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 .
Information Disclosure Statement
The information disclosure statement filed November 19, 2024 is acknowledged and has been considered by the examiner.
Drawings
Color photographs and color drawings are not accepted in utility applications unless a petition filed under 37 CFR 1.84(a)(2) is granted. Any such petition must be accompanied by the appropriate fee set forth in 37 CFR 1.17(h), one set of color drawings or color photographs, as appropriate, if submitted via the USPTO patent electronic filing system or three sets of color drawings or color photographs, as appropriate, if not submitted via the via USPTO patent electronic filing system, and, unless already present, an amendment to include the following language as the first paragraph of the brief description of the drawings section of the specification:
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Color photographs will be accepted if the conditions for accepting color drawings and black and white photographs have been satisfied. See 37 CFR 1.84(b)(2).
Specification
The abstract of the disclosure is objected to because it is too short (26 words). A corrected abstract of the disclosure is required and must be presented on a separate sheet, apart from any other text. See MPEP § 608.01(b).
Claim Objections
Claim 21 is objected to because of the following informalities
Claim 21 recites a list of chelator options. In view of the lists as a whole, it appears that options within the list are separated by semicolons. However, the first option in the list, deferoxamine, is only separated from the other items in the list by a comma. In view of the disclosure as a whole and claim 6, it appears that deferoxamine is intended to be a unique member of the claimed list and should thus be separated from the following options by a semicolon instead of a comma.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
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 6 and 21 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.
Both claims 6 and 21 recite a list of chelators. However, for most of the options of these lists, the chelator is described as a chelator “or one of its derivatives.” This phrase renders the claims indefinite. It is not clear what structures the chelator derivatives may possess and how different the derivative may be from the parent chelator. Therefore, the scopes of these claims are indefinite.
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:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. 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.
Claims 1-3, 5, 7-8, 10, 12-14, and 18-22 are rejected under 35 U.S.C. 103 as being unpatentable over Bansal (Bansal, A.; et al., Sci. Rep., 2022) in view of Baskin (Baskin, J. M.; et al., PNAS, 2010) and Pochon (Pochon, S.; et al., Int. J. Cancer, 1989), as evidenced by STEMCELL® (LYMPHOPREP® Product Sheet, 2018).
Bansal teaches a method of labeling cells with 89Zr and tracking them in vivo (pg. 1, Abstract). More specifically, Bansal teaches labels comprising 89Zr coordinated by deferoxamine (DFO) or Hy3ADA5 chelators conjugated to isothiocyanato-benzyl or squaramide ester groups (pg. 3, Figure 1). Bansal teaches complexing 89Zr in these chelating groups (pg. 4, Figure 2; and pg. 9, last paragraph through pg. 10 first paragraph). Bansal teaches labeling stem cells and white blood cells with these radiolabeled chelating groups (pg. 4, Figures 3 and 4; and pg. 10, paragraph 3). Bansal teaches using these radiolabeled cells to track cells in vivo (pg. 5, Figure 5; pg. 7, Figure 8; and pg. 10, paragraph 6). Bansal teaches that this method is an effective means of using PET-based cell radiolabeling to noninvasively track cells that can be used to understand the safety, efficacy, distribution, and clearance of cell-based therapies (pg. 8, last paragraph).
Bansal does not teach a method of labeling a cell comprising generating an aldehyde group on a cell surface glycan via reaction with an oxidizing agent and reacting the aldehyde group with an aldehyde-reactive bifunctional chelator.
Baskin teaches a method of fluorescently imaging cells for imaging in vivo (pg. 10360, Abstract). Specifically, Baskin teaches methods of labeling sialic acid and sialic acid derivatives with fluorophores (pg. 10361, Figure 1; and pg. 10363, Figure 3). In the nonmetabolic approach, Baskin teaches oxidizing sialylated cell surface glycans with sodium periodate to modify the sialic acid to have aldehyde groups and subsequently reacting the cells with an aminooxy-derivatized fluorophore (pg. 10362, right column, third paragraph; and Figure 3). Baskin teaches that this approach for tagging sialylated glycans did not result in any noticeable toxicity (pg. 10363, left column, first paragraph). Baskin teaches that this nonmetabolic approach is an effective means of labeling cells for imaging (Figure 3; and pg. 5, right column, second paragraph).
Pochon teaches the labeling of an antibody with an aminooxyacetyl deferoxamine chelator and radiolabeling with 67Ga (pg. 1188, Title and Abstract). Pochon teaches the synthesis of this chelator by the addition of an aminooxyacetyl group to ferrioxamine (pg. 1189. Left column, fourth paragraph) followed by removal of the chelated iron (pg. 1189, right column, fourth paragraph) and labeling with 67Ga (pg. 1189-1190, Labelling of the chelator). Pochon also teaches the oxidation of an antibody with periodate (pg. 1189, Oxidation) and teaches that periodate oxidation is known to generate aldehyde groups on oligosaccharides on antibodies (pg. 1188, left column, last paragraph, lines 15-18) and that aminooxy groups are known to be reactive with aldehydes (pg. 1188, left column, last paragraph, lines 26-29). Pochon teaches conjugating the oxidized antibody with the 67Ga-labeled aminooxy-deferoxamine (pg. 1190, Coupling with labelled chelator) and with the unlabeled aminooxy-deferoxamine followed by labeling of the ligated complex (pg. 1190, Post-conjugation labelling). Pochon teaches using the 67Ga-aminooxy deferoxamine-labeled antibody conjugates for in vivo distribution in mice after i.v. injection (pg. 1190, Imaging study; and pg. 1192, Figure 6). Pochon teaches that this radionuclide labeling through an aminooxy deferoxamine chelator reduced liver and spleen accumulation of radiolabeled antibody compared to chloramine-T 125I labeled antibodies, providing an advantage for the periodate oxidation method (pg. 1191, In vivo testing of the radiolabelled conjugates).
