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 .
This Action is in response to the papers filed October 20, 2025.
Claims 36-61 are pending in the application and claims 36, 51-53 and 60 are amended in accordance with the claim set filed October 20, 2025. No claims were canceled or newly added. Claims 36, 57, 58 and 60 are independent claims.
Claims 59 and 61 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made with traverse in the reply filed on 08/24/2021.
Thus, claims 36-58 and 60 are examined on the merits.
Priority
The present application is a 35 U.S.C. 371 national stage filing of International Application No. PCT/CH2017/000080, filed August 29, 2017.
Applicant’s claim for the benefit of prior-filed Swiss foreign patent 1109/16 filed August 29, 2016 is acknowledged.
Thus, the earliest possible priority for the instant application is August 29, 2016.
Claim Interpretation
Claims 36 and 60 recite the limitation of “non-hematopoietic stem cells comprising non-hematopoietic progenitor or stem cells, multipotent stem cells, and pluripotent stem cell.” In light of Table 8.1, this limitation is interpreted as a population of cells comprising:
non-hematopoietic progenitor cells, non-hematopoietic pluripotent stem cells, and non-hematopoietic multipotent stem cells.
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Claim 37 recites “wherein the suspension of the original population of cells is washed and res-suspended as a washed and/or single cell suspension prior to the in vitro depletion.” The in vitro depletion is interpreted as the in vitro immuno-depletion of hematopoietic stem cells and cells of hematopoietic lineage disclosed in independent claim 36 since that is the only in vitro depletion recited in the claim.
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Response to arguments
Maintained Objections/Rejections in response to Applicants’ arguments or amendments
Claim Rejections - 35 USC § 112(b)
Claims 36-58 and 60 remain 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.
This rejection has been modified as necessitated by amendment of the claims in the response filed 10/20/2025.
Regarding claim 36, the term “a significant portion” in that last wherein clause is a relative term which renders the claim indefinite. The term “a portion” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention.
A “significant portion” of cells could be anywhere from 1% to 99% of a composition. Therefore, the metes and bounds of the invention would be unclear to one of ordinary skill in the art.
Regarding newly amended claim 36, the term “significant” is recited to further describe “portion.” This is a relative term which renders the claim indefinite. The term “significant” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. Significant could still mean any percentage within the “portion” range.
Thus, at least claim 36 is rejected as being indefinite and all dependent claims are additionally rejected.
Claims 36 and 60 remain rejected for being in improper Markush form. Claims 36 and 60 require that antigen comprises CD14, CD34, CD45, and at least one member of the CD45 family. The claims also require species of the CD45 family. Thus, it is unclear which species are included in the Markush group. Moreover, the claim remains indefinite because they recite a genus of CD45 and also specific species of the genus which are the narrower statements of the range/limitation. Thus, the metes and bounds of the claims are indefinite.
Claims 37-58 are indefinite insofar as they depend from claim 36.
In response to Applicant’s arguments and amendments filed 10/20/2025 regarding the 112b rejection,
Applicant’s arguments and amendments are not persuasive regarding the term “a portion” in claim 36. Applicant has added a relative term to the previous term and therefore, the claim is still indefinite as any part of “a portion” could be significant. Significance is not defined by a percentage of the population.
Moreover, Applicants argue that “Claims 36, 52, and 60 are amended to properly reflect Markush form. Such is not persuasive. Though claim 36 and 60 have been amended to recite “and” instead of “or” the claims remain indefinite in relation to the recitation of “ at least one member of the CD45 family” which is a broad statement of the claimed species of said family in the same claim.
Note that a broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) is considered indefinite, since the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). Note the explanation given by the Board of Patent Appeals and Interferences in Ex parte Wu, 10 USPQ2d 2031, 2033 (Bd. Pat. App. & Inter. 1989), as to where broad language is followed by "such as" and then narrow language. The Board stated that this can render a claim indefinite by raising a question or doubt as to whether the feature introduced by such language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims. Note also, for example, the decisions of Ex parte Steigewald, 131 USPQ 74 (Bd. App. 1961); Ex parte Hall, 83 USPQ 38 (Bd. App. 1948); and Ex parte Hasche, 86 USPQ 481 (Bd. App. 1949).
