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 .
Claim Status
The amendments submitted on 11/17/2025 have been entered.
Claims 1-20 are pending.
Claims 7-20 are withdrawn from examination as being drawn to non-elected inventions.
Claims 1-6 are being examined.
All previous objections and rejections not set forth below have been withdrawn in view
of applicant’s amendments to the claims. Due to applicant’s amendments, the rejection is modified from the rejection set forth in the Office action dated 08/21/2025.
Claim Rejections - 35 USC § 112(b)
Response to Applicant’s arguments: Amendments made to the claims filed on 11/17/2025 overcame the rejections of record under 35 USC § 112(b).
Claim Rejections - 35 USC § 103
Claims 1-3 remain rejected under 35 U.S.C. 103 as being unpatentable over Pitino et al. (Plant production of high affinity nanobodies that block SARS-CoV-2 spike protein binding with its receptor, human angiotensin converting enzyme, 2022, Front. Bioeng. Biotechnol. 10:1045337) in view of Dimitrov et al. (US 10,822,379 B1, published on 14th May 2020) and Schillberg et al. (Plant molecular farming for the production of valuable proteins – Critical evaluation of achievements and future challenges, 2021, Journal of Plant Physiology, 258-259:153359).
Claim 1 is drawn to a transgenic plant or plant tissue or plant cell, or progeny thereof, comprising at least one polynucleotide integrated into the nuclear genome of the plant or plant tissue or plant cell, or progeny thereof. The at least one polynucleotide comprising at least one nucleotide sequence encoding a single variable domain of a heavy-chain antibody fragment (VHH) that specifically binds to a receptor binding domain of SARS-CoV-2 spike protein, wherein the VHH comprises amino acid sequence SEQ ID NO: 1 that is expressed in the transgenic plant or plant tissue or plant cell, or progeny thereof.
Pitino et al. describes production of functional nanobodies blocking a process necessary to initiate SARS-CoV-2 infection and can be produced in plants to rapidly respond to therapeutic needs for emerging pathogens in human medicine and agriculture (abstract). The term, “nanobody” reads on to “variable domain of a heavy-chain antibody”, “VHH” as defined in the instant specification which are used interchangeably (page 7, para 0022, line 1-3). Pitino et al. also describes, “nanobodies (VHH antibodies), as small peptides that represent the antigen binding domain, VHH of unique single domain antibodies (heavy chain only antibodies, HcAb)” (Abstract, line 1-3). Pitino et al. teaches producing transgenic plants via agrobacterium infiltration (page 3, right column, para 1, line 3) and describes transgenic plant tissues (e.g., apoplast) expressing at least one polynucleotide sequence encoding a nanobody or a single variable domain of a heavy-chain antibody (VHH) (page 4, para 2). The recombinant nanobodies produced in plants specifically binds to SARS-CoV2 spike protein (page 6, left column, para 2, line 6-8), as checked by a competitive binding assay to measure inhibition of the interaction between the receptor binding domain (RBD) of the SARS-CoV-2 spike protein with the ACE2 receptor in the presence of the purified nanobodies introduced into the plant leaves using agrobacterium infiltration (page 04, left column, para 3, line 1-5).
However, Pitino et al. does not describe a nanobody or VHH comprising amino acid sequence of SEQ ID NO: 1 or explicitly state that the nucleotide sequence encoding the nanobody is integrated into the host plant nuclear genome.
Dimitrov et al. (US 10822379 B1) describes a polynucleotide sequence encoding the variable domain of a heavy chain (SEQ ID NO: 96) (column 4, line 42-47; column 47, line 5-8) having the ability to bind the spike (S) protein of SARS-CoV-2 (column 46, line 58-65). The SEQ ID NO: 96, as described by Dimitrov et al., comprises 100% sequence identity to instant SEQ ID NO: 1, as shown below.