STEMCELL® provides a product description and manual for use for a LYMPHOPREP® kit. The kit is described as a density gradient medium for the isolation of mononuclear cells from peripheral blood, cord blood, and bone marrow (pg. 1, first paragraph). The directions for use (pg. 1-2) teach a method of isolation of mononuclear cells.
A person of ordinary skill in the art would have recognized that both Bansal and Baskin teach methods of labeling the surface of cells with a label for in vivo imaging. It would also be recognized that Baskin and Pochon teach labeling of biomolecules using the same periodate oxidation followed by reaction with an aminooxy conjugated labeling group and using the labeled materials for in vivo imaging. Therefore, it would be understood that the method of Baskin could be used to radiolabel cells with the bifunctional chelator of Pochon. It would further be recognized that both Bansal and Pochon teach covalently labeling biomolecules with the same deferoxamine chelator (only differing in the attachment linker group) bound to a radionuclide for in vivo imaging applications.
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the cell labeling method of Bansal by substituting the protein labeling with to isothiocyanato-benzyl-deferoxamine with the periodate sialic acid oxidation and aminooxy-deferoxamine method taught by the combination of Baskin and Pochon because these methods are alternative means of labeling cells for in vivo imaging applications (MPEP § 2143(I)(B)). This substitution would predictably result in a method of radiolabeling cells in which a cell surface glycan is oxidized to produce an aldehyde and a bifunctional chelator ligates to the cells by reacting with the aldehyde.
A person of ordinary skill in the art would have had a reasonable expectation of success in modifying the method of Bansal by substituting the labeling method in this way because Baskin teaches that periodate oxidation of cell surface glycan sialic acids is an effective means of attaching imaging labels to cells. Additionally, Pochon teaches that the periodate oxidation of sugars can be effectively used to label biomolecules with a radiolabeled deferoxamine chelator modified with an aminooxy group.
The skilled artisan would have been motivated to make this modification because the periodate oxidation and aminooxy-label conjugation system of Baskin and Pochon labels glycans instead of amino acid sidechains of proteins, which could prevent any negative effects of direct protein modification on protein structure and function. Furthermore, Pochon teaches that the aldehyde conjugation system is compatible with radiolabeling both before and after conjugation whereas Bansal only teaches radionuclide binding before chelator conjugation; thus, the modified method may provide more flexibility the method of preparation of labeled cells.
Regarding claim 1, Bansal teaches a method of labeling the surface of stem cells and white blood cells using radiolabeled bifunctional chelators including 89Zr-DFO-Bn-NCS (Figure 1; and pg. 10, third paragraph). Bansal teaches using the radiolabeled cells for PET imaging in vivo to track the cells (Figure 5; Figure 8; pg. 6, first paragraph, lines 7-9; and pg. 10, paragraph 6). Additionally, Baskin teaches a method of labeling sialic acids on cell surface glycans by oxidizing the sialic acid groups with periodate to produce aldehydes and subsequently labeling the aldehydes with an aminooxy-modified label for in vivo imaging (pg. 10362, right column, third paragraph; and Figure 3). Furthermore, Pochon teaches a method of radiolabeling sugars oxidized by periodate to form aldehydes with an aminooxy-deferoxamine chelator and the radiolabeling of the chelator with a radionuclide (pg. 1190, Coupling with labelled chelator; and Post-conjugation labelling), enabling in vivo imaging (Figure 6). As described above, it would have been prima facie obvious to modify the method of Bansal labeling cell surfaces with deferoxamine through the isothiocyanato reactive group by substituting the aminooxy-deferoxamine labeling method taught by the combination of Baskin and Pochon. The resulting method would be a method of labeling stem cells or white blood cells with deferoxamine through cell surface glycan oxidation by the addition of periodate oxidizing agent to form aldehydes and conjugation of an aminooxy-deferoxamine bifunctional chelator to the aldehyde groups to ligate the bifunctional chelator to the cell surface glycan to form a labeled cell. As indicated by Bansal and Pochon, such a radiolabeled cell could be used for in vivo tracking. Therefore, the combined teachings of Bansal, Baskin, and Pochon render claim 1 obvious.
Regarding claims 2, 3, 5, and 7; Pochon (pg. 1890, paragraphs 3-5) teaches the preparation of aminooxy-deferoxamine. Pochon teaches that deferoxamine is a chelator (pg. 1188, right column, second paragraph, first sentence). Pochon also teaches that aminooxy groups readily react with aldehydes (pg. 1188, left column, last paragraph, lines 28-30). Thus, the examiner interprets the conjugation of an aminooxy containing group to deferoxamine to be the generation of a bifunctional chelator by conjugating an aldehyde reactive group to a chelator. Additionally, Bansal teaches a cell surface label with a deferoxamine chelator (Figure 1). As described above, the combined method of the teachings of Bansal, Baskin, and Pochon would result in labeling cells with aminooxy-deferoxamine. Therefore, the combined teachings of Bansal, Baskin, and Pochon render claims 2, 3, 5, and 7 obvious.
Regarding claim 8, Baskin teaches oxidizing cell surface glycans with NaIO4 (Figure 3). Additionally, Pochon teaches oxidizing sugars with NaIO4 prior to conjugation with aminooxy-deferoxamine (pg. 1189, Oxidation). Therefore, the combined teachings of Bansal, Baskin, and Pochon render claim 8 obvious.
Regarding claims 10 and 12, Pochon teaches conjugating a 67Ga radionuclide to the bifunctional chelator aminooxy-deferoxamine both before and after ligation to the oxidized sugar aldehyde groups (pg. 1190, Coupling with labelled chelator; and Post-conjugation labeling). Additionally, Bansal teaches radiolabeling cells with a 89Zr radionuclide conjugated deferoxamine bifunctional chelator (pg. 10, third paragraph). Therefore, the combined teachings of Bansal, Baskin, and Pochon render claims 10 and 12 obvious.