Claim Rejections - 35 USC § 112(d)
Claims 53-54 remain rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 53 recites “further comprises CD45RO.” Claim 36 on which the claim depends already recites “at least one member of the CD45 family selected from CD45R, CD45RB, CD45RC, CD45RAB, CD45RAC, CD45RBC, CD45RO or CD45R (ABC).” Therefore Claim 54 does not further limit Claim 36.
Claim 54 recites “prepared from a tissue donation from a subject.” Claim 36 on which the claim depends already recites “cell product is prepared ex vivo from a tissue donation.” Ex vivo implicitly indicates that the tissue donation is from a subject. Therefore, claim 54 does not further limit claim 36.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
In response to Applicant’s arguments and amendments filed 10/20/2025 regarding the 112d rejection,
Applicant’s arguments and amendments are not persuasive regarding claims 53 and 54.
Applicant states that claim 53 further limits because it requires a specific species. However, the selection of CD45RO is an option within the grouping of Claim 36. Therefore, to say that it further comprises CD45RO is redundant as it is indicating that CD45RO is an additional marker to the grouping recited. Furthermore, claim 36 remains rejected under 35 U.S.C. 112(b) as stated above because of the recitation of “at least one member of the CD45 family” which is a broad statement of the claimed species of CD45R, CD45RA, CD45RB, CD45RC,….. and CD45R(ABC) in the same claim.
Applicant states that claim 54 further limits the claim as it is not indicated that the tissue donation is obtained by a subject. Again, an artisan would know that it is implicitly indicated that a tissue donation is obtained from some kind of source (i.e. a subject). Therefore, this limitation does not further limit what is encompassed in claim 36.
Claim Rejections - 35 USC § 103
Claims 36, 38, 39, 42, 43, and 50-58 remain rejected under 35 U.S.C. 103 as being unpatentable over Centeno (WO2007087519; IDS filed 12/05/2019) in view of Booth (J Immunol (2010) 184 (8): 4317–4326) as evidenced by Shaikh et al. (2012. Blood Cell: An Overview of Studies in Hematology, Chapter 4; previously cited) and Kayis (AMIA Annu Symp Proc. 2012;2012:456-462; previously cited)
Regarding claims 36, 52, 53 and independent claim 57, Centeno teaches a method for obtaining mesenchymal stem cells (i.e. non-hematopoietic stem cells) and other bone marrow stromal cell through negatively selecting hematopoietic cells expressing CD19, CD14, CD34 and CD45 from bone marrow to remove from the population for autologous transplantation (Abstract, Claims 1 and 4). Centeno further teaches that their experimental techniques are described to determine which bone marrow cells should be removed via negative selection to generate a helper cell population most likely to regenerate tissues (Abstract). They teach that bone marrow can be collected from the patient (i.e. tissue donation in the form of a suspension of cells), centrifuged to separate the cells from plasma, and then subjected to an isolation column or device containing antibodies to the specific cells which need to be removed by negative selection (i.e. immuno-depletion) (para. 0021). Centeno teaches that the negative selection column includes antibodies against antigens such as CD14, CD19, CD34 and CD45 to exclude all non-MSCs (p. 9-10, para. 0021-0023; p. 14, bullet e). Targeting these cell markers as well as others disclosed for negative selection results in a population comprised primarily of mesenchymal stem cells (MSCs, i.e. non-hematopoietic stem cells/multipotent stem cells) and other bone marrow stromal cells while cells such as lymphocytes, monocytes, and granulocytes (i.e. hematopoietic cells) and CD34+ heme progenitors are removed from the cell population as they are positive for CD19, CD14, CD34, and CD45 (p. 14, bullet e; Claims 1, 6 and 14, para. 0004). Though p. 14 of Centeno states it excludes all non-MSCs, within the context, it excludes all hematopoietic lineage progenitor cells within the sample and therefore MSCs and other cells which are non-hematopoietic remain. As evidenced by Shaikh et al., the pluripotent stem cells in bone marrow do not have the surface antigen markers of CD14, CD19, CD34, and CD45 (Table 2) which are negatively selected in the method of Centeno. Thus, the resulting cell product would have pluripotent stem cells in the non-hematopoietic stem cells in addition to MSCs, non-hematopoietic progenitors, and multipotent cells.
However, Centeno does not teach immunodepleting specific species of the CD45 family in addition to CD14, CD34, and CD45.
