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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on Sept. 2, 2025, has been entered. Claims 31-40 have been newly added. Claims 5, 7, 15, and 19-30 have been canceled. Claims 1-4, 6, 8-14, 16-18, and 31-40 are pending.
Supplemental Restriction
Newly added claims 31-40 introduce new inventions and non-elected species, and this requires a supplemental restriction requirement for the newly added claims.
Newly restricted inventions starting where the original restriction left off:
XVIII. Claim 31, drawn to a genetically modified plant comprising at least one cell expressing simultaneously at least two milk proteins from a cow, the at least two milk proteins selected from the group consisting of serum albumin, alpha-S1-casein, alpha-S2-casein, beta-casein, kappa-casein, beta-lactoglobulin, and alpha-lactalbumin wherein expression of each of said milk proteins is independently under control of a promoter suitable to achieve a weight-to-weight (w/w) content of each of said proteins such that, for two expressed milk proteins, a proportion of a relative abundance of the first protein to the second protein in the plant is at least 70% of the corresponding proportion of the same proteins in the liquid milk of said cow, thereby facilitating imitation of the protein content of the milk of said cow, wherein the two milk proteins is achieved using a single vector comprising multiple expression cassettes encoding the two proteins, classified in C12N 15/63, for example.
XIX. Claims 1 and 34, drawn to a genetically modified plant comprising at least one cell expressing simultaneously at least two milk proteins from a cow, the at least two milk proteins selected from the group consisting of serum albumin, alpha-S1-casein, alpha-S2-casein, beta-casein, kappa-casein, beta-lactoglobulin, and alpha-lactalbumin wherein expression of each of said milk proteins is independently under control of a promoter suitable to achieve a weight-to-weight (w/w) content of each of said proteins such that, for two expressed milk proteins, a proportion of a relative abundance of the first protein to the second protein in the plant is at least 70% of the corresponding proportion of the same proteins in the liquid milk of said cow, thereby facilitating imitation of the protein content of the milk of said cow, wherein the proportion of kappa casein/alpha S2 casein expressed in the plant is at least 70% of the kappa casein/alpha S1 casein proportion in the milk of the cow, classified in A61K 38/018, for example.
XX. Claims 32 and 35, drawn to a genetically modified plant comprising at least one cell expressing simultaneously at least two milk proteins from a cow, the at least two milk proteins selected from the group consisting of serum albumin, alpha-S1-casein, alpha-S2-casein, beta-casein, kappa-casein, beta-lactoglobulin, and alpha-lactalbumin wherein expression of each of said milk proteins is independently under control of a promoter suitable to achieve a weight-to-weight (w/w) content of each of said proteins such that, for two expressed milk proteins, a proportion of a relative abundance of the first protein to the second protein in the plant is at least 70% of the corresponding proportion of the same proteins in the liquid milk of said cow, thereby facilitating imitation of the protein content of the milk of said cow, wherein there are more than two milk proteins; including wherein they are alpha S1 casein, alpha S2 casein, and kappa casein, classified in C07K 14/4732, for example.
XXI. Claims 32, and 39, drawn to a genetically modified plant comprising at least one cell expressing simultaneously at least two milk proteins from a cow, the at least two milk proteins selected from the group consisting of serum albumin, alpha-S1-casein, alpha-S2-casein, beta-casein, kappa-casein, beta-lactoglobulin, and alpha-lactalbumin wherein expression of each of said milk proteins is independently under control of a promoter suitable to achieve a weight-to-weight (w/w) content of each of said proteins such that, for two expressed milk proteins, a proportion of a relative abundance of the first protein to the second protein in the plant is at least 70% of the corresponding proportion of the same proteins in the liquid milk of said cow, thereby facilitating imitation of the protein content of the milk of said cow, wherein there are more than two milk proteins; including wherein they are alpha S1 casein, alpha S2 casein, and beta casein, classified in A23C 11/08, for example.
Claim 32 links XX and XXI
XXII. Claims 1 and 36, drawn to a genetically modified plant comprising at least one cell expressing simultaneously at least two milk proteins from a cow, the at least two milk proteins selected from the group consisting of serum albumin, alpha-S1-casein, alpha-S2-casein, beta-casein, kappa-casein, beta-lactoglobulin, and alpha-lactalbumin wherein expression of each of said milk proteins is independently under control of a promoter suitable to achieve a weight-to-weight (w/w) content of each of said proteins such that, for two expressed milk proteins, a proportion of a relative abundance of the first protein to the second protein in the plant is at least 70% of the corresponding proportion of the same proteins in the liquid milk of said cow, thereby facilitating imitation of the protein content of the milk of said cow, wherein the proportion of alpha S2 casein/alpha S1 casein expressed in the plant is at least 70% of the kappa casein/alpha S1 casein proportion in the milk of the cow, classified in A61K 38/018, for example.