Regarding claim 13, Bansal teaches radiolabeling white blood cells with a radiolabeled deferoxamine bifunctional chelator (pg. 10, third paragraph). Bansal teaches that the white blood cells were isolated from peripheral blood using a LYMPHOPREP® kit per the manufacturer instructions (pg. 10, second paragraph). As evidenced by STEMCELL®, the LYMPHOPREP® kit is used for the isolation of mononuclear cells from peripheral blood (pg. 1, first paragraph). As Bansal teaches using this kit for the isolation of white blood cells from peripheral blood, the examiner interprets the white blood cells of Bansal to be mononuclear cells or be a population of cells including mononuclear cells. Thus, these cells read on the cell type option of peripheral blood mononuclear cells. Therefore, the combined teachings of Bansal, Baskin, and Pochon, as evidenced by STEMCELL®, render claim 13 obvious.
Regarding claim 14, Bansal teaches a method of labeling the surface of stem cells and white blood cells using radiolabeled bifunctional chelators including 89Zr-DFO-Bn-NCS (Figure 1; and pg. 10, third paragraph). Bansal specifically teaches adding the bifunctional chelator to populations of 6x106 cells (pg. 10, third paragraph), which the examiner interprets to be a population of cells. Bansal teaches using the radiolabeled cells for PET imaging in vivo to track the cells (Figure 5; Figure 8; pg. 6, first paragraph, lines 7-9; and pg. 10, paragraph 6). Additionally, Baskin teaches a method of labeling sialic acids on cell surface glycans by oxidizing the sialic acid groups with periodate to produce aldehydes and subsequently labeling the aldehydes with an aminooxy-modified label for in vivo imaging (pg. 10362, right column, third paragraph; and Figure 3). Furthermore, Pochon teaches a method of radiolabeling sugars oxidized by periodate to form aldehydes with an aminooxy-deferoxamine chelator and the radiolabeling of the chelator with a radionuclide (pg. 1190, Coupling with labelled chelator; and Post-conjugation labelling), enabling in vivo imaging (Figure 6). As described above, it would have been prima facie obvious to modify the method of Bansal labeling cell surfaces with deferoxamine through the isothiocyanato reactive group by substituting the aminooxy-deferoxamine labeling method taught by the combination of Baskin and Pochon. The resulting method would be a method of labeling stem cells or white blood cells with deferoxamine through cell surface glycan oxidation by the addition of periodate oxidizing agent to form aldehydes and conjugation of an aminooxy-deferoxamine bifunctional chelator to the aldehyde groups to ligate the bifunctional chelator to the cell surface glycan wherein the chelator may be labeled with a radionuclide either before or after conjugation of the chelator to the sugar to form a labeled cell population. As indicated by Bansal and Pochon, such a radiolabeled cell could be used for in vivo tracking. Therefore, the combined teachings of Bansal, Baskin, and Pochon render claim 14 obvious.
Regarding claim 18, Bansal teaches a method of labeling the surface of stem cells and white blood cells using radiolabeled bifunctional chelators including 89Zr-DFO-Bn-NCS (Figure 1; and pg. 10, third paragraph). Bansal teaches using the radiolabeled cells for PET imaging in vivo to track the cells (Figure 5; Figure 8; pg. 6, first paragraph, lines 7-9; and pg. 10, paragraph 6). Additionally, Baskin teaches a method of labeling sialic acids on cell surface glycans by oxidizing the sialic acid groups with periodate to produce aldehydes and subsequently labeling the aldehydes with an aminooxy-modified label for in vivo imaging (pg. 10362, right column, third paragraph; and Figure 3). Furthermore, Pochon teaches a method of radiolabeling sugars oxidized by periodate to form aldehydes with an aminooxy-deferoxamine chelator and the radiolabeling of the chelator with a radionuclide (pg. 1190, Coupling with labelled chelator; and Post-conjugation labelling), enabling in vivo imaging (Figure 6). As described above, it would have been prima facie obvious to modify the method of Bansal labeling cell surfaces with deferoxamine through the isothiocyanato reactive group by substituting the aminooxy-deferoxamine labeling method taught by the combination of Baskin and Pochon. The resulting method would be a method of labeling stem cells or white blood cells with deferoxamine through cell surface glycan oxidation by the addition of periodate oxidizing agent to form aldehydes and conjugation of an aminooxy-deferoxamine bifunctional chelator to the aldehyde groups to ligate the bifunctional chelator to the cell surface glycan wherein the chelator may be labeled with a radionuclide either before or after conjugation of the chelator to the sugar to produce radionuclide labeled cells. The product of this method would be a radionuclide labeled cells wherein a radionuclide conjugated bifunctional aminooxy-deferoxamine chelator is ligated to a cell surface glycan. Therefore, the combined teachings of Bansal, Baskin, and Pochon render claim 18 obvious.
Regarding claim 19, Baskin teaches that the periodate oxidation and aminooxy-label conjugation method labels sialic acid in cell surface glycans (pg. 10362, right column; and Figure 3). Therefore, the combined teachings of Bansal, Baskin, and Pochon render claim 19 obvious.
Regarding claims 20 and 21, Pochon (pg. 1890, paragraphs 3-5) teaches the preparation of aminooxy-deferoxamine. Pochon teaches that deferoxamine is a chelator (pg. 1188, right column, second paragraph, first sentence). Additionally, Bansal teaches a cell surface label with a deferoxamine chelator (Figure 1). Furthermore, Baskin teaches labeling cell surface glycans with an aminooxy-modified label (Figure 3). As described above, the combined method of the teachings of Bansal, Baskin, and Pochon would result in cells radiolabeled with aminooxy-deferoxamine. Therefore, the combined teachings of Bansal, Baskin, and Pochon render claims 20, and 21 obvious.