Booth teaches CD45RO and CD45RA are both expressed in T lymphocyte regulatory cells (Tregs) in both peripheral blood and bone marrow (p. 4317, 2nd column; p. 4323, last paragraph). Booth additionally teaches that these cells expressing CD45RO and CD45RA can be negatively selected within a population of cells via directly conjugated microbeads with anti-CD45RO and anti-CD45RA (p. 4318, Isolation of cells).
It would have been obvious to one of ordinary skill in the art at the time of the effective filing date to add microbeads negatively selecting CD45RO and CD45RA as taught by Booth in as the species of the CD45 family of Centeno to further separate Treg from bone marrow with a reasonable expectation of success. An artisan would have been motivated to add negative selection for CD45RO and CD45RA to Centeno’s cell sorting column as CD45RO and CD45RA are markers for Tregs in both bone marrow and peripheral blood as taught by Booth, and the aim of Centeno is to exclude cell types which are not progenitors or stem cells from a population of bone marrow cells in order to enrich the population for stem cells/progenitors and remove cells which dilute the active agent (MSCs) within the cell population according to Centeno. Thus, CD45RO and CD45RA further remove undesired hematopoietic cell types (Tregs) which dilute the active agent of MSCs in Centeno’s method for negative selection. In addition to this, the addition of CD45RA and CD45RO to CD19, CD14, CD34 and CD45 would be combining known methods of selecting different hematopoietic cell markers for the same purpose of separation from a population.
Regarding claim 38 and 42, the combined teachings of Centeno and Booth render obvious claim 36. Moreover, Centeno teaches that negative selection can occur through the use of immunoadsorption techniques where antibodies bind to the surface antigens present on cells (i.e. immune-labeling) (para. 0008). The antibodies are attached to a surface of either a bead, heavier chain of molecules, or a magnetic particle (i.e. magnetic tag) (para. 0008). Centeno teaches that negative selection columns can be prepared using beads, microspheres, microbeads, alginate gels, and/or magnetic separation technologies (i.e. separation procedure for the removal of cells) (para. 0023). Additionally, Centeno teaches that cell isolation can be achieved by using a fluorescence activated cell sorter (i.e. antibody conjugated to a fluorescent tag) (para. 0027, Example 1).
Regarding claim 39 and 43 , the combined teachings of Centeno and Booth render obvious claim 36. Though Centeno does not explicitly teach that the process of immunolabeling is direct or indirect in conjugating antibodies and tags to the microbeads, Booth teaches directly conjugating microbeads with antibodies and florescent tags before introducing the beads and incubating which thereby would bind to the surface antigens (p. 4318, 1st column).
It would have been obvious to one of ordinary skill in the art at the time of the effective filing date to utilize the known method of directly conjugated antibodies to microbeads as taught by Booth in the method of negative selection utilizing microbeads taught by Centeno with a reasonable expectation of success. An artisan would be utilizing known methods of immunolabeling for the same purpose of immunodepleting.
Regarding claim 50, the combined teachings of Centeno and Booth render obvious claim 36. Moreover, Centeno teaches that their method is intended for operating room staff in a clinical setting to isolate a MSC population during the same surgical procedure as transplantation (Abstract). As the length of surgeries are under 10 hours, as evidenced by Kayis (Figure 1 of Kayis et al), the teachings of Centeno are considered to meet the claimed limitation.
Regarding claim 51, the combined teachings of Centeno and Booth render obvious claim 36. Moreover, Centeno teaches that several surface antigens can be used for negative selection, including CD14, CD11a, CD45, glycophorin A, CD3, CD19, CD34, CD39, and CD66b (Example 1).
Regarding claims 54 and 55, the combined teachings of Centeno and Booth render obvious claim 36. Moreover, Centeno teaches that the bone marrow is harvested from the patient (para. 0021), inherently, the cell product obtained from the bone marrow is autologous to the patient as the patient was the source of the sample.
Regarding claim 56, the combined teachings of Centeno and Booth render obvious claims 36 and 54. Moreover, Centeno teaches that the bone marrow which is utilized to provide cells which are sorted into the targeted cell product are harvested from a donor in need of cartilage repair in order to be put back in as an autologous transplant (Claim 1). This is for the purpose of at least one of degenerated intervertebral discs, spinal joints, or peripheral joints (i.e. lost or damaged tissue) (Claim 6).
