DETAILED CORRESPONDENCE
Status of the Application
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 September 26, 2025 has been entered.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 1-5, 12, 13, 27, 28, and 30-33 are pending in the application.
Applicant’s amendment to the claims, filed September 26, 2025, is acknowledged. This listing of the claims replaces all prior versions and listings of the claims.
Applicant’s remarks filed September 26, 2025 in response to the final rejection mailed July 3, 2025 have been fully considered.
Rejections previously applied to claims 7, 8, and 29 are withdrawn in view of applicant’s amendment to cancel these claims.
Restriction/Election
In response to a requirement for restriction/election mailed on December 27, 2024, applicant elected the following invention and species without traverse in the reply filed on January 16, 2025:
Group I, pending claims 1-5, 12, and 13, drawn to the technical feature of a cell-free system for glycosylating a peptide or polypeptide sequence in vitro and a lysate,
Species (1A), Actinobacillus pleuropneumoniae (ApNGT) or a modified form thereof (claim 1),
Species (2A), SEQ ID NO: 1 (claim 1),
Species (3B), a β1-4 galactosyltransferase including Neisseria gonorrhoeae β1-4 galactosyltransferase LgtB (NgLGtB) and Neisseria meningitidis β1-4 galactosyltransferase LgtB (NmLGtB) (claim 1), and
Species (4G), an α2-6 sialyltransferase including Homo sapiens α2-6 sialyltransferase (HsSIAT1), Photobacterium damselae α2-6 sialyltransferase (PdST6), and Photobacterium leiognathid α2-6 sialyltransferase (P1ST6) (claim 12).
Claims 27, 28, and 30-33 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to nonelected inventions, there being no allowable generic or linking claim.
Claims 1-5, 12, and 13 are being examined on the merits with claims 1 and 12 being examined only to the extent the claims read on the elected subject matter as set forth above.
Claim Objections
Claim 1 is objected to in the recitation of “the second glycosyltransferases” in lines 5-6 of part (iii) and in the interest of improving grammar, it is suggested that the term “glycosyltransferases” be replaced with the singular “glycosyltransferase.”
Claim Rejections - 35 USC § 103
The rejection of claims 1-5 and 12 under 35 U.S.C. 103 as being unpatentable over Jewett et al. (WO 2019/035916 A1; cited on the IDS filed on June 8, 2023; hereafter “Jewett”) in view of Wetter et al. (WO 2017/093291 A1; cited on Form PTO-892 mailed on February 24, 2025; hereafter “Wetter”), and
the rejection of claim 13 under 35 U.S.C. 103 as being unpatentable over Jewett and Wetter as applied to claims 1-5 and 12 above, and further in view of Advantages
are withdrawn as being substantially redundant to the rejection of claims 1-5 and 12 under 35 U.S.C. 103 as being unpatentable over Wetter et al. (WO 2017/093291 A1; cited on Form PTO-892 mailed on February 24, 2025; hereafter “Wetter”) in view of CN105505959A (cited on the IDS filed on August 10, 2023) as set forth below.
Claims 1-5 and 12 are rejected under 35 U.S.C. 103 as being unpatentable over Wetter in view of CN105505959A. In the interest of clarity, it is noted that the instant rejection has been modified from its previous version to address the amendment to claim 1 and to remove reference to the evidentiary reference Dictionary because Dictionary is no longer considered necessary to support obviousness.