Regarding claim 22, Pochon teaches conjugating a 67Ga radionuclide to the bifunctional chelator aminooxy-deferoxamine both before and after ligation to the oxidized sugar aldehyde groups (pg. 1190, Coupling with labelled chelator; and Post-conjugation labeling). Additionally, Bansal teaches radiolabeling cells with a 89Zr radionuclide conjugated deferoxamine bifunctional chelator (pg. 10, third paragraph). Therefore, the combined teachings of Bansal, Baskin, and Pochon render claim 22 obvious.
Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over Bansal, Baskin, and Pochon, as applied to claims 1-3, 5, 7-8, 10, 12-14, and 18-22 above, and further in view of De Bank (De Bank, P. A.; et al., Biotechnol. Bioeng., 2003) and Kolmel (Kolmel, D. K.; and Kool, E. T., Chem. Rev., 2017 – provided by applicant in IDS filed November 19, 2024).
As described above, the combined teachings of Bansal, Baskin, and Pochon teach a method of radiolabeling cells for in vivo tracking wherein cell surface glycans are oxidized by periodate to generate aldehyde groups on sialic acid and the aldehyde-modified sialic acids are reacted with an aminooxy-deferoxamine bifunctional chelator to label the cells. Pochon also teaches that aminooxy groups readily react with aldehydes (pg. 1188, left column, last paragraph, lines 28-30). Additionally, Baskin teaches that such aldehydes may be targeted by oxime/hydrazone-forming reactions (pg. 10362, right column, third paragraph).
The combined teachings of Bansal, Baskin, and Pochon do not teach the use of a bifunctional chelator wherein the aldehyde/ketone-reactive group is a hydrazine group.
De Bank teaches a method of modifying a cell surface through periodate oxidation (pg. 800, Title and Abstract). More specifically, De Bank teaches periodate oxidation of cell surface sialic acids (pg. 801, right column, third paragraph). De Bank teaches further labeling the cells with biotin hydrazide (pg. 802, paragraphs 1 and 4). De Bank teaches that the biotin hydrazide is ligated to the non-native aldehydes on sialic acid groups formed by the periodate oxidation via covalent hydrazone bond (pg. 803, Results, first paragraph; and Figure 1). De Bank teaches using this biotin group to fluorescently label cells with FITC-avidin (pg. 802, first paragraph; and pg. 804, Figure 3).
Kolmel provides a review on oximes and hydrazones and their use in bioconjugation (pg. 10358, Abstract). Kolmel teaches that alkoxyamines and hydrazines can analogously be reacted with aldehydes or ketones to form oximes or hydrazones (pg. 10359, Scheme 1). Kolmel teaches that in order to produce oximes or hydrazones, biomolecules must contain either a carbonyl moiety or an α-nucleophile (pg. 10359, right column, paragraph 5). Kolmel teaches that periodate oxidation to generate aldehyde or ketone carbonyl groups is a widely used method (pg. 10359, right column, paragraph 4; and pg. 10360, Scheme 1 and Scheme 2). Kolmel teaches that the mechanism of formation of oximes from alkoxyamines and hydrazones from hydrazines via reaction with aldehydes or ketones is the same (pg. 10362, Scheme 9).
A person of ordinary skill in the art would have recognized that De Bank and Baskin both teach labeling cell surface sialic acid glycans via periodate oxidation and reaction with an aldehyde reactive group. It would also be recognized that Kolmel describes hydrazines and alkoxyamines as similarly reactive with aldehyde and ketone carbonyls on biomolecules.
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the aminooxy-deferoxamine cell surface labeling method taught by the combination of Bansal, Baskin, and Pochon by substituting the aminooxy group in the bifunctional chelator with a hydrazine group because this reactive group serves the same purpose in labeling oxidized sialic acid glycans (as taught by De Bank) (MPEP § 2143(I)(B)) and because Kolmel teaches that aminooxy groups and hydrazine groups are useful for the same types of reactions in bioconjugation (MPEP § 2143(I)(G)). This substitution would have predictably resulted in a method of labeling a cell for in vivo tracking using a hydrazine-modified chelator.
A person of ordinary skill in the art would have had a reasonable expectation of success in making this modification because Kolmel teaches that hydrazine and aminooxy groups can perform the same types of reactions using the same mechanism and are both suitable for reaction with oxidized sugar aldehydes. Additionally, De Bank teaches successfully using a hydrazine group to label cell surface sialic acid glycans. Furthermore, Baskin teaches that periodate oxidation of sialic acid generates and aldehyde that can be subsequently targeted by oxime or hydrazone forming reactions (pg. 10362, right column third paragraph).
The skilled artisan would have been motivated to make this substitution because De Bank teaches that hydrazine is an effective reactive group for labeling aldehyde modified cell surface glycans and because Kolmel teaches that a hydrazine group can perform the same labeling reaction as the aminooxy group.
Regarding claim 4, as described above, the combined teachings of Bansal, Baskin, and Pochon teach a method of labeling stem cells or white blood cells with deferoxamine through cell surface glycan oxidation by the addition of periodate oxidizing agent to form aldehydes and conjugation of an aminooxy-deferoxamine bifunctional chelator to the aldehyde groups to ligate the bifunctional chelator to the cell surface glycan to form a labeled cell for in vivo tracking. Additionally, Baskin teaches that such aldehydes may be targeted by oxime/hydrazone-forming reactions (pg. 10362, right column, third paragraph). Furthermore, Pochon (pg. 1890, paragraphs 3-5) teaches the preparation of aminooxy-deferoxamine. Pochon teaches that deferoxamine is a chelator (pg. 1188, right column, second paragraph, first sentence). Pochon also teaches that aminooxy groups readily react with aldehydes (pg. 1188, left column, last paragraph, lines 28-30). Additionally, De Bank teaches a method of labeling periodate oxidized sialic acid glycans with aldehyde groups with a hydrazide modified molecule (Figure 1). Kolmel teaches that aminooxy and hydrazide groups are similar in that they can react with aldehydes and ketones to form oximes or hydazones (Scheme 1). As described above, the substitution of the aminooxy group in the bifunctional chelator of the combination of Bansal, Baskin, and Pochon with the hydrazine group of De Bank and Kolmel is prima facie obvious. The resulting method would thus use a hydrazine-modified deferoxamine chelator for cell labeling. Therefore, the combined teachings of Bansal, Baskin, Pochon, De Bank and Kolmel render claim 4 obvious.