Regarding claim 58, the pharmaceutical composition comprises the product produced by the method of claim 36. As claim 36 is made obvious by the references above, so is its product and thus the pharmaceutical formulation comprising said product. No further limitations which provide structure are recited in claim 58 and therefore the method recited in claim 36 will yield both the product of claim 57 and the pharmaceutical formulation of claim 58.
Therefore, the invention would be prima facie obvious to one of ordinary skill in the art at the time of the effective filing date.
Response to Applicants’ Arguments as they apply to rejection of claims 36, 38, 39, 42, 43, and 50-58 under 35 USC § 103 in relation to the teachings of Centeno et al.,
Applicant’s arguments and amendments filed 10/20/2025 have been considered, however they are not persuasive.
Applicant reiterates arguments previously addressed by the Office Action filed 04/18/2025 such as Centeno contains a long list of antigens that are known not to be expressed by MSCs and paragraph [0009] expressly notes that “it is not known which cells should be selected out to produce the best clinical result.”
Examiner reiterates, Centeno specifically singles out utilizing CD19, CD14, CD34, and CD45 for negative selection in a single example, (Cell Sort #5 on page 14). Therefore, providing a teaching for the combination. While Applicant has pointed out paragraph 0009, the context of this statement is to establish the issue that Centeno aims to solve in their methods by listing relevant prior art. On the contrary, Centeno later on teaches those specific markers in question and decides from that data to create Cell Sort #5 (para. 0025, Example 1) which includes the same negative sorting markers of the present invention.
Applicant argues that this is merely removal of all MSCs and that the aim of Centeno is not the same as the present invention.
The examiner states that the intended use of the invention or method steps is not patentably distinct if the combination of method steps are the same and a motivation, teaching or rationale to combine is present. A recitation of the intended use of the claimed invention must result in a structural difference between the claimed invention and the prior art in order to patentably distinguish the claimed invention from the prior art. If the prior art structure is capable of performing the intended use, then it meets the claim.
The examiner states that while Applicant points to parts of their specification, if there is specification components not listed in the claims which would distinguish the claims from the combination of references set forth, Applicant is advised to amend the claims to show the difference between their invention and the prior art such as Applicant pointing out particular process parameters in para. 0131. The examiner notes that independent claims 36 and 60 require the group of surface antigens CD14, CD34, CD45, but the instant claims are not so limited such that this is the only possible surface antigens’ makers from the claimed non-hematopoietic progenitor or stem cells, multipotent stem cells, and pluripotent stem cells. The claims are "open" and thus lend themselves to surface antigens, or additional therapeutically active cell product which contains pluripotent stem cells in the non-hematopoietic stem cells in addition to MSCs, non-hematopoietic progenitors, and multipotent cells
Applicant argues that while Booth discloses the utilization of CD45RA and CD45RO this is not in the same context as the present application and does not show the combination of recited markers an method steps.
In response to applicant's argument, 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). Again, the notion of intended use is reiterated as seen above.
Applicant argues against the combination of Centeno and Bernstein on p. 11, this is interpreted as a typographical error and that the argument is against the combination of Centeno and Booth. Applicant argues that impermissible hindsight reasoning is utilized to combine the two references.
In response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, 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).
Against these arguments of lack of motivation to combine and hindsight reasoning, the motivation and rationale of the above 103 rejection is reiterated. As CD45RO and CD45RA are markers for Tregs in both bone marrow and peripheral blood as taught by Booth, and the aim of Centeno is to exclude cell types which are not progenitors or stem cells from a population of bone marrow cells in order to enrich the population for stem cells/progenitors and remove cells which dilute the active agent (MSCs) within the cell population according to Centeno. Thus, CD45RO and CD45RA further remove undesired hematopoietic cell types (Tregs) which dilute the active agent of MSCs in Centeno’s method for negative selection. In addition to this, the addition of CD45RA and CD45RO to CD19, CD14, CD34 and CD45 would be combining known methods of selecting different hematopoietic cell markers for the same purpose of separation from a population.
Therefore, the 103 rejection is maintained.
Claims 37, 40, 41, 44, 45, 47-49, and 60 remain rejected under 35 U.S.C. 103 as being unpatentable over Centeno (supra) in view of Booth (supra) as evidenced by Shaikh et al. (supra) and Kayis (supra) as applied to claims 36, 38, and 39, and in further view of Basu et al. (J Vis Exp. 2010;(41):1546) as evidenced by Abcam (Indirect Flow Cytometry (FACS) protocol; Wayback Machine: May 29, 2015).
