employs only cell-produced soluble factors is mandatory to overcome the major cell therapy complications and to overcome drawbacks derived from the use of synthetic factors [column 3 ¶ 4]. Also, Veronesi teaches that conditioned medium (CM) comprises trophic factors released by cells in culture into the medium, and that the soluble factors contained in the CM could initiate regenerative signaling events without the use of cells [column 3 ¶ 4]. Veronesi further teaches that the use of CM for tissue regeneration could eliminate the negative aspects associated with the use of cells and also help to concentrate paracrine factors at physiological levels into the lesion site, reducing the use of recombinant factors and thus reducing the inflammation rate [column 3 ¶ 5]. Veronesi also teaches that CM obtained from macrophages or from CD14 monocytes improved osteoblast migration, which was enhanced by CM of cells cultured on bone [column 9 ¶ 1, column 13 ¶ 3], and that CM from macrophages increased muscle recovery in vitro and in vivo [column 9 ¶ 2, column 13 ¶ 3].
Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to administer a conditioned media comprising secreted factors beneficial to tendon healing, such as the conditioned media taught by Stolk, Schoenenberger, da Silva, Sardana, Lehmann, de Santa, and Committee, to a subject in need thereof. An ordinarily skilled artisan would have been further motivated to administer the CM composition to the subject, wherein the CM was derived from a cell source which is accessible and amendable to isolation protocols, including sources which are allogenic or xenogenic to the subject such that the subject is different from the donor of the monocytes and/or the tenocytes.
Therefore, Applicant’s amendments and arguments do not overcome a finding of obviousness over Stolk, Schoenenberger, da Silva, Sardana, Lehmann, de Santa, and Committee for Proprietary Medicinal Products & Committee for Veterinary Medicinal Products, further in view of Veronesi and Lipman, and the rejection under 35 U.S.C. 103 is maintained.
Conclusion
No claim is allowed.
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DR. KATIE L. PENNINGTON
Examiner
Art Unit 1634
/KATIE L PENNINGTON/Examiner, Art Unit 1634
Dr. A.M.S. Wehbé
/ANNE MARIE S WEHBE/Primary Examiner, Art Unit 1634
Prosecution Insights