Claim 6 is rejected under 35 U.S.C. 103 as being unpatentable over Bansal, Baskin, and Pochon, as applied to claims 1-3, 5, 7-8, 10, 12-14, and 18-22 above, and further in view of Lu (Lu, D.; et al., J. Nanobiotechnol., 2021).
As described above, the combined teachings of Bansal, Baskin, and Pochon teach a method of radiolabeling cells for in vivo tracking wherein cell surface glycans are oxidized by periodate to generate aldehyde groups on sialic acid and the aldehyde-modified sialic acids are reacted with an aminooxy-deferoxamine bifunctional chelator to label the cells. Pochon also teaches that aminooxy groups readily react with aldehydes (pg. 1188, left column, last paragraph, lines 28-30). Additionally, Baskin teaches the periodate/aminooxy method of labeling native sialic acid groups on cell surface glycans as a nonmetabolic alternative to metabolic labelling of cells treated with N-azidoacetylmannosamine to generate azide-modified glycans to be reacted with an alkyne ring-linked labeling molecule (Figure 1; Figure 3; and pg. 10362, right column).
The combined teachings of Bansal, Baskin, and Pochon do not teach the use of a bifunctional chelator wherein the chelator is one of the chelators listed in claim 6.
Lu teaches metabolic radiolabeling and PET imaging of radiolabeled lymphocytes in vivo using azidosugars and dibenzocyclooctyne-modified NOTA (pg. 1, Title and Abstract; and pg. 3, Figure 1). Specifically, Lu teaches treating cells with N-azidoacetylmannosamine (pg. 10, right column, third paragraph) and subsequently reacting the cell surface azides with 64Cu-NOTA-DBCO (pg. 10-11, Metabolic radiolabeling of the CTLs with 64Cu; and Figure 1). Lu teaches using the 64Cu-NOTA labeled cells for PET imaging of injected cells in vivo (pg. 12, first paragraph; and pg. 5, Figure 3).
A person of ordinary skill in the art would have recognized that the metabolic azide-alkyne labeling method of Lu is nearly identical to the metabolic method of Baskin in that cells are treated with N-azidoacetylmannosamine to generate azide-presenting cell surface glycans and the azide is subsequently reacted with a cyclic alkyne for labeling. It would be recognized that Lu uses this technique to modify the cell surface with a radionuclide conjugated chelator for in vivo imaging. It would also be recognized that Baskin describes the nonmetabolic periodate/aminooxy labeling method as an alternative to the azide/alkyne metabolic method. In view of the rejections above, it would be recognized that the combined method of Bansal, Baskin, and Pochon is similar to that of Lu in that a radionuclide conjugated chelator is ligated to cell surface glycans.
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the aminooxy-deferoxamine cell surface labeling method taught by the combination of Bansal, Baskin, and Pochon by substituting the deferoxamine chelator in the bifunctional chelator with a NOTA chelator group as taught by Lu because this chelator group serves the same purpose in binding to radionuclides enabling in vivo imaging (as taught by Lu) (MPEP § 2143(I)(B)). This substitution would have predictably resulted in a method of labeling a cell for in vivo tracking using an aminooxy-modified NOTA chelator.
A person of ordinary skill in the art would have had a reasonable expectation of success in making this modification because Baskin teaches that metabolic azide/alkyne glycan labeling through the use of N-azidoacetylmannosamine is analogous to and an alternative to the periodate/aminooxy labeling method. Baskin teaches that the cyclic alkyne attached to an imaging label can be substituted for an aminooxy group (Figures 1, 3, and 4), allowing the same label to be attached to glycans in different ways. Additionally, Pochon and Bansal teach deferoxamine as a radionuclide chelator and Lu teaches NOTA as a radionuclide chelator. Thus, the skilled artisan would recognize that either the cyclic alkyne in the bifunctional NOTA could be substituted to be an aminooxy group or that the deferoxamine chelator of the combination of Bansal, Baskin, and Pochon could be substituted for the alternative radionuclide chelator NOTA.
The skilled artisan would have been motivated to make this substitution because Lu teaches NOTA as an effective chelator for the labeling of cell surface glycans for in vivo PET imaging. Furthermore, Lu teaches that NOTA can be used to bind 64Cu, a radionuclide useful for PET imaging.
Regarding claim 6, as described above, the combined teachings of Bansal, Baskin, and Pochon teach a method of labeling stem cells or white blood cells with deferoxamine through cell surface glycan oxidation by the addition of periodate oxidizing agent to form aldehydes and conjugation of an aminooxy-modified bifunctional chelator to the aldehyde groups to ligate the bifunctional chelator to the cell surface glycan to form a labeled cell for in vivo tracking. Additionally, Baskin teaches that the periodate/aminooxy labelling method is an alternative to metabolic N-azidoacetylmannosamine azide/alkyne labeling of glycans and that the label of one system can be used in combination with the reactive group of the other (pg. 10362, right column; and Figures 1 and 3). Lu teaches labeling cell surface glycans using an alkyne-modified NOTA chelator (Figure 1). As described above, it would have been prima facie obvious to substitute the deferoxamine chelator in the aminooxy-deferoxamine bifunctional chelator used in the combined method of Bansal, Baskin, and Pochon with the NOTA of Lu. This modification would result in the use of an aminooxy-modified NOTA chelator. Therefore, the combined teachings of Bansal, Baskin, Pochon, and Lu render claim 6 obvious.