RESULT 1
US-15-931-939-96
Sequence 96, US/15931939
Patent No. 10822379
GENERAL INFORMATION
APPLICANT: University of Pittsburgh - Of the Commonwealth System of Higher Education
TITLE OF INVENTION: MOLECULES THAT BIND TO SARS-CoV-2
FILE REFERENCE: 48881-0007001
CURRENT APPLICATION NUMBER: US/15/931,939
CURRENT FILING DATE: 2020-05-14
PRIOR APPLICATION NUMBER: US 62/988,856
PRIOR FILING DATE: 2020-03-12
NUMBER OF SEQ ID NOS: 533
SEQ ID NO 96
LENGTH: 124
TYPE: PRT
ORGANISM: Artificial
OTHER INFORMATION: heavy chain variable region of an antigen binder
Query Match 100.0%; Score 657; Length 124; Best Local Similarity 100.0%;
Matches 124; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWIGRMYNNGRTSYN 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWIGRMYNNGRTSYN 60
Qy 61 PSLKSLVTISRDNSKNTLYLQMNSLRAEDTATYYCARDNLGYRPSENLYGMDVWGQGTTV 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 PSLKSLVTISRDNSKNTLYLQMNSLRAEDTATYYCARDNLGYRPSENLYGMDVWGQGTTV 120
Qy 121 TVSS 124
||||
Db 121 TVSS 124
Before the effective filing date of the invention, it would have been obvious to an ordinarily skilled artisan to modify the method of expressing a VHH that binds to the SARS-CoV-2 spike protein in a plant, as described by Pitino et al., by expressing the VHH of SEQ ID NO: 96 of Dimitrov et al., as these two proteins (VHH that binds to the SARS-CoV-2 spike protein of Pitino et al and the VHH of SEQ ID NO: 96 of Dimitrov et al.) are functionally equivalent to each other. The SEQ ID NO: 96 of Dimitrov et al. inherently has the property of binding to the receptor binding domain of the SARS-CoV-2 spike protein (instant SEQ ID NO: 3), as evidenced by the instant specification (spec, page 2, para 0006, line 8-10).
Pitino et al. does not explicitly state that the nucleotide sequence encoding the VHH integrates into the nuclear genome, although it teaches Agrobacterium infiltration (page 3, right column, para 1, line 3). However, it is a standard practice to use Agrobacterium mediated plant transformation to introduce any polynucleotide sequence (transgene) into a plant and expressing the protein encoded by the nucleotide sequence. It is well known in the art that the transgene is delivered into the host cell nucleus and finally integrates into the host nuclear genome and with chromatin1.
Before the effective filing date, one with ordinary skill in the art would have been motivated to express a nucleotide sequence encoding a single variable domain of a heavy-chain antibody fragment (VHH) polypeptide having 100% identity to instant SEQ ID NO: 1 in a plant to rapidly respond to therapeutic needs for emerging pathogens in human medicine and agriculture. The plant-expressed polypeptide would specifically bind to the receptor binding domain (RBD) of SARS-CoV-2 spike protein and, thus, able to block a process necessary to initiate SARS-CoV-2 infection.
Regarding claim 2, claim 2 depends from claim 1 and is drawn to the transgenic tobacco plant or plant tissue or plant cell, or progeny thereof, wherein the at least one nucleotide sequence comprises SEQ ID NO: 2, the receptor binding domain of SARS-CoV-2 spike protein comprises the sequence of SEQ ID NO: 3.
Pitino et al. in view of Dimitrov et al. describes a transgenic plant tissue comprising at least one polynucleotide sequence integrated into the nuclear genome of the plant tissue, and the at least one polynucleotide sequence encoding a single variable domain of a heavy-chain antibody fragment (VHH) specifically binds to a receptor binding domain of SARS-CoV-2 spike protein, wherein the VHH comprises amino acid sequence SEQ ID NO: 1 that is expressed in the transgenic plant tissue, as discussed above. Pitino et al. also teaches using tobacco plants (Nicotiana benthamiana) to produce heterologous proteins including monoclonal antibodies and recombinant antibodies (page 2, right column, para 3, line 8-11), besides producing VHH nanobodies against the SARS-CoV-2 spike proteins (abstract, line 3-4).
However, Pitino et al. in view of Dimitrov et al. do not explicitly describe SEQ ID NO: 2.
The polypeptide sequence encoded by (the polynucleotide of) instant SEQ ID NO: 2 comprises 100% sequence identity to instant SEQ ID NO: 1. The SEQ ID NO: 96 of Dimitrov et al. (which has 100% sequence identity to instant SEQ ID NO: 1) inherently has the property of binding to the receptor binding domain of instant SEQ ID NO: 3 of the SARS-CoV-2 spike protein, as discussed above.