The combined teachings of Centeno and Booth teach a method of negative selection from a tissue sample obtained from a donor comprising adding to a suspension of cells a directly conjugated microbead or magnetic particle which binds to CD14, CD19, CD34, CD45, CD45RO and CD45RA which resulted in a therapeutically active cell product comprising non-hematopoietic progenitor and stem cells as iterated above in the 103 rejection the content of which is incorporated herein, in its entirety.
Regarding claim 37, Centeno does not teach that the cells were washed and resuspended as a washed and/or single cell suspension prior to the in vitro depletion.
Basu teaches generating a single cell suspension of the starting population before staining in a protocol for FACS, then the cells are washed with stain buffer (i.e. population of cells washed prior to the in vitro depletion as a washed suspension) which is then resuspended in the staining buffer (Step 4-5).
It would have been obvious to one of ordinary skill in the art at the time of the effective filing date to wash the cells of Centeno with a stain buffer prior to the depletion as taught by Basu with a reasonable expectation of success. An artisan would have been motivated to wash the population of cells before the in vitro depletion steps, as Basu teaches that it is a known protocol during FACS and Centeno’s method of cell sorting with the claimed antibodies is via FACS (para. 0036).
Regarding claims 40, 45, 47 and 48, Centeno fails to teach in the direct immuno-labeling procedure, limiting incubation conditions allow for only a partial saturation of the antigenic binding sites of the selected surface antigens of CD14, CD19, CD34, or CD45 family on the cells by tag-conjugated antibodies, conditions are adjusted such that cells which are labeled with two or more tags are removed from the original cell population whereas cells comprising fewer tags remain in the original cell population,
Basu et al teaches that FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker, or when separation is based on differential marker density (Abstract; Discussion, 1st paragraph). Basu et al teaches that, during sorting, gating tools and subsetting methods should be used to define the populations of interest (p. 2, step 13). Once the gates have been determined, the gates can be selected for sorting the cells into external collection tubes (p. 2, steps 13-14). Fig. 1A demonstrates that the fluorescent signal between two populations does not have a distinct cut off, and that some partial overlap may exist (i.e. partial saturation). Basu et al teaches that FACS can also be used for negative selection, therefore the opposite conditions could also be applied in Fig. 1 (i.e. retaining cells with low B220 staining and removing those with high B220 staining) (Abstract; Fig. 1). Basu et al teaches that once the antibodies have been chosen, their activity should be optimized by performing a titration analysis (i.e. incubating the cells under limiting conditions for both primary and secondary antibodies) (Discussion, 5th paragraph). This step is important as the use of suboptimal concentrations of staining can result in poor separation of the desired cell population while the use of high concentrations increases the chance for non-specific staining (Discussion, 5th paragraph) (i.e. some cells are either partially saturated or express lower levels of antigen) (Fig. 1A). Additionally, Fig.1A shows that cells which demonstrated higher fluorescent signals for the antigen B220 (i.e. more antigens or higher saturation of antigens) were preferentially separated from those with low or no signal (Bottom left quadrant of Fig. 1A). As mentioned above, Basu et al also teaches that for multicolor staining, antibody selection is a critical step and fluorochromes are chosen based on the detection system, brightness of fluorophores, and the expression level of target antigens (Discussion, 3rd paragraph). Lastly, Basu et al teaches that once the antibodies have been chosen, their activity should be optimized by performing a titration analysis (i.e. incubating the cells under limiting conditions for both primary and secondary antibodies) (Discussion, 5th paragraph). This step is important as the use of suboptimal concentrations of staining can result in poor separation of the desired cell population while the use of high concentrations increases the chance for non-specific staining (Discussion, 5th paragraph).
It is noted that typically antibody binding to a target antigen is partially dependent on the concentration of the antibody, therefore performing a titration analysis would result in both the partial and complete saturation of antigenic binding sites (lower concentrations resulting in less saturation, higher concentrations resulting in greater saturation). This is true whether the immuno-labeling procedure is direct or indirect. Therefore, cells which are labeled with less antibodies (low fluorescence) would be separated from those with more antibodies (high fluorescence). Lastly, as the separation of cells by FACS limits the cells which are sorted according to their fluorescent intensity FACS is carried out under limiting conditions.