Claim 15 is rejected under 35 U.S.C. 103 as being unpatentable over Bansal, Baskin, and Pochon, as applied to claims 1-3, 5, 7-8, 10, 12-14, and 18-22 above, and further in view of Zeng (Zeng, Y.; et al., Nat. Methods, 2009 – provided by applicant in IDS filed November 19, 2024; but as the examiner cites a Supplementary figure, the examiner has provided a copy with the supplemental materials appended).
As described above, the combined teachings of Bansal, Baskin, and Pochon teach a method of radiolabeling cells for in vivo tracking wherein cell surface glycans are oxidized by periodate to generate aldehyde groups on sialic acid, the aldehyde-modified sialic acids are reacted with an aminooxy-deferoxamine bifunctional chelator, and the chelator is conjugated to a radionuclide. Additionally, Baskin teaches that this labeling method did not result in any noticeable toxicity in the cells labeled with the periodate oxidation and aminooxy-label reaction (pg. 10363, left column, first paragraph). Baskin also teaches that the aminooxy-aldehyde ligation reaction can be performed in the presence of aniline, which increased cell surface fluorescence (pg. 10363, left column, first paragraph). Furthermore, Bansal teaches that 89Zr-conjugated deferoxamine chelator ligated cells showed 90-95% viability (pg. 2, last paragraph).
The combined teachings of Bansal, Baskin, and Pochon do not explicitly teach a cell viability of greater than 90% after generating aldehyde groups on cell surface glycans via reaction with an oxidizing agent.
Zeng teaches a method of labeling cell surface sialic acid containing glycans using an aminooxy modified label (pg. 207, Title and Abstract). More specifically, Zeng teaches oxidizing sialic acids on the cell surface with periodate to introduce aldehyde groups and subsequently reacting the aldehyde with aminooxy-biotin (pg. 207, right column, paragraphs 2 and 3; and pg. 208, Figure 1a). Zeng teaches that this ligation reaction can also be done in the presence of aniline, which significantly increased cell labeling (pg. 207, right column, paragraph 3, lines 1-7). Zeng teaches that the periodate, aminooxy-biotin, and aniline combination labeling method results in cell viability of ~88-93% (pg. 208, left column, second paragraph; and Supplementary Figure 3). Additionally, Zeng teaches that cell viability after incubation with periodate alone at 1 mM concentration at 4°C for 5 minutes is about 100% (Supplementary Figure 3).
A person of ordinary skill in the art would have recognized that Zeng and Baskin both teach labeling cell surface sialic acid glycans via periodate oxidation and reaction with an aminooxy-modified label. It would be recognized that Baskin teaches that no noticeable toxicity was observed for such a labeling reaction and Zeng teaches reaction conditions that quantifiably demonstrate low toxicity and high cell viability. It would also be recognized that both Baskin and Zeng teach that aniline can be used to catalyze the ligation reaction and that Zeng teaches that this does not significantly reduce cell viability.
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the aminooxy-deferoxamine cell surface labeling method taught by the combination of Bansal, Baskin, and Pochon by using the reaction conditions of Zeng because Zeng demonstrates that these conditions lead to high oxime formation labeling and has limited toxicity to cells (MPEP § 2143(I)(C) and 2143(I)(G)). This substitution would have predictably resulted in a method of labeling a cell for in vivo tracking wherein cell viability is greater than about 90% after periodate oxidation.
A person of ordinary skill in the art would have had a reasonable expectation of success in making this modification because the reactions performed by Baskin and Zeng are nearly identical in that periodate oxidized sialic acid glycans displaying aldehyde groups are reacted with an aminooxy-modified labeling group and this ligation may be performed with or without aniline catalyst.
The skilled artisan would have been motivated to make this modification because Zeng demonstrates that the reaction conditions allow for maintaining high cell viability and allow for high labeling efficiency.
Regarding claim 15, as described above, the combined teachings of Bansal, Baskin, and Pochon teach a method of labeling stem cells or white blood cells with deferoxamine through cell surface glycan oxidation by the addition of periodate oxidizing agent to form aldehydes and conjugation of an aminooxy-deferoxamine bifunctional chelator to the aldehyde groups to ligate the bifunctional chelator to the cell surface glycan to form a labeled cell for in vivo tracking. Additionally, Baskin teaches that such reactions may be performed in the presence of aniline (pg. 10363, left column, first paragraph). Furthermore, Zeng teaches that performing the oxidation reaction with 1 mM NaIO4 at 4°C for 5 minutes results in cell viability of about 100% (Supplementary Figure 3). The examiner interprets this to read on the viability of the cell population being greater than 90% after the generation of aldehyde groups on cell surface glycans via reaction with an oxidizing agent. The examiner notes that Zeng also teaches that further reactions with the aminooxy-biotin with or without aniline also results in a cell viability of at least ~88% (Supplementary Figure 3; and pg. 208, left column, second paragraph); which the examiner interprets to read on greater than about 90%, as the Applicant defines in [0052] of the instant specification that “about” can mean ±10%, which means “greater than about 90%” can mean greater than 81%. Therefore, the combined teachings of Bansal, Baskin, Pochon, and Zeng render claim 15 obvious.
Claims 16 and 17 are rejected under 35 U.S.C. 103 as being unpatentable over Bansal in view of Baskin, Pochon, and Gildehaus (Gildehaus, F. J.; et al., Mol. Img. Biol., 2011).