Before the effective filing date of the invention, it would have been obvious to an ordinarily skilled artisan to modify the method described by Pitino et al. by replacing the nucleotide sequence encoding the VHH with a functional equivalent polynucleotide (e.g., instant SEQ ID NO: 2) encoding a polypeptide (e.g. SEQ ID NO: 96, as described by Dimitrov et al.) having 100% sequence identity to instant SEQ ID NO: 1. Using any specific polynucleotide sequence is an experimental design choice of an ordinarily skilled artisan so long the nucleotide sequence encodes a polypeptide having 100% sequence identity to instant SEQ ID NO: 1.
Before the effective filing date, one with ordinary skill in the art would have been motivated to express a nucleotide sequence encoding a single variable domain of a heavy-chain antibody fragment (VHH) polypeptide encoding instant SEQ ID NO:1 in a tobacco plant to rapidly respond to therapeutic needs for emerging pathogens in human medicine and agriculture. The plant expressed VHH polypeptide needs to specifically bind to SARS-CoV-2 spike protein (SEQ ID NO: 3) to be used a therapeutic agent.
Regarding claim 3, claim 3 depends from claim 2 and is drawn to a transgenic Nicotiana tabacum plant.
Pitino et al. used tobacco (Nicotiana benthamiana) plants (abstract) to express the recombinant proteins, as described above. However, neither Pitino et al. nor Dimitrov et al. describe using Nicotiana tabacum, as recited in claim 3.
Schillberg et al. describes that hundreds of different proteins have been produced successfully in plants, including pharmaceutical proteins such as antibodies, vaccines, hormones and enzymes, as well as proteins for diagnostic, research and cosmetic applications (page 2, left column, para 2, first 4 lines). Schillberg et al. teaches that both the species of tobacco plant- Nicotiana tabacum and Nicotiana benthamiana- are widely being used to produce heterologous proteins as part of molecular farming (page 2, left column, para 3, line 7-10). Thus, Nicotiana tabacum and Nicotiana benthamiana are functional equivalent of each other in terms of producing heterologous pharmaceutical proteins.
Claims 4-6 are rejected under 35 U.S.C. 103 as being unpatentable over Pitino et al. in view of Dimitrov et al. and Schillberg et al. as applied to reject claims 1-3 under 35 U.S.C. 103 above, and further in view of Efferth T. (Biotechnology Applications of Plant Callus Cultures, 2019, Engineering, 5:50–59).
Claim 4 depends from claim 1 and is drawn to a tissue culture, produced from protoplasts or cells or callus tissue from the transgenic plant or plant tissue or plant cell, or progeny thereof, of claim 1, wherein the protoplasts or cells or callus tissue are produced from a plant part selected from the group consisting of: leaves, pollen, embryos, cotyledon, hypocotyl, meristematic cells, roots, root tips, pistils, anthers, flowers, stems, glumes and panicles.
Pitino et al. in view of Dimitrov et al. describes a transgenic plant tissue (leaf) (Pitino et al., page 3, right column, last para, line 8) comprising at least one polynucleotide sequence integrated into the nuclear genome of the plant tissue, the at least one polynucleotide sequence encodes a single variable domain of a heavy-chain antibody fragment (VHH) that specifically binds to a receptor binding domain of SARS-CoV-2 spike protein, wherein the VHH comprises amino acid sequence SEQ ID NO: 1 that is expressed in the transgenic plant tissue, as discussed above. Pitino et al. also describes plant cell tissue culture systems to produce nanobody (page 6, left column, para 2, line 28-29).
. However, Pitino et al. in view of Dimitrov et al. do not explicitly describe a tissue culture produced from protoplasts or cells or callus tissue from a transgenic plant or progeny thereof.
Efferth T. describes genetically engineered plants producing desirable features including synthesis of bioactive secondary metabolites and for the generation of plants with improved traits (abstract). It also describes that callus cultures and suspension cell cultures (both come under tissue culture) offer a wide range of usages in pharmacology and pharmacy, as well as in agriculture and horticulture (abstract). Efferth T. describes callus culture from tobacco (Nicotiana tabacum) (as recited in claims 5-6) leaf (mesophyll protoplasts) (page 56, left column, para 4-5) and using transgenic tobacco plants for producing therapeutic proteins like single-chain antibody (page 54, left column, para 2, line 1-4). Plant callus culture and plant suspension cell culture based production systems offer advantages in terms of cost-effectiveness, large-scale production, and safety as compared with other (bacterial, fungal, or mammalian cell culture based) production systems (page 53, right column, last para).