It would have been prima facie obvious to one of ordinary skill in the art, prior to the effective filing date of the claimed invention, to modify the method of isolating MSCs taught by Centeno utilizing CD14, CD34, CD45 and CD19 as antigen markers for negative selection to include titrating the antibodies (i.e. immuno-label under limiting conditions) with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to do so to find the optimal concentration of antibodies for distinguishing a target population of cells from an undesired cell population as taught by Basu et al.; (p. 1, step 3). Additionally, it would have been prima facie obvious to one of ordinary skill in the art to modify the method of isolating MSCs taught by Centeno to include establishing fluorescent gates for cell sorting (i.e. immuno-depletion under limiting conditions) as taught by Basu et al. which separate cells by saturation with a reasonable expectation of success. One of ordinary skill would have been motivated to do so to differentiate between a desired and undesired cell population, and to sort the cells accordingly (Basu et al.; Discussion, 5th paragraph).
Regarding claim 41, Centeno teaches that antibodies are attached to a surface of either a bead, heavy chain of molecules, or a magnetic particle, indicating that the immuno-labeling procedure in their method utilized direct immuno-labeling (p. 3, para 0008). Additionally, Centeno teaches that negative selection can be achieved through fluorescence activated cell sorting (p. 12, para. 0027; p. 13-14, example 1).
However, Centeno does not teach that the procedure is an indirect immune-labeling procedure and tag is conjugated to the antibody after the specific binding of the antibodies to the surface antigens.
Basu teaches that both directly conjugated antibodies and indirect secondary antibodies are utilized in the protocol for FACS via repeating the step of antibody conjugation originally done with the first antibody where it binds to the cell (i.e. tag is conjugated to the antibody after the specific binding of the antibodies to the surface antigens) (p. 2, step 8).
It would have been obvious to one of ordinary skill in the art at the time of the effective filing date to utilize an indirect method as taught by Basu for the direct method taught by Centeno of negatively selecting a population of cells via FACs with a reasonable expectation of success. An artisan would have been motivated to do so as both indirect and direct methods of immune-selection are well known in the art to be utilized with FACS as evidenced by Centeno, Booth and Basu.
Regarding claim 44, as discussed above in its entirety, Centeno, Booth and Basu teach indirect immunolabeling procedures for the isolation of a specific population for negative selection. Basu further teaches that this indirect method would at step 8, repeat step 7 which involves two washes with staining buffer after already washing with the primary antibody (step 7-8). Therefore, Basu teaches a first step of incubating primary antibodies, and then incubating those same cells with secondary antibodies which bind to the primary antibodies with washes of stain in between. As evidenced by Abcam, indirect flow cytometry utilizing FACS is carried out with a centrifugation step in between the primary antibody binding and the secondary antibody binding and the cells are resuspended (Step 6).
Regarding claim 49, Centeno teaches that the negative selection method could include antibodies against antigens such as CD14, CD11a, CD45, glycophorin A, CD3, CD19, CD34, CD39, and CD66b (p. 9-10, para. 0021 and 0023; p. 14, bullet e). Centeno teaches that negative selection columns can be prepared using beads, microspheres, microbeads, alginate gels, and/or magnetic separation technologies (p. 9, para. 0023). Additionally, Centeno teaches that cell isolation can be achieved by using a fluorescence activated cell sorter (i.e. antibody conjugated to a fluorescent tag) (p. 12, para. 0027; p. 13-14, example 1).
Centeno fails to teach wherein the immuno-labeling is performed in at least two stages, wherein in a first stage cells are labeled with antibodies against selected surface antigens except for antibodies against CD34, and wherein in a second stage performed after the first stage, cells are labeled with antibodies against CD34.
Basu et al teaches that prior to FACS a starting cell suspension can be enriched for a desired cell population by a bulk purification method such as magnetic sorting (p. 1, step 3). Basu et al teaches that introducing the additional enrichment step is beneficial because it can reduce sorting time (p. 1, step 3).
It would have been prima facie obvious to one of ordinary skill in the art, prior to the effective filing date of the claimed invention, to modify the method of isolating MSCs taught by Centeno to include a bulk enrichment step (targeting a desired cell population to be retained or depleted) prior to further cell isolation and/or purification with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to do so to reduce cell sorting time (Basu et al.; p. 1, step 3).