As described above, Bansal teaches a method of labeling cells with 89Zr and tracking them in vivo (pg. 1, Abstract). More specifically, Bansal teaches labels comprising 89Zr coordinated by deferoxamine (DFO) or Hy3ADA5 chelators conjugated to isothiocyanato-benzyl or squaramide ester groups (pg. 3, Figure 1). Bansal teaches complexing 89Zr in these chelating groups (pg. 4, Figure 2; and pg. 9, last paragraph through pg. 10 first paragraph). Bansal teaches labeling mesenchymal stem cells and white blood cells with these radiolabeled chelating groups (pg. 4, Figures 3 and 4; and pg. 10, paragraph 3). Bansal teaches using these radiolabeled cells to track cells in vivo (pg. 5, Figure 5; pg. 7, Figure 8; and pg. 10, paragraph 6). Specifically, Bansal teaches injecting radiolabeled cells into the tail vein of mice and performing PET imaging (pg. 10, paragraph 6). Bansal teaches that this method is an effective means of using PET-based cell radiolabeling to noninvasively track cells that can be used to understand the safety, efficacy, distribution, and clearance of cell-based therapies (pg. 8, last paragraph). Additionally, Bansal teaches that PET and SPECT are advantageous imaging modalities over other techniques and are often integrated with MRI or CT to provide quantitative and temporal distributions of cells in vivo (pg. 1, Introduction, second paragraph).
Bansal does not teach radiolabeled cells made by the generation of aldehyde groups on cell surface glycans, ligation of a bifunctional chelator to the aldehydes, and conjugation of a radionuclide to the chelator or imaging radiolabeled cells in vivo by PET/CT, SPECT/CT, or Planar scan of a subject.
As described above, Baskin teaches a method of fluorescently imaging cells for imaging in vivo (pg. 10360, Abstract). Specifically, Baskin teaches methods of labeling sialic acid and sialic acid derivatives with fluorophores (pg. 10361, Figure 1; and pg. 10363, Figure 3). In the nonmetabolic approach, Baskin teaches oxidizing sialylated cell surface glycans with sodium periodate to modify the sialic acid to have aldehyde groups and subsequently reacting the cells with an aminooxy-derivatized fluorophore (pg. 10362, right column, third paragraph; and Figure 3). Baskin teaches that this approach for tagging sialylated glycans did not result in any noticeable toxicity (pg. 10363, left column, first paragraph). Baskin teaches that this nonmetabolic approach is an effective means of labeling cells for imaging (Figure 3; and pg. 5, right column, second paragraph).
As described above, Pochon teaches the labeling of an antibody with an aminooxyacetyl deferoxamine chelator and radiolabeling with 67Ga (pg. 1188, Title and Abstract). Pochon teaches the synthesis of this chelator by the addition of an aminooxyacetyl group to ferrioxamine (pg. 1189. Left column, fourth paragraph) followed by removal of the chelated iron (pg. 1189, right column, fourth paragraph) and labeling with 67Ga (pg. 1189-1190, Labelling of the chelator). Pochon also teaches the oxidation of an antibody with periodate (pg. 1189, Oxidation) and teaches that periodate oxidation is known to generate aldehyde groups on oligosaccharides on antibodies (pg. 1188, left column, last paragraph, lines 15-18) and that aminooxy groups are known to be reactive with aldehydes (pg. 1188, left column, last paragraph, lines 26-29). Pochon teaches conjugating the oxidized antibody with the 67Ga-labeled aminooxy deferoxamine (pg. 1190, Coupling with labelled chelator) and with the unlabeled aminooxy deferoxamine followed by labeling of the ligated complex (pg. 1190, Post-conjugation labelling). Pochon teaches using the 67Ga-aminooxy deferoxamine-labeled antibody conjugates for in vivo distribution in mice after i.v. injection (pg. 1190, Imaging study; and pg. 1192, Figure 6). Pochon teaches that this radionuclide labeling through an aminooxy deferoxamine chelator reduced liver and spleen accumulation of radiolabeled antibody compared to chloramine-T 125I labeled antibodies, providing an advantage for the taught periodate oxidation method (pg. 1191, In vivo testing of the radiolabelled conjugates).
Gildehaus teaches a method of in vivo tracking of radiolabeled cells (pg. 1204, Title and Abstract). Specifically, Gildehaus teaches radiolabeling mesenchymal stem cells (pg. 1205, left column, last paragraph) with 111In-oxine (pg. 1205, 111In-oxine Labelling). Gildehaus teaches using these radiolabeled cells for in vivo SPECT/CT imaging (pg. 1207, left column, third paragraph; and pg. 1212, Figure 8). Gildehaus teaches that this method is effective at monitoring transplanted cells by serial SPECT/CT imaging (pg. 1213, first paragraph).
A person of ordinary skill in the art would have recognized that both Bansal and Gildehaus teach methods of radiolabeling cells for in vivo tracking. It would also be recognized that while Bansal teaches using PET imaging, Bansal describes PET/CT, SPECT/CT, and PET/MRI as advantageous imaging methods for tracking cells in vivo. As described above, it would also be recognized that both Bansal and Baskin teach methods of labeling the surface of cells with a label for in vivo imaging. It would also be recognized that Baskin and Pochon teach labeling of biomolecules using the same periodate oxidation followed by reaction with an aminooxy conjugated labeling group and using the labeled materials for in vivo imaging. Therefore, it would be understood that the method of Baskin could be used to radiolabel cells with the bifunctional chelator of Pochon. It would further be recognized that both Bansal and Pochon teach covalently labeling biomolecules with the same deferoxamine chelator (only differing in the attachment linker group) bound to a radionuclide for in vivo imaging applications.
As described above, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the cell labeling method of Bansal by substituting the protein labeling with to isothiocyanato-benzyl-deferoxamine with the periodate sialic acid oxidation and aminooxy-deferoxamine method taught by the combination of Baskin and Pochon because these methods are alternative means of labeling cells for in vivo imaging applications (MPEP § 2143(I)(B)). This substitution would predictably result in a method of radiolabeling cells in which a cell surface glycan is oxidized to produce an aldehyde and a bifunctional chelator ligates to the cells by reacting with the aldehyde.