XXIII. Claims 1 and 37, drawn to a genetically modified plant comprising at least one cell expressing simultaneously at least two milk proteins from a cow, the at least two milk proteins selected from the group consisting of serum albumin, alpha-S1-casein, alpha-S2-casein, beta-casein, kappa-casein, beta-lactoglobulin, and alpha-lactalbumin wherein expression of each of said milk proteins is independently under control of a promoter suitable to achieve a weight-to-weight (w/w) content of each of said proteins such that, for two expressed milk proteins, a proportion of a relative abundance of the first protein to the second protein in the plant is at least 70% of the corresponding proportion of the same proteins in the liquid milk of said cow, thereby facilitating imitation of the protein content of the milk of said cow, wherein the proportion of beta-Lactoglobulin/alpha S1 casein expressed in the plant is at least 70% of the kappa casein/alpha S1 casein proportion in the milk of the cow, classified in A61K 38/38, for example.
XXIV. Claims 1 and 38, drawn to a genetically modified plant comprising at least one cell expressing simultaneously at least two milk proteins from a cow, the at least two milk proteins selected from the group consisting of serum albumin, alpha-S1-casein, alpha-S2-casein, beta-casein, kappa-casein, beta-lactoglobulin, and alpha-lactalbumin wherein expression of each of said milk proteins is independently under control of a promoter suitable to achieve a weight-to-weight (w/w) content of each of said proteins such that, for two expressed milk proteins, a proportion of a relative abundance of the first protein to the second protein in the plant is at least 70% of the corresponding proportion of the same proteins in the liquid milk of said cow, thereby facilitating imitation of the protein content of the milk of said cow, wherein the proportion of alpha S2 casein/kappa casein expressed in the plant is at least 70% of the kappa casein/alpha S1 casein proportion in the milk of the cow, classified in C12N 15/8257, for example.
XXV. Claim 40, drawn to a genetically modified plant comprising at least one cell expressing simultaneously at least two milk proteins from a cow, the at least two milk proteins selected from the group consisting of serum albumin, alpha-S1-casein, alpha-S2-casein, beta-casein, kappa-casein, beta-lactoglobulin, and alpha-lactalbumin wherein expression of each of said milk proteins is independently under control of a promoter suitable to achieve a weight-to-weight (w/w) content of each of said proteins such that, for two expressed milk proteins, a proportion of a relative abundance of the first protein to the second protein in the plant is at least 70% of the corresponding proportion of the same proteins in the liquid milk of said cow, thereby facilitating imitation of the protein content of the milk of said cow, wherein at least five proteins are expressed: alpha S1 casein, alpha S2 casein, beta casein, kappa-casein, and beta-lactoglobulin, classified in C07K 16/04, for example.
Claim 32 link(s) inventions XX and XXI. The restriction requirement between the linked inventions is subject to the nonallowance of the linking claim(s), claim 32. Upon the indication of allowability of the linking claim(s), the restriction requirement as to the linked inventions shall be withdrawn and any claim(s) depending from or otherwise requiring all the limitations of the allowable linking claim(s) will be rejoined and fully examined for patentability in accordance with 37 CFR 1.104. Claims that require all the limitations of an allowable linking claim will be entered as a matter of right if the amendment is presented prior to final rejection or allowance, whichever is earlier. Amendments submitted after final rejection are governed by 37 CFR 1.116; amendments submitted after allowance are governed by 37 CFR 1.312.
Applicant(s) are advised that if any claim presented in a divisional application is anticipated by, or includes all the limitations of, the allowable linking claim, such claim may be subject to provisional statutory and/or nonstatutory double patenting rejections over the claims of the instant application. Where a restriction requirement is withdrawn, the provisions of 35 U.S.C. 121 are no longer applicable. In re Ziegler, 443 F.2d 1211, 1215, 170 USPQ 129, 131-32 (CCPA 1971). See also MPEP § 804.01.
Inventions XVIII-XXV are directed to related products. The related inventions are distinct if: (1) the inventions as claimed are either not capable of use together or can have a materially different design, mode of operation, function, or effect; (2) the inventions do not overlap in scope, i.e., are mutually exclusive; and (3) the inventions as claimed are not obvious variants. See MPEP § 806.05(j). In the instant case, the inventions as claimed each have a different design and effect which has been shown in bold in the descriptions for each group, above. Furthermore, the inventions as claimed do not encompass overlapping subject matter and there is nothing of record to show them to be obvious variants.