Regarding claim 60, as discussed in the above 103 rejection and incorporated herein in its entirety Centeno and Booth teach a method of negative selection from a tissue sample obtained from a donor comprising adding to a suspension of cells a directly conjugated microbead or magnetic particle which binds to CD14, CD19, CD34, CD45, CD45RO and CD45RA which resulted in a therapeutically active cell product comprising non-hematopoietic progenitor and stem cells.
However, Centeno and Booth do not teach wherein the in vitro depletion of hematopoietic cells in an original population of cells results in a reduction of at least one hematopoietic surface antigen by a factor of at least 2, 3, 5, 10, or 50 in the final cell preparation when compared to the original population of cells.
Basu et al teaches that FACS is a highly sophisticated technique for purifying cell populations of interest, in which a very high purity (95-100%) of the sorted population can be obtained (Discussion, 1st paragraph). As an example, they teach that TCRβ+ B220- cells were separated from TCRβ-B220+ cells using FACS (Fig. 1A). Following cell sorting, the population of TCRβ+ B220- cells was reduced by a factor of 59.5 (Fig. 1A).
It would have been prima facie obvious to one of ordinary skill in the art, prior to the effective filing date of the claimed invention, to modify the method of Centeno which negatively selects CD19, CD14, CD34 and CD45 to confirm that the presence of a cell population expressing at least one hematopoietic marker was reduced by a factor of at least 2 to 50 with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to do so to confirm that the isolated cell population was free of undesired cells (such as hematopoietic cells) and therefore enriched for the desired population (such as MSCs) (Basu; Discussion, 5th paragraph) Additionally, one would expect this reduction as Basu demonstrated the reduction of one undesired cell population by a factor of 59.5. (Fig 1A).
Therefore, the invention would be prima facie obvious to one of ordinary skill in the art at the time of the effective filing date.
Claim 46 remains rejected under 35 U.S.C. 103 as being unpatentable over Centeno (supra) in view of Booth (supra), Basu (supra) as evidenced by Shaikh et al. (supra), Kayis (supra), and Abcam (supra) as applied to claims 36 and 44, and further in view of Koguchi et al. (J Vis Exp. 2016;(108):e53485; previously cited)
Centeno, Booth, Basu and evidentiary references provide teachings for a method of preparing a cell product comprising non-hematopoietic stem cells comprising non-hematopoietic progenitor cells, multipotent cells, and pluripotent cells from bone marrow in a patient via immune-depletion of CD19, CD14, CD34, CD45RA, CD45RO and CD45 as iterated above and incorporated here in its entirety.
However, Centeno fails to teach wherein, in the first step of the immuno-labeling, the cell population is incubated with primary antibodies against at least 3 different selected surface antigens at the same time, and each of the primary antibodies is incubated according to standard incubation conditions.
Koguchi et al teaches a method for preparing antibody cocktails for immunophenotypic analysis of human peripheral blood with flow cytometry (Abstract). They teach that antibody cocktails (i.e. a mixture of multiple antibodies) used in the clinical laboratory are typically less complex and available pre-titered and pre-mixed from the manufacturer (Introduction, 3rd paragraph). Only a single transfer of pre-mixed antibody cocktail is then required (Introduction, 3rd paragraph). They further teach that in a research setting, antibody cocktails of 10-16 antibodies are typical (introduction, 3rd paragraph). Once the antibody cocktail is prepared it can be mixed with a sample of cells for labeling (p. 5, steps 14-22).
It would have been prima facie obvious to one of ordinary skill in the art, prior to the effective filing date of the claimed invention, to modify the method of isolating MSCs taught by Centeno, Booth and Basu et al to include labeling cell populations by incubating them with multiple primary antibodies with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to do so to as Koguchi et al. teaches that antibody cocktails (i.e. a mixture of multiple antibodies) used in the clinical laboratory are typically less complex and available pre-titered and pre-mixed from the manufacturer (Introduction, 3rd paragraph). Only a single transfer of pre-mixed antibody cocktail is then required (Introduction, 3rd paragraph).
Therefore, the invention would be prima facie obvious to one of ordinary skill in the art at the time of the effective filing date.
Conclusion
No claim is allowable.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
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/ALEXANDRA F CONNORS/Examiner, Art Unit 1634
/MARIA G LEAVITT/Supervisory Patent Examiner, Art Unit 1634