Before the effective filing date of the invention, it would have been obvious to an ordinarily skilled artisan to establish a tissue/cell culture including callus culture and cell suspension culture based protein production system, as described by Efferth T., from transgenic tobacco (Nicotiana tabacum) leaves expressing the heavy-chain antibody fragment (VHH) that specifically binds to a receptor binding domain of SARS-CoV-2 spike protein, as described by Pitino et al. in view of Dimitrov et al., to efficiently produce therapeutically important heavy-chain antibody fragment (VHH) protein at large scale while the plant produced protein would be safer for therapeutic use in animals, as described by Efferth T.
Before the effective filing date, one with ordinary skill in the art would have been motivated to use a tissue/cell culture including callus culture based therapeutic protein production system from transgenic tobacco leaves expressing the heavy-chain antibody fragment (VHH) that specifically binds to a receptor binding domain of SARS-CoV-2 spike protein to efficiently produce the heavy-chain antibody fragment (VHH) protein at large scale while the plant produced protein would be safer for therapeutic use in animals.
Response to Applicants’ arguments
The argument set forth in the Applicant’s reply on 11/17/2025 to the rejection of the claims under 35 U.S.C. 103 has been fully considered but not found persuasive.
The Applicant asserts that, “…the Office incorrectly asserts that Pitino et al., plants are transgenic... “ (page 8, last para, line 4) and indicates that Pitino et al. does not describe any transgenic plants.
The Applicant argues that transient expression in plants is fundamentally different from stable transformation (page 9, para 2, line 1-2) and the present invention uses stable transformation (page 9, last para, line 1). The Applicant continues to argue that, “it is no any possibility to produce transgenic plants without using vectors for stable transformation, without using sterile conditions and tissue culture methods, without selection of transgenic cells and induction of plant regeneration by plant hormones. Pitino et al. did not demonstrate any of these approaches” (page 10, last para and page 11, first para) and Pitino et al. “does not provide any evidence that the particular Agrobacterium used actually integrates into the nuclear genome” (page 11, last para, line 3-4).
The Examiner disagrees. Pitino et al. teaches producing transgenic plants via Agrobacterium infiltration (page 3, right column, para 1, line 3) and describes transgenic plant tissues (e.g., apoplast) expressing at least one polynucleotide sequence encoding a nanobody or a single variable domain of a heavy-chain antibody (VHH) (page 4, para 2).
It is important to mention that both stable transformation and transient expression using Agrobacterium are standard and a well-known practice in the art. Neither of the processes alter protein structure and/or its biological function. In this context of heterologous protein production, transient expression is generally used to quickly verify if the plant expressed protein retains its biological function while using plants as a bioreactor. Stable transformation can be used once the proof of concept is verified. However, there is no difference in terms of particular Agrobacterium strain including EHA105, as used by Pitino et al. (page 3, right column, para 1, line 3), or plant expression vectors used in stable transformation and transient expression. The difference, however, lies in the method of using it, which depends on the objective.
In the art, Agrobacterium strain EHA105 is known to be highly efficient to produce transgenic plants including tobacco where the transgene is stably integrated into the host genome (Reddy et al. Spearmint R2R3-MYB transcription factor MsMYB negatively regulates monoterpene production and suppresses the expression of geranyl diphosphate synthase large subunit (MsGPPS.LSU), 2017, Plant Biotechnology Journal, 15:1105–1119; page 1115, left column, first para, last 3 lines).
In response to Applicants’ arguments against the cited references individually, these are not persuasive, because they are attacks on the Pitino et al. reference individually rather than considering the combined teachings as a whole. One cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
It is also important to note that the test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981).
Conclusion
No claim is allowed.
THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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Jay Chatterjee
Patent Examiner
Art Unit 1662
/Jay Chatterjee/Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Units 1661 & 1662
1 Lacroix et al. (Agrobacterium aiming for the host chromatin Host and bacterial proteins involved in interactions between T-DNA and plant nucleosomes, 2009, Communicative & Integrative Biology, 1:42-45) provides evidence that agrobacterium mediated plant transformation enables the transgene to be delivered into the host cell nucleus and finally integrates into the host nuclear genome and with chromatin (abstract; page 42, right column, para 1, line 1).
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