Additionally, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of imaging radiolabeled cells of Bansal by using 111In as the radionuclide and performing SPECT/CT as taught by Gildehaus because this is an alternative imaging technique that enables similar tracking of cells in vivo (MPEP § 2143(I)(B)) and because Bansal teaches that combination methods such as SPECT/CT have advantages over PET alone due to the anatomical information provided by the CT component (pg. 1, Introduction, second paragraph) (MPEP § 2143(I)(G)). This modification would predictably result in a method of tracking and imaging a radiolabeled cell in vivo using SPECT/CT.
As described above, a person of ordinary skill in the art would have had a reasonable expectation of success in modifying the cell labeling method of Bansal by substituting the isothiocyanato method for the periodate/aminooxy method because Baskin teaches that periodate oxidation of cell surface glycan sialic acids is an effective means of attaching imaging labels to cells. Additionally, Pochon teaches that the periodate oxidation of sugars can be effectively used to label biomolecules with a radiolabeled deferoxamine chelator modified with an aminooxy group. Furthermore, a person of ordinary skill in the art would have had a reasonable expectation of success in modifying the imaging method of Bansal with the SPECT/CT imaging as taught by Gildehaus because Bansal teaches that deferoxamine can chelate 89Zr and Pochon teaches that deferoxamine can chelate 67Ga. Therefore, it would be expected that deferoxamine could chelate the 111In radionuclide of Gildehaus. Additionally, Gildehaus teaches that 111In labeled cells are effective at in vivo imaging and tracking cells.
As described above, the skilled artisan would have been motivated to use the labeling method of Baskin and Pochon because the periodate oxidation and aminooxy-label conjugation system labels glycans instead of amino acid sidechains of proteins, which could prevent any negative effects of direct protein modification on protein structure and function. Furthermore, Pochon teaches that the aldehyde conjugation system is compatible with radiolabeling both before and after conjugation whereas Bansal only teaches radionuclide binding before chelator conjugation; thus, the modified method may provide more flexibility the method of preparation of labeled cells. Additionally, the skilled artisan would have been motivated to use SPECT/CT imaging for in vivo tracking because Bansal teaches that combination of SPECT and CT is advantageous over PET or SPECT alone because it provides both quantitative imaging of radiolabeled cell distribution and anatomical information together.
Regarding claim 16, as described above (see rejections of claims 14 and 18), the combined teachings of Bansal, Baskin, and Pochon render claim 14 and thus the radiolabeled cells produced by the method obvious. Additionally, Bansal teaches administration of radiolabeled cells by injection into the tail vein of mice (pg. 10, paragraph 6). Furthermore, Gildehaus teaches tracking and imaging radiolabeled cells in a subject by SPECT/CT (Figure 8). Therefore, the combined teachings of Bansal, Baskin, Pochon, and Gildehaus render claim 16 obvious.
Regarding claim 17, Bansal teaches administration of radiolabeled cells by injection into the tail vein of mice (pg. 10, paragraph 6). The examiner interprets this to be intravenous administration of radionuclide labeled cells. Therefore, the combined teachings of Bansal, Baskin, Pochon, and Gildehaus render claim 17 obvious.
Pertinent Art
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
As pertinent art, the examiner cites Kang (Kang, S. W.; et al., Theranostics, 2014). Kang teaches a method of labeling sialic acid glycans on the surface of cells with a fluorophore and performing in vivo tracking imaging of the labeled cells (pg. 420, Title and Abstract). More specifically, Kang teaches metabolic labeling of cells by incubation with N-azidoacetylmannosamine, generating azide-modified sialic acid glycans, and reacting the azide groups with a cyclic alkyne-modified fluorescent dye (pg. 421, Figure A). Kang teaches administering labeled cells into a mouse and using fluorescence to track the location of the cells in vivo (pg. 427, Figure 6). The examiner notes that in view of this reference, the Baskin reference above is understood to belong in the art of in vivo cell tracking and imaging.
As pertinent art, the examiner cites Perk (Perk, L. R.; et al., Eur. J. Nucl. Med. Mol. Imaging, 2010). Perk teaches labeling antibodies with 89Zr-conjugated p-isothiocyanatobenzyl-deferoxamine and performing PET imaging (pg. 250, Title and Abstract; pg. 251, right column, fourth paragraph; pg. 252, left column, second paragraph; pg. 253, Evaluation of in vivo biodistribution; pg. 253, Figure 1; and pg. 256, Figure 5). The examiner notes that in view of Perk and Pochon (as cited in above rejections), it can be considered that periodate oxidation coupled with aminooxy chelator conjugation (Pochon) and isothiocyanato-benzyl conjugation(Perk) are alternative labeling methods of biomolecules.
As pertinent art, the examiner cites Gahmberg (Gahmberg, C. G. and Andersson, L. C., J. Biol. Chem., 1977). Gahmberg teaches a method of radiolabeling cells by first reacting cells with sodium metaperiodate and subsequently adding tritiated sodium borohydride (pg. 5889, right column, first paragraph). Gahmberg teaches that this method results in rapid oxidation of sialic acids (pg. 5889, right column, paragraph 5). Gahmberg teaches that this radiolabeling method has “obvious advantages” including cheap reagents, short reaction times, and the use of chemical probes instead of enzymes (pg. 5894, left column, third paragraph). The examiner notes that Gahmberg demonstrates that it has been understood since at least 1977 that cells can be radiolabeled through sialic acid oxidation by periodate and subsequent reactions with the generated aldehyde groups.
Conclusion
No claim is allowed.
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/E.P.M./Examiner, Art Unit 1612
/SAHANA S KAUP/Supervisory Primary Examiner, Art Unit 1612
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