Inventions XX and XXI are related to Invention II (claim 1) as combinations and a subcombination. Inventions in this relationship are distinct if it can be shown that (1) the combination as claimed does not require the particulars of the subcombination as claimed for patentability, and (2) that the subcombination has utility by itself or in other combinations (MPEP § 806.05(c)). In the instant case, the combination as claimed does not require the particulars of the subcombination as claimed because the subcombination of claim 1 can be any combination of two of the recited proteins; whereas the combinations claimed in invention XX and XXI require a specific list of three proteins. The subcombination has separate utility such as the utility of claim 1, a transgenic plant expressing two milk proteins which can be used for plant-based milk substitutes.
Inventions XXV is related to Inventions XX and XXI as combination and subcombinations. Inventions in this relationship are distinct if it can be shown that (1) the combination as claimed does not require the particulars of the subcombination as claimed for patentability, and (2) that the subcombination has utility by itself or in other combinations (MPEP § 806.05(c)). In the instant case, the combination as claimed does not require the particulars of the subcombinations as claimed because the combination only requires the 70% proportion for one of three different choices of protein pairs without, specifically, requiring the proportion associated with one particular subcombination. The subcombinations have separate utilities such as the utility of claim 35 or claim 39, a transgenic plant expressing three milk proteins which can be used for plant-based milk substitutes.
The examiner has required restriction between combination and subcombination inventions. Where applicant elects a subcombination, and claims thereto are subsequently found allowable, any claim(s) depending from or otherwise requiring all the limitations of the allowable subcombination will be examined for patentability in accordance with 37 CFR 1.104. See MPEP § 821.04(a). Applicant is advised that if any claim presented in a divisional application is anticipated by, or includes all the limitations of, a claim that is allowable in the present application, such claim may be subject to provisional statutory and/or nonstatutory double patenting rejections over the claims of the instant application.
Restriction for examination purposes as indicated is proper because all these inventions listed in this action are independent or distinct for the reasons given above and there would be a serious search and examination burden if restriction were not required because one or more of the following reasons apply:
(a) the inventions have acquired a separate status in the art in view of their different classification;
(b) the inventions require a different field of search (for example, searching different classes/subclasses or electronic resources, or employing different search queries); and,
(c) the inventions are likely to raise different non-prior art issues under 35 U.S.C. 101 and/or 35 U.S.C. 112, first paragraph.
Claims 1-4, 6, 8-14, 16-18, and 31-40 are pending. Claims 2-4, 6, 8-14, 16-18, and 31, 32, and 34-40 are withdrawn. Claims 1 and 33 are examined in this Office Action.
Objections and Rejections That Are Withdrawn
The objection to claim 1 is withdrawn in light of Applicant’s amendments to the claims.
Claim Interpretation
Claim 1, as amended, is interpreted to require expression of two recombinant milk proteins in a plant wherein the expression level of each of the two proteins reported as g particular protein/100 g harvested plant tissue results in a proportion of a relative abundance of the first milk protein to the second milk protein in the plant of at least 70% of the corresponding proportion of the same proteins expression levels in cow’s milk. For example, if the first protein is alpha S1 casein and the second protein is alpha S2 casein, and if the cow’s milk being compared to were the cow’s milk described in Table 7 of the specification (Spec 172), then the proportion in the cow’s milk is 28/14 or 2. In this case, the plant would need to express alpha S1 casein and alpha S2 casein at levels that would result in a proportion of at least 1.4 to be “at least 70%”.
Claim 1 concludes with “thereby facilitating imitation of the protein content of the milk of said cow”, and this is interpreted to merely reflect the fact that the “at least two milk proteins” are expressed in the plant in a ratio that is at least 70% of the ratio between the same two proteins that is found in cow’s milk.
Claim Rejections - 35 USC § 112
Indefiniteness
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 and 33 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. All dependent claims are included in these rejections unless they include a limitation that overcomes the deficiencies of the parent claim.
Claim 1 requires that a proportion of a relative abundance of a first milk protein to a second milk protein is at least 70% of the corresponding proportion of the same proteins in the liquid milk of a cow. This causes indefiniteness because the relative abundance of different milk proteins differs from one cow to another (Roin et al. J. Dairy Sci. (2022) Vol. 105; pp. 1014-1027; especially page 1022), and differs seasonally throughout the year (Timlin et al. Foods (2021) Vol. 10; pp. 1-32; especially page 13), and differs due to the stage of lactaction and other environmental factors (McDermott et al. J. Dairy Sci. (2016) Vol. 100; pp. 6272-6284; especially pp. 6277-6278).
