Patent Application 17626515 - ASSAY FOR IDENTIFYING COLONY-FORMING CELLS

Title: ASSAY FOR IDENTIFYING COLONY-FORMING CELLS

Application Information

Invention Title: ASSAY FOR IDENTIFYING COLONY-FORMING CELLS
Application Number: 17626515
Submission Date: 2025-05-16T00:00:00.000Z
Effective Filing Date: 2022-01-12T00:00:00.000Z
Filing Date: 2022-01-12T00:00:00.000Z
National Class: 435
National Sub-Class: 007210
Examiner Employee Number: 97184
Art Unit: 1677
Tech Center: 1600

Rejection Summary

102 Rejections: 1
103 Rejections: 3

Cited Patents

The following patents were cited in the rejection:

US 0059778🔗
US 0255359🔗

Office Action Text

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 .

Information Disclosure Statement
The information disclosure statement filed January 12, 2023 fails to comply with 37 CFR 1.98(a)(2), which requires a legible copy of each cited foreign patent document; each non-patent literature publication or that portion which caused it to be listed; and all other information or that portion which caused it to be listed. It has been placed in the application file, but the information referred to therein has not been considered.

Drawings
The drawings are objected to because Fig. 10 is missing.
New drawings in compliance with 37 CFR 1.121(d) are required in this application. Applicant is advised to employ the services of a competent patent draftsperson outside the Office, as the U.S. Patent and Trademark Office no longer prepares new drawings. The corrected drawings are required in reply to the Office action to avoid abandonment of the application. The requirement for corrected drawings will not be held in abeyance.

Claim Objections
Claims 2 and 8 are objected to because of the following informalities:
Claim 2 contains abbreviations CFU-GEMM, CFU-GM, CFU-M, BFU-E and CFU-G. These should be completely spelled out in its first occurrence.
Claim 8 recites “an fragmented antibody, an fragmented antibody derivative”. Should be “an antibody fragment, an antibody derivative fragment”.
Appropriate correction is required.

Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL. —The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.

Claim 2 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claim 2 recites relative amounts of three markers used for identification of the differentiated cells. Fig. 3 legend discloses that there were other antigens detected in the same experiment – “The difference to 100% relates to other antigens which are not of interest for the method of the invention” ([0031]). The specification fails to disclose contribution of other markers to the total count. In the absence of the total count for all markers/antigens the disclosed relative amounts are meaningless as quantitative limitations. Therefore, the actual percent values of claim 2 lack support from the disclosure.
Claim 2 recites characterization of differentiated hematopoietic stem cells using CD14, CD15, and CD235a specific conjugates. Applicant discloses that conventional analysis of differentiated cells is highly unreliable for identification purposes (Fig. 3ab and [0008]). The specification is silent on Applicant using any standardized or reference cell materials for disclosed experiment. Therefore, it is not clear what cell types actually contributed to the measured relative amounts and whether these cell types were pure or mixed individual types.
Applicant discloses a seeding cell concentration of 2.5 cells per well ([0047]), which is too high to ensure that a cell colony grown in each well would originate from a single seed cell. The issue of pure/mixed cultures is not addressed by the specification. Therefore, in the absence of well-characterized or pure cell culture samples used in FACS experiments disclosed in Fig.6-Fig.10, disclosed percents of different markers (Table 3 and claim 2) are meaningless as quantitative limitations.
Based on the above findings, one of ordinary skill in the art would conclude that Applicant did not have possession of the claimed invention at the time the application was originally filed.

The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION. —The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.

The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.