As amended, the claims are drawn to (in relevant part) a cell-free system for glycosylating a target peptide or polypeptide sequence in vitro, the target peptide or polypeptide sequence comprising an asparagine residue, the system comprising, in a single vessel:
a first cell-free protein synthesis reaction mixture comprising a lysate prepared from E. coli and a first glycosyltransferase which is an N-glycosyltransferase (NGT) that catalyzes transfer to an amino group of the asparagine residue a monosaccharide to provide an N-linked glycan, or an expression vector that expresses the NGT in the first cell-free protein synthesis (CFPS) reaction mixture, wherein the first glycosyltransferase is a bacterial N-linked glycosyltransferase (NGT) having the amino acid sequence of SEQ ID NO: 1;
a glycosylation mixture comprising a monosaccharide donor, optionally a monosaccharide;
a second cell-free protein synthesis reaction mixture comprising a lysate prepared from E. coli and a second glycosyltransferase that catalyzes transfer to the N-linked glycan a monosaccharide, or an expression vector that expresses the second glycosyltransferase in the second cell-free protein synthesis (CFPS) reaction mixture, wherein the second glycosyltransferases is Neisseria gonorrhoeae β1-4 galactosyltransferase LgtB (NgLGtB) or Neisseria meningitidis β1-4 galactosyltransferase LgtB (NmLGtB).
According to the instant specification, in certain exemplary embodiments, one or more of the methods [of glycoprotein synthesis] described herein are performed in a vessel, e.g., a single, vessel (paragraph [00112]). In accordance with the disclosure of the specification, the claimed cell-free system is interpreted as encompassing an aqueous, homogenous mixture of the components (i), (ii), and (iii) in a single vessel.
According to the instant specification, a “cell-free protein synthesis reaction mixture” (alternatively referred to as “CFPS reaction mixture”) typically may contain one or more of a crude or partially-purified cell extract, an RNA translation template, and a suitable reaction buffer for promoting cell-free protein synthesis from the RNA translation template (paragraph [0098]).
Regarding claims 1-5 and 12, Wetter teaches N-glycosyltransferases (NGT) are capable of adding a glucose to an amino acid present in an N-glycosylation consensus sequence, e.g., NGTs N-glycosylate the asparagine (Asn) residue present in the N-glycosylation consensus sequence Asn-X-Ser(Thr) (paragraph [0095]). Wetter teaches NGTs use UDP-Glc (a monosaccharide donor) as a donor substrate (paragraphs [0095] and [00168]). Wetter teaches a method of producing glycosylated target proteins in vitro, said method comprising (i) using an NGT to add a glucose to a target protein that comprises one or more of N-glycosylation consensus sequences (paragraph [0125]; Figure 1). Wetter teaches the NGT is the NGT of Actinobacillus pleuropneumoniae (paragraph [00125]).
Wetter teaches (ii) using a glycosyltransferase (e.g., a galactosyltransferase) to add a monosaccharide (e.g., galactose) to said glucose (paragraph [00125]; Figure 1). Wetter further teaches the galactosyltransferase is LgtB of Neisseria gonorrhoeae (paragraph [00107]).
Wetter teaches (iii) using one or more additional glycosyltransferases to generate an oligosaccharide or polysaccharide on the glucose-monosaccharide primer (paragraph [00125]; Figure 1). Wetter teaches a sialyltransferase capable of adding one or more sialic acid residues to the monosaccharide (e.g., galactose) linked to the glucose that is linked to the Asn residue can be used in accordance with the methods described herein (paragraph [00109]). Wetter teaches the sialyltransferase is CstI or CstII of Campylobacter jejuni (paragraph [00111]). Although CstI or CstII of Campylobacter jejuni correspond to non-elected species (4I) and (4J), the non-elected species of (4I) and (4J) have yet to be searched and examined on the merits because the reference of Wetter was identified during a search and examination of the elected species of (4G).