For example, the instant specification shows a ratio of alpha S1 casein proportion/alpha S2 casein proportion in cow to be 2.0 (Spec 172, ¶ 444, Table 7). However, Roin found the ratio of alpha S1 casein proportion/alpha S2 casein proportion in different breeds of cows to be different: Doela (DF) 4.8, Norwegian Red cattle (NRF) 4.0, Eastern Red Polled cattle (ORA) 4.4, Blacksided Tronder and Nordland cattle (STN) 4.6, Telemark cattle (TF) 4.0, Western Fjord (VFF) 4.4, Western Red Polled (VR) 4.0 (Roin 1022, Table 6). Similarly, Roin found the ratio of kappa casein/alpha S1 casein in the same cow varieties to be 0.25, 0.22, 0.27, 0.27, 0.28, 0.27, and 0.29, respectively; whereas Table 7 from the specification found the ratio of kappa casein/alpha S1 casein to be 0.32.
Because the relative proportion changes from cow to cow and changes due to other environmental factors, the requirement that the plant will have a ratio that is at least 70% of the ratio is undefined, and this causes the claim to be indefinite.
Lack of Written Description – NEW MATTER
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.
Claims 1 and 33 are 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. All dependent claims are included in these rejections unless they include a limitation that overcomes the deficiencies of the parent claim.
Claim 1 has been to recite “a proportion of a relative abundance of a first milk protein to a second milk protein is at least 70% of the corresponding proportion of the same proteins in the liquid milk of a cow”, and this introduces NEW MATTER. Claim 33 comprises the same NEW MATTER issue.
The specification contains the following statement about relative protein content: ““relative protein content” of a protein may encompass a proportion (or percentage) of that specific protein within the total protein measured” (Spec 5 ¶ 16). This makes it clear that “relative protein content” can be the amount of that particular protein as a percentage of total protein. All references in the specification to “at least 70%” that are directed to protein content are in the context of the percentage of total protein (Id. 30 ¶ 84). The specification even provides a sample calculation showing that if alpha-S1-casein is present in cows’ milk at the level of 38% of total protein, then “at least 70%” would mean at least 26% of total protein (Id. ¶ 85).
The instant claim is applying the limitation of “at least 70%” to the proportion or ratio of relative abundance between two proteins. For example, Table 7 shows alpha-S1-casein at 28% of total protein and alpha-S2-casein at 14% of total protein (Spec 172), therefore, the proportion or ratio of alpha-S1-casein to alpha-S2-casein would be 2. At least 70% of 2 is 1.4. There is nothing in the originally filed specification, abstract, drawings, or claims to support applying “at least 70%” to the ratio. It is only applied to the percent of total protein. For this reason, this limitation is NEW MATTER.
Inadequate Written Description for Breadth of Claims
Claims 1 and 33 are 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.
The claims are broadly drawn to a genetically modified plant comprising at least one cell expressing simultaneously at least two milk proteins from a cow, the at least two milk proteins selected from the group consisting of serum albumin, alpha-S1-casein, alpha-S2-casein, beta-casein, kappa-casein, beta-lactoglobulin, and alpha-lactalbumin wherein expression of each of said milk proteins is independently under control of a promoter suitable to achieve a weight-to-weight (w/w) content of each of said proteins such that, for two expressed milk proteins, a proportion of a relative abundance of the first protein to the second protein in the plant is at least 70% of the corresponding proportion of the same proteins in the liquid milk of said cow, thereby facilitating imitation of the protein content of the milk of said cow (claim 1); including wherein the proportion of kappa casein/alpha S1 casein expressed in the cow is at least 70% of the kappa casein/alpha S1 casein in the milk of a cow (claim 33).
Applicant describes N. benthamiana plants transfected with 7 different plasmids for transient expression of serum albumin (ALB), alpha-S1-casein (CSN1S1), alpha-S2-casein (CSN1S2), beta casein (CSN2), kappa-casein (CSN3), beta-lactoglobulin (LGB or LACB), and alpha-lactalbumin (LALBA) (Spec 126-133). Applicant describes seven T-DNA binary vectors each expressing one of these seven cow’s milk proteins (Id. 134). Expression of five of the seven proteins was detected by LC-MS/MS in the N. benthamiana leaves; the five proteins were CSN1S1, ALB, CSN2, LALBA, and LGB (Id. 155-6). These plants were only expressing one milk protein.