Claims 1-11 are 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.
Claim 1 recites “isolation of undifferentiated hematopoietic stem cells in groups of 1 - 1000 cells on a support.” It is unclear if the cells are being isolated or merely seeded “in groups of 1 - 1000 cells”, because the claim fails to recite any isolation method steps.
The metes and bounds of the limitation “1 - 1000 cells on a support” are not clear because “support” is not a quantitative parameter, therefore a number of seeded cells without any indication of the support surface area or volume of a culture medium is an indefinite limitation.
Claim 1 recites in step (d) “detecting the relative amount of differentiated hematopoietic stem cells in a cell colony labelled with the marker conjugates.” It is unclear how the relative amounts of can be detected if the claim does not require detection of all three markers: CD14, CD235a and CD15 - claim 1 recites “at least one antigen recognizing moiety.”
Claim 1 recites in step (d) “detecting the relative amount of differentiated hematopoietic stem cells in a cell colony labelled with the marker conjugates.” It is unclear how the relative amount of differentiated hematopoietic stem cells (i.e., different cell kinds or types) can be detected if the specification fails to disclose simultaneous quantitation of different cell kinds or types. Table 3 discloses relative amounts of CD14, CD15, and CD235a markers for identification of individual cell kinds (i.e., CFU-GEMM, CFU-GM, CFUM, BFU-E and CFU-G). The relative amount of differentiated hematopoietic stem cells of five kinds cannot be determined from experiments with only three marker conjugates (a system of five equations with five unknowns and only three known parameters). The specification and the prior art are silent on such detecting.
Claim 2 recites “relative amount in % more than” in the column header. It is unclear how to interpret ranges using “% more than” criterion. For example, any measured value above 0% is more than the lower range value of 0% in a 0-5% range, and therefore meets the claim.
Claim 2 recites that for CFU-GEMM cells CD14 should be more than 15%. The specification discloses over 15% in [0057] and at least 14% in [0058]. It is unclear which relative amount for CD14 is correct. Also, the terms “over” and “at least” are not similar. For example, a relative amount of 10% would meet the limitation of at least 10%, but fail to meet the limitation of over 10%. The specification and claim 2 show different relative amounts and use different terminology.
Claim 2 recites relative amounts of three markers and presents them in a table. Where possible, claims are to be complete in themselves. Incorporation by reference to a specific figure or table "is permitted only in exceptional circumstances where there is no practical way to define the invention in words and where it is more concise to incorporate by reference than duplicating a drawing or table into the claim. Incorporation by reference is a necessity doctrine, not for applicant’s convenience." Ex parte Fressola, 27 USPQ2d 1608, 1609 (Bd. Pat. App. & Inter. 1993). See MPEP 2173.05(s).
Claim 5 recites “a cell sample 25”. It is unclear what the cell sample 25 is.
Claims 7-8 recite “one more marker conjugates.” The meaning of “one more” is unclear, because both claims are independent claims.
Claim 9 recites “the differentiation status”. There is insufficient antecedent basis for this limitation in the claim, because claim 1 does not recite any “differentiation status”.
Additionally, it is unclear what “stem cells” and “a cell sample” are recited by the claim. Claim 1 recites “hematopoietic stem cells”. It is unclear if the cell sample is taken before or after step (b).
Claim 9 recites “Use of the method according to claim 1.” It is unclear what method steps are recited in the claim.

Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.

Claim 9 is rejected under 35 U.S.C. 101 because the claimed invention is directed to non-statutory subject matter. The claim does not fall within at least one of the four categories of patent eligible subject matter because a “use” is not a process, a machine, a manufacture, or a composition of matter. A “use” is not one of the statutory categories of invention.

Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale or otherwise available to the public before the effective filing date of the claimed invention.

(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.