Wetter teaches the exemplified embodiment of an E. coli comprising plasmid pLMTB4250 (paragraph [00156]) and co-expressing a plasmid for a target protein for glycosylation (paragraph [00192]). Wetter teaches plasmid pLMTB4250 comprises an ApNGT gene (i.e., gene for NGT of Actinobacillus pleuropneumoniae), a LgtB gene, and a CstII gene (p. 45, Table 1, description of “pLMTB4250”) and shows the pathway for polysialylation comprises the enzymes ApNGT, LgtB, and CstII, and the respective donor substrate UDP-glucose, UDP-galactose, and CMP-Neu5Ac (Figure 1). Wetter teaches preparing an extract for glycosylation analysis (paragraphs [00156] and [00157]) and that the target protein in the extract was polysialylated when co-expressed with pLMTB4250 in E. coli (paragraphs [00192] and [0193]). Given that polysialylation of the target protein in the E. coli cell of Wetter involves ApNGT, LgtB, and CstII and the respective donor substrate UDP-glucose, UDP-galactose, and CMP-Neu5Ac, and given that the target protein is polysialylated in the extract of Wetter, it follows that the extract of Wetter comprises at least ApNGT, LgtB, CstII, UDP-glucose, UDP-galactose, and CMP-Neu5Ac.
The difference between claims 1-5 and 12 and the extract of Wetter is that while Wetter teaches the amino acid sequence of ApNGT (p. 46, Table 2), which has 99% sequence identity to instant SEQ ID NO: 1 (see Appendix of Office action mailed February 24, 2025 for sequence alignment), Wetter does not teach or suggest ApNGT that comprises the amino acid sequence of the elected species of instant SEQ ID NO: 1.
CN105505959A teaches a ApNGT comprising a mutation such that the mutant NGT has the advantage of increasing the efficiency of peptide glucosylation by more than 100 times (Translation at the paragraph bridging pp. 2-3 and p. 4, top). The amino acid sequence of the mutant NGT of CN105505959A is identical to instant SEQ ID NO: 1 (see Appendix of Office action mailed July 3, 2025 for sequence alignment).
In view of the combined teachings of Wetter and CN105505959A, it would have been obvious to one of ordinary skill in the art before the effective filing date to use the mutant NGT of CN105505959A in the glycosylation of Wetter. One would have been motivated and would have expected success to do so because CN105505959A taught the mutant NGT has the advantage of increasing the efficiency of peptide glucosylation by more than 100 times.
Therefore, the cell-free system of claims 1-5 and 12 would have been obvious to one of skill in the art before the effective filing date.
Claim 13 is rejected under 35 U.S.C. 103 as being unpatentable over Wetter in view of in view of CN105505959A as applied to claims 1-5 and 12 above, and further in view of Advantages.
The relevant teachings of Wetter and CN105505959A as applied to claims 1-5 and 12 are set forth above. The combination of Wetter and CN105505959A does not teach or suggest the cell extract of the combined teachings of Wetter and CN105505959A is in a freeze-dried form (i.e., lyophilized).
Advantages teaches a variety of industries including biotechnology use lyophilization for long-term storage because it helps with sample stability and purity (sentence bridging pp. 1-2). According to Advantages, despite the involved process, pharmaceutical and biotechnology industries employ lyophilization as a standard practice because heat can adversely affect samples (p. 2, top). Advantages teaches that given the adverse effects of heat and water on long-term storage of sample, lyophilization is often the best solution for long-term sample storage, noting that from sample stability and purity to increased shelf life and reduced costs, lyophilization is a convenient, safe and efficient method for long-term storage of lab samples (p. 3, middle).
In view of the combined teachings of Wetter, CN105505959A, and Advantages, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to lyophilize the cell extract of the combined teachings of Wetter and CN105505959A. One would have been motivated and would have expected success to lyophilize the cell extract of the combined teachings of Wetter and CN105505959A because Advantages acknowledges the use of lyophilization of samples in biotechnology, and Advantages taught benefits of lyophilization such as sample stability.
Therefore, the cell-free system of claim 13 would have been obvious to one of skill in the art before the effective filing date.
RESPONSE TO REMARKS: Applicant argues Wetter does not teach or suggest combining two separate CFPS reaction mixtures into a single vessel and instead teaches a lysate derived from a cell co-expressing the glycosyltransferases. Applicant argues that in contrast to Wetter, the amended claims recite at least a first and a second reaction mixture, with the first and second glycosyltransferases originating from the separate reaction mixtures.