Applicant has described the construction of a single T-DNA binary vector comprising expression cassettes for all seven of the milk proteins under the control of the constitutive S1PUbq10 promoters, and they refer to this vector as pDGB-W1 (Spec 156, Example 4). Applicant describes transformation of Nicotiana benthamiana, Oryza sativa, and Glycine max (soybean) with this vector (Id.).
Applicant describes using pDGB-W1 vector for transfection of Nicotiana benthamiana, rice, and soybean (Spec 170, Examples 5-7). In transgenic soybean production of three milk proteins (CSN2 (b casein), LALBA (a-lactalbumin), and LGB (b-lactoglobulin)) was confirmed (Id. 171). Approximately 40 independent transgenic soybean lines were generated, and some lines expressed multiple milk proteins in their leaves (Fig. 6). Six transgenic lines were shown to express more than one of the milk proteins in their seeds with line #9 showing significant expression of CSN1S1, CSN2, CSN3, and CSN1S2, and showing low expression of LALBA (Spec 171, Figure 7).
Applicant describes sequences for six seed-specific promoters chosen for expression of the seven milk proteins in soybean seeds with “relative predicted abondance” (Spec 172-6). Applicant describes a plan to use CRISPR/Cas9 technology to knock out the expression of endogenous soybean genes encoding seed proteins (Id. 176) and endogenous FAD2-1A, FAD2-1B and SACPD-C genes (Id. 177).
Applicant describes the relative abundance in cow’s milk for seven of the milk proteins relative to the total of those seven (Spec 172, Table 7). Using the data in this table to calculate the proportion of relative abundance for the five proteins that are shown to be expressed in Figure 7, the following proportions/ratios are determined:
CSN1S1: 28% CSN2: 34% CSN3: 9% CSN1S2: 14% LALBA: 5%
CSN1S1 (a-S1-casein): 28% 1.00
CSN2 (b casein): 34% 0.82 1.00
CSN3 (k-casein): 9% 3.11 3.78 1.oo
CSN1S2 (a-S2-casein): 14% 2.00 2.43 0.64 1.00
LALBA (a-lactalbumin): 5% 5.6 6.8 1.80 2.80 1.00
The specification describes the amount of protein in the seeds of these transgenic soybeans as being from 1% to 34%. Looking at Figure 7, the Examiner will approximate the ratios assuming that the values on the Y-axis are linear. Here is Figure 7:
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For transgenic plant #9, it seems to have approximately 33% CSN1S1, 6% CSN2, 34% CSN3, and 34% CSN1S2.
The CSN1S1/CSN2 ratio appears to be about 5, but in cow’s milk from Table 7, this should be 0.82.
The CSN1S1/CSN3 ratio is about 1.0, but in cow’s milk this should be 3.11.
The CSN1S1/CSN1S2 ratio is about 1.0, but in cow’s milk this should be 2.0.
The same calculation can be performed for each of the six lines described in Figure 7. None of these meet the limitation required by claim 1 because the relative abundances are so dramatically different when comparing expression in the soybeans to expression in the cow of Table 7.
This data analysis shows that Applicant was not in possession of the claimed invention at the time of filing. Furthermore, Applicant has not described any particular combination of promoter, coding sequence, and plant species that will provide the necessary structures to confer the function of providing the required proportion of relative abundance. For these reasons, the instant application does not provide an adequate written description to support the breadth of the claims.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claim(s) 1 and 33 are rejected under 35 U.S.C. 103 as being unpatentable over El-Richani et al. (US Pre-Grant Publication US 2018/0291392; published on Oct. 11, 2018) in view of Mayfield et al. (US Pre-Grant Publication, US 2016/0257730; published on Sept. 8, 2016), further in view of Huang et al. (US Pre-Grant Publication US 2004/0111766, published on June 10, 2004), and further in view of Peach et al. (Plant Molecular Biology (1991) Vol. 17; pp. 49-60). This rejection has been modified to include Peach to address the amendments to the claims. Applicant’s arguments in the response received on Sept. 2, 2025, have been considered and were not found to be persuasive.
The claims are directed to a genetically modified plant comprising at least one cell expressing simultaneously at least two milk proteins from a cow, the at least two milk proteins selected from the group consisting of serum albumin, alpha-S1-casein, alpha-S2-casein, beta-casein, kappa-casein, beta-lactoglobulin, and alpha-lactalbumin wherein expression of each of said milk proteins is independently under control of a promoter suitable to achieve a weight-to-weight (w/w) content of each of said proteins such that, for two expressed milk proteins, a proportion of a relative abundance of the first protein to the second protein in the plant is at least 70% of the corresponding proportion of the same proteins in the liquid milk of said cow, thereby facilitating imitation of the protein content of the milk of said cow (claim 1); including wherein the proportion of kappa casein/alpha S1 casein expressed in the cow is at least 70% of the kappa casein/alpha S1 casein in the milk of a cow (claim 33).