Claims 1-3 and 5-8 are rejected under 35 U.S.C. 102(a)(1) and 35 U.S.C. 102 (a)(2) as being anticipated by Wognum et al. (IDS; PGPub 2007/0059778).
Regarding claim 1, Wognum teaches colony forming cell (CFC) assays applicable to hematopoietic CFC assays (Abstract) comprising BFU-E, CFU-G, CFU-M, CFU-GM, and CFU-GEMM cells ([0005]). Specifically, Wognum teaches a method comprising:
proliferating the isolated cells to form cell colonies of differentiated hematopoietic cells - specifically, Wognum teaches culturing the cell preparation in a culture medium suitable to promote the growth and differentiation of the progenitor cells into colonies containing a specific cell type [0021];
cell media comprising a growth factor – specifically, invention also comprises modifications to the culture medium to selectively promote the development of one colony type (Abstract) and using different cytokines such as GM-colony stimulating factor, IL-3, IL-6, G-CSF, and Stem Cell Factor ([0005]);
contacting the cell colonies with one or more marker conjugates comprising at least one detection moiety – specifically, adding a detection reagent that can detect the progenitor cells or the specific cell type ([0022]). “The invention comprises adding a detection reagent, most likely a fluorescently labeled antibody, that is specific for antigens” (Abstract); and
detecting the differentiated hematopoietic stem cells in a cell colony labelled with the marker conjugates – specifically, detecting progenitor cells or the specific cell type ([0023]).
Regarding marker conjugates comprising antigen recognizing moieties against CD14, CD235a, and CD15, Wognum teaches that erythroid colonies can be stained with a FITC-conjugated antibody against Glycophorin-A (another name for CD235a) ([0066]), monocytes can be selectively stained with antibodies against CD14, and granulocytes can be selectively stained with antibodies against CD15 ([0050]).
Regarding claim 2, Wognum teaches that:
erythroid colonies can be stained with an antibody against Glycophorin-A (CD235a) ([0066]). “E” in CFU-GEMM stands for erythrocyte and erythrocyte is an erythroid cell type. Therefore, CFU-GEMM cells can be detected using CD235a marker;
monocytes can be selectively stained with antibodies against CD14 ([0050]). “M” in CFU-GEMM stands for monocyte. Therefore, CFU-GEMM cells can be detected using CD14 marker;
granulocytes can be selectively stained with antibodies against CD15 ([0050]). “G” in CFU-GEMM stands for granulocyte. Therefore, CFU-GEMM cells can be detected using CD15 marker;
Therefore, CFU-GEMM cells are CD15+, CD14+, and CD235a+, meeting all claim 2 limitations for this cell type.
Please, note that rejection of claim 2 does not address specific relative amounts of the markers. See 112(a) rejection above for details.
Regarding claim 3, Wognum teaches that “[l]ess widely used are CFC assays that contain agar or collagen as the semisolid matrix” [0005], meeting the limitation of claim 3 reciting the method is performed in absence of methyl cellulose.
Regarding claim 5, Wognum teaches that there is a variability in the types of different colony types that can be reduced by modifying the preparation of the cell suspension ([0058]). This can be achieved by purifying colony forming cells, by removing mature cells and non-colony-forming cells that produce factors that can affect the growth of colony-forming cells. Colony-forming cells can be purified by staining cell preparations with antibodies or antibody conjugates that are specific against antigens, such as CD34 (id.).
Regarding claim 6, Applicant has not disclosed that the specific limitations recited in instant claims (that the CD34+ cells of the cell sample are enriched to a purity of at least 50%) are for any particular purpose or solve any stated problem and the prior art teaches that colony forming cells can benefit from an enrichment, it would have been obvious for one of ordinary skill to discover the optimum workable range of purity in order to achieve such enrichment of CFCs that provides desirable improvement to the contrast between colonies and background and between colony types, and reduces variability by introducing a more uniform population of cells (Wognum, [0080]).
Regarding claims 7-8, Wognum teaches that “the invention comprises adding a detection reagent, most likely a fluorescently labeled antibody, that is specific for antigens” (Abstract), meeting the limitation of claim 7 reciting the detection moiety is fluorescent moiety and the limitation of claim 8 reciting the antigen recognizing moiety is an antibody.

Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.

The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
Determining the scope and contents of the prior art.
Ascertaining the differences between the prior art and the claims at issue.
Resolving the level of ordinary skill in the pertinent art.
Considering objective evidence present in the application indicating obviousness or nonobviousness.

This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.

Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over Wognum et al. (IDS; PGPub 2007/0059778), as applied to claim 1 above, in view of Perry et al. (PGPub 2014/0255359).
The teachings of Wognum have been set forth above.
Wognum fails to teach lysing red blood cells prior to step (a) of claim 1.
Regarding claim 4, Perry teaches that prior to analysis of hematopoietic stem cells red blood cells are lysed using hemolysis buffer ([0273]).
It would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Wognum by employing red blood cells lysis as taught by Perry, in order to provide the sample free of interfering cell types. One having ordinary skill in the art would have been motivated to make such a change to be able to remove mature cells and non-colony-forming cells that produce factors that can affect the growth of colony-forming cells (Wognum, [0058]). The use of such combination would have been desirable to those of ordinary skill in the art for the reason mentioned above.
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references because Perry teaches red blood cells lysis performed before analysis of hematopoietic stem cells; therefore, the hematopoietic stem cells are not damaged by the lysis step.

Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Wognum (IDS; PGPub 2007/0059778), as applied to claims 1-2 above.
The teachings of Wognum have been set forth above.
Wognum fails to teach a marker cocktail comprising more than one marker conjugate comprising at least one detection moiety and at least one antigen recognizing moiety against CD14, CD235a and CD15.
Regarding claim 10, in a separate embodiment, Wognum teaches three marker conjugates comprising at least one detection moiety and at least one antigen recognizing moiety against CD14, CD235a and CD15. See, claims 1-2 above for CD14, CD235a and CD15. The detection moiety is a fluorescent group (Abstract). The antigen recognizing moieties are antibodies (Abstract).
It would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Wognum by combining more than one marker conjugate against CD14, CD235a and CD15 as taught by Wognum, in order to provide a convenient master cocktail, as an obvious matter of combining prior art elements according to known methods to yield predictable results. The individual marker conjugates against CD14, CD235a and CD15 when combined in a master cocktail would still perform their binding functions with corresponding markers, and the combination would not produce a ‘new’ or ‘different function’.

Claim 11 is rejected under 35 U.S.C. 103 as being unpatentable over Wognum (IDS; PGPub 2007/0059778), as applied to claims 1-2 above.
The teachings of Wognum have been set forth above.
Wognum fails to teach a kit comprising cell media with at least one growth factor and a marker cocktail comprising marker conjugates each comprising at least one detection moiety and at least one antigen recognizing moiety against CD14, CD235a and CD15.
Regarding claim 11, in a separate embodiment, Wognum teaches three marker conjugates comprising at least one detection moiety and at least one antigen recognizing moiety against CD14, CD235a and CD15. See, claims 1-2 above for CD14, CD235a and CD15. The detection moiety is a fluorescent group (Abstract). The antigen recognizing moieties are antibodies (Abstract).
Additionally, Wognum teaches culturing the cell preparation in a culture medium suitable to promote the growth and differentiation of the progenitor cells into colonies containing a specific cell type ([0021]). Invention also comprises modifications to the culture medium to selectively promote the development of one colony type (Abstract) and using different cytokines such as GM-colony stimulating factor, IL-3, IL-6, G-CSF, and Stem Cell Factor ([0005]);
It would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Wognum by combining a cell media with at least one growth factor and a marker cocktail comprising marker conjugates each comprising at least one detection moiety and at least one antigen recognizing moiety against CD14, CD235a and CD15 as taught by Wognum, in order to make a kit, as an obvious matter of combining prior art elements according to known methods to yield predictable results. The individual components when combined in the kit would still perform their corresponding functions, and the combination would not produce a ‘new’ or ‘different function’: the marker conjugates would bind and detect their markers; the cell media would support growth of the cells; and the growth factor would stimulate growth of a selected cell type. Organizing several reagents as a kit is very well-known in the art and widely used because of its convenience.

Subject Matter Free of the Prior Art
Claim 9 is free of prior art. Please, see 112(b) and 101 rejections above for details.

Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Alexander Volkov whose telephone number is (571) 272-1899. The examiner can normally be reached M-F 9:00AM-5:00PM (EST).
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Bao-Thuy Nguyen can be reached on (571) 272-0824. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/ALEXANDER ALEXANDROVIC VOLKOV/
Examiner, Art Unit 1677

/REBECCA M GIERE/Primary Examiner, Art Unit 1677