Applicant’s argument is not found persuasive. The instant claims are drawn to a product – not a method – and thus do not require an active step of combining two separate CFPS reaction mixtures into a single vessel. Regarding a “single vessel,” the specification discloses the embodiment of methods [of glycoprotein synthesis] being performed in a single vessel (specification at paragraph [00112]) and in accordance with the disclosure of the specification, the claimed cell-free system is interpreted as encompassing an aqueous, homogenous mixture of the components (i), (ii), and (iii) in a single vessel. In view of this broad, yet reasonable interpretation, the claims encompass the cell extract of the combined teachings of Wetter and CN105505959A.
Even assuming arguendo the claims recited a step of combining two separate CFPS reaction mixtures into a single vessel, such a step would be interpreted as a “product-by-process” limitation (MPEP 2113) and would not structurally and/or functionally distinguish the claimed cell-free system from the cell extract of the combined teachings of Wetter and CN105505959A.
For these reasons, the claimed cell-free system would have been obvious to one of ordinary skill in the art before the effective filing date.
Claims 1-5 and 12 are newly rejected under 35 U.S.C. 103 as being unpatentable over Natarajan et al. (Emerg. Top. Life Sci. 2:419-432, 2018; cited on the attached Form PTO-892; hereafter “Natarajan”) in view of Wetter and CN105505959A.
Natarajan teaches cell-free metabolic engineering (CFME) of glycoprotein biosynthesis using cell extracts enriched with glycosylation enzymes such as glycosyltransferases (GTs) (pp. 425-427). According to Figure 2(b) of Natarajan, the cell extract-based CFME proceeds by preparing individual crude extracts enriched with synthesized glycosylation enzymes and combining the crude extracts and substrate to assemble a glycosylation pathway for glycoconjugate production (p. 425). Natarajan teaches that currently, the best-characterized and most widely adopted crude extract CFME is from E. coli (p. 426, bottom). Natarajan teaches the crude extract system bypasses protein production and purification (p. 426, bottom) and allows real-time monitoring and fine-tuning of conditions (p. 425, Figure 2). Figure 2 of Natarajan is reproduced below for convenience.
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The difference between Natarajan and claims 1-5 and 12 is that Natarajan does not teach or suggest the first, second, and third glycosyltransferases and donor substrates as recited in the claims.
Figure 1 of Wetter shows an engineered pathway for N-linked polysialylation of proteins including the glycosyltransferases ApNGT, LgtB, CstII, and polyST and the relevant donor substrates. A relevant portion of Figure 1 of Wetter is reproduced below for convenience.
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Regarding the ApNGT of Figure 1, Wetter teaches N-glycosyltransferases (NGTs) are capable of adding a glucose to an amino acid present in an N-glycosylation consensus sequence, e.g., NGTs N-glycosylate the asparagine (Asn) residue present in the N-glycosylation consensus sequence Asn-X-Ser(Thr) (paragraph [0095]). Wetter teaches NGTs use UDP-Glc (a monosaccharide donor) as a donor substrate (paragraphs [0095] and [00168]). While Wetter teaches the NGT is the NGT of Actinobacillus pleuropneumoniae, i.e., ApNGT (paragraph [00125]), CN105505959A teaches a ApNGT comprising a mutation such that the mutant NGT has the advantage of increasing the efficiency of peptide glucosylation by more than 100 times (Translation at the paragraph bridging pp. 2-3 and p. 4, top). The amino acid sequence of the mutant NGT of CN105505959A is identical to instant SEQ ID NO: 1 (see Appendix of Office action mailed July 3, 2025 for sequence alignment).
Regarding the LgtB of Figure 1, Wetter teaches using a glycosyltransferase (e.g., a galactosyltransferase) to add a monosaccharide (e.g., galactose) to said glucose (paragraph [00125]; Figure 1). Wetter further teaches the galactosyltransferase is LgtB of Neisseria gonorrhoeae (