El-Richani teaches transgenic plants expressing bovine milk protein(s) by introducing a recombinant DNA construct expressing a bovine milk protein into a plant (El-Richani, abstract). El-Richani uses the Bos taurus (cow) protein sequence when they are referring a “bovine” sequence (Id. 8 ¶¶ 79-82).
El-Richani claims a transgenic plant expressing alpha-S1-casein, alpha-S2-casein, beta-casein, kappa-casein, alpha-lactalbumin, beta-lactoglobulin, serum albumin, lactoferrin, lysozyme, lactoperoxidase, immunoglobulin-A, and lipase (Id. 83, claim 1). This list of proteins claimed by El-Richani includes all seven of the proteins recited in the instant claim 1 with five additional milk proteins.
El-Richani claims transgenic plants comprising expression constructs that utilize codon-optimized sequences, and specifically claims expression of alpha-S1 casein and alpha-S2 casein (Id., claims 8 and 9). El-Richani claims this plant wherein “the bovine milk protein comprises alpha-S1-casein, alpha-S2-casein, beta-casein, kappa-casein, alpha-lactalbumin, beta-lactoglobulin, and lysozyme” (Id., claim 21; emphasis added), and this appears to be claiming co-expression of seven milk proteins together. El-Richani claims a method of producing such a transgenic plant that involves introducing “at least one expression cassette” capable of expressing a bovine milk protein (Id. 84, claim 24).
El-Richani does not reduce to practice co-expression of two of the seven proteins recited in the instant claim 1 each under the control of an independent promoter, nor does El-Richani teach specific yields of protein expressed as grams of protein per 100 grams of plant tissue or calculate proportions/ratios between the different proteins.
Huang teaches expressing milk proteins in transgenic monocot seeds, and claims alpha-casein (Huang 71, claim 2); and claims 3%, 5%, 10%, and 20% total soluble protein (Id. claims 1 and 13-15). Huang reduces to practice the production of lysozyme with up to 50% total soluble protein (TSP) (Id. 8, Figure 7).
Huang teaches and reduces to practice the use of multiple different promoters to drive expression in plants: Gt1 promoter for lysozyme (Id. 5 ¶ 145), Gt1 promoter for lactoferrin (Id. 6 ¶ 153), Glb promoter, Gt1 promoter, and Bx7 promoter for AAT protein (Id. 6 ¶ 159), actin promoter for Reb protein (Id. 6 ¶ 163), Glb promoter for lysozyme (Id. 6 ¶ 165), Glb promoter for haptocorrin protein (Id. 7 ¶ 166), Gt1 promoter for haptocorrin protein (Id. 7 ¶ 167), Glb promoter for kappa-casein protein (Id. 7 ¶ 168), Gt1 promoter for kappa-casein protein (Id. 7 ¶ 169), Gt1 promoter for lactadherin protein (Id. 7 ¶ 170), Gt1 promoter for lactoperoxidase protein (Id. 7 ¶ 171), Bx7 promoter for lysozyme protein (Id. 7 ¶ 173), Glb promoter for lactoferrin protein (Id. 7 ¶ 174), Glb promoter for lysozyme protein (Id. 7 ¶ 174), GT-3 promoter for lysozyme protein (Id. 7 ¶ 175), and RP-6 promoter for lysozyme protein (Id. 7 ¶ 176).
Huang teaches co-expression of lysozyme under the control of the Glb promoter and Reb under the control of its own Reb promoter (Id. 6 ¶ 165, Figure 20).
Mayfield claims a chloroplast co-expressing at least two mammalian milk or colostrum proteins (Mayfield, claim 11). Mayfield claims this chloroplast wherein the two or more proteins further comprises a protein from a list recited in claim 13; and this list includes all seven proteins recited in instant claim 1. Mayfield claim a cell comprising this chloroplast (Id. 53, claims 32 and 40). Mayfield teaches colostrum and milk polypeptides recombinantly expressed in photosynthetic organisms (abstract). Mayfield teaches the desirability of producing milk proteins from photosynthetic organisms, including algae and higher plants, so that these proteins can be delivered orally without the need for extracting or purifying out of the host organism (Mayfield 1). Mayfield specifically suggests Brassicaceae, Solanaceae, Phaseoleae, Zea, and Oryzea as examples of higher plants that could be used (Id.).
Peach teaches variability in expression levels for transgenes due to position effects (Peach 49, abstract). Peach teaches co-expression of two reporter proteins in transgenic tobacco (Id. 52). Peach uses the relative enzymatic activities of the two reporter proteins to calculate a ratio of expression levels between the two proteins for 45 different individual transformants (Id. 53, Table 1). The ratio of GUS/CAT ranges from 0.05 for transformant GC4-00.39 up to 48.99 for transformant GC4-00.20 (Id.). This demonstrates that the effects of the transgenes incorporating in different locations in the genome can cause orders of magnitude differences in expression levels that can severely alter the ratio of expression between the two proteins even when using the exact same expression constructs for each of the transformed plant cells.
At the time the instant application was filed, it would have been obvious and within the scope of one of ordinary skill in the art to follow the teachings of El-Richani to produce a transgenic plant by introducing at least one expression cassette comprising a codon-optimized sequence encoding a desired milk protein operably linked to a promoter. Because El-Richani mentions protein(s) in the abstract and includes the introduction of “one or more” coding sequences in claim 24, it is clear that El-Richani has contemplated the expression of more than one protein in the transgenic plants. Because Mayfield expressly claims the co-expression of “at least two” mammalian milk or colostrum proteins, one would have been motivated to express at least two of the proteins recited by El-Richani. In the absence of any showing of criticality or unexpected results, any combination of two proteins recited the claims is an obvious variation over the prior art, including co-expression of both kappa casein and alpha-S1-casein as claimed in instant claim 33.
The teachings of Peach demonstrate that transformation with two expression constructs would necessarily generate multiple individual transformants in which the respective transgenes are incorporated into different locations within the genome, and this will necessarily result in a wide range of ratios for the respective expression levels.
At the time the instant application was filed, it would have been obvious and within the scope of one of ordinary skill in the art to use one of the promoters taught by Huang that provided high levels of expression for each of the proteins desired to be expressed. This would have necessarily resulted in at least one transformed plant producing the proteins at levels that would have resulted in a ratio that is least 70% of the proportion of relative abundance for the pair of proteins in mammalian milk.
Applicant points out that Table 7 of the specification discloses the relative abundance of each of the milk proteins of interest in cow milk and its predicted abundance in soybeans when using specifically assigned promoters (Resp 21). Here is a screen shot of Table 7:
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The ratio of kappa casein/alpha-S1-casein is 9/28 which is calculated to be 0.32.
Roin teaches different relative abundances in different cow varieties, and the kappa casein/alpha-S1-casein ratios for these cow varieties is 0.25, 0.22, 0.27, 0.27,0.28, 0.27, and 0.29 (calculated from numbers in Table 6; Roin 1022). The claims would require plants to express the proteins at a ratio that is at least 70% of one of these ratios or some other ratio from a different cow. The range would be somewhere around 0.15 – 0.22 or a 6.7-fold to a 4.5-fold difference in expression level with there being more alpha-S1-casein than kappa casein.
Applicant’s own data show that there are large differences in ratios between two milk proteins when comparing one transgenic line to another even when they were transformed with the same construct (Figure 6). Furthermore, in comparing Figure 6 to Figure 7, one can see that the ratio between the same milk proteins in the same transgenic line can be completely opposite when comparing expression levels in leaves to expression levels in seeds (Figure 6 is leaves and Figure 7 is seeds; especially plant line #9). There are definitely protein pairs that have 5-fold differences in expression levels (see line #9). In these transgenic soybean plants all of the proteins were expressed under the control of the ubiquitin promoter, so the differences observed were due to something else besides the identity of the promoter. It is common to generate a population of independent transformants when a transgene is introduced into a plant. The instant specification states that they isolated 40 separate independent transformants. Larger numbers can be generated for screening if the desired outcome is expected to be infrequent. For this reason, if one were to co-express kappa casein and alpha-S1-casein in a population of transgenic plants, one would expect there to be at least a 4.5-fold higher expression level of alpha-S1-casein in at least one tissue of at least one plant in the population of plants. The teachings of Peach reinforce this concept.
Furthermore, with regard to claim 1, if one were to coexpress kappa casein and beta-Lactoglobulin, then any ratio obtained would satisfy the current claim limitation, because the ratio of kappa casein/beta-Lactoglobulin is 1.0 and the ratio of beta-Lactoglobulin/kappa casein is also 1.0, therefore, any ratio obtained would be at least 70% of one of those ratios.
For these reasons, the combination of the indefiniteness regarding what ratio is used to calculate “at least 70%” and the variability amongst a population of transgenic plants transformed with the exact same construct renders the instant claims unpatentable over the prior art.
Applicant’s Arguments
Applicant argues that the current claim language is clear when read in light of the specification as filed and the Declaration of Dr. Michal Shahar submitted on June 30, 2025 (Resp 11-13). The Examiner agrees that the amendments make it clear that the required proportion is comparing expression levels between protein pairs. This ground of indefiniteness has been resolved. However, the ground of indefiniteness directed the variability of the ratios/proportions between protein pairs in cows remains.
The Shahar Declaration provides evidence that the ratios are different depending on which cows are used or which study is cited (Shahar Dec, pages 3-4 and Annex A2). This moving target for comparing the calculated proportion in the transgenic plants to 70% of the corresponding proportion in cows causes indefiniteness, because the cut-off for “at least 70%” is unclear.
Applicant argues that the data in the Shahar declaration addresses the written description concerns (Resp 13-14). This is not persuasive, however, because the data in the declaration is only for a few selected protein pairs. The Examiner has pointed out, above, that the data in the specification demonstrates protein pairs from individual transformants that would not satisfy the instant claims.
Applicant argues that they have amended to claims to limit the bovine species to cows only, and that the Shahar Declaration provides evidence of well-established ranges for relative abundance of the principal bovine milk proteins (Resp 14). This is not persuasive, however, because the fact that the ratios between the pairs of proteins have ranges between different cow species is evidence that the cut off for what ratio will satisfy “at least 70%” is a moving target and this renders the metes and bounds of the claim unclear.
Applicant argues that the data in the Shahar Declaration demonstrates that in soybean, tobacco, and rice, the observed ratios fell within 70-100% of the values calculated for different cows from the literature and the cows from Table 7 of the instant application (Resp 14-15). Applicant posits this data provides adequate written description and definiteness for the claims (Id.). This is not persuasive, however, because, as the Examiner points out, the data in the instant specification does not provide evidence of transformants having the required ratios.
The Declaration states that tobacco leaves were transformed with a T-DNA binary vector coding for four of the milk proteins under the control of 35S constitutive promoters which resulted in the required ratios for kappa casein/alpha S1 casein, kappa casein/alpha S2 casein, and alpha S2 casein/alpha S1 casein (Shahar Dec 2-3). The Declaration states that rice plants were transfected with the vector from Figure 4 which comprises multiple expression cassettes to encode 7 milk proteins each under the control of a ubiquitin promoter, and the Declaration provides data showing the alpha S2 casein/alpha S1 casein and beta Lactoglobulin/alpha S1 casein ratios satisfy the requirements of the claims (Id. 4). The Declaration states that soy plants were transfected with the same vector as the rice plants and the alpha S2 casein/kappa casein ratio satisfies the requirements of the claims (Id. 5); the soybean ratios for alpha S1 casein/beta casein, kappa casein/beta Lactoglobulin, and alpha S2 casein/alpha S1 casein also satisfy the requirements of the claims (Id. 6).
This is not persuasive, however, because it is only data for vectors using the 35S promoter or the ubiquitin promoter, and the correct ratios were only demonstrated for a small subset of the protein pairs encompassed by the claims. There are 21 possible protein pairs for the seven claimed milk proteins, and Applicant has only demonstrated success with 6 out of the possible 21 pairs. Furthermore, the data in the specification shows multiple non-compliant protein pairs. In addition, it appears that the experiments from the declaration were transient expression, because soybean and rice were “transfected” which is different than being transformed and tobacco leaves were inoculated and harvested without regenerating stably transformed tobacco plants. The instant claims encompass both stable transformation and also transient expression. Stable transformation can lead to the issue of position effects as taught by Peach (see above). It is also unclear if the pooled tobacco leaves all came from the same plant or if the pooled rice or soy seeds all came from the same plant. The claims are directed to an individual plant.
For these reasons, the described experiments are not representative across the breadth of the claims, and it does not appear that the full scope of the claimed invention was in possession of Applicant at the time of filing.
Applicant incorporates arguments against the obviousness rejection that were presented in the June 30, 2025, response. These arguments were all directed to the 103 rejection that was made in the final rejection (mailed on April 3, 2025) over the Feb. 22, 2024 claim set. The amendments to the claims required a modification to the rejection, including an additional reference, and these arguments are not applicable to the new rejection over the amended claims.
Summary
No claim is allowed.
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CATHY KINGDON
Primary Examiner
Art Unit 1662
/CATHY KINGDON/Primary Examiner, Art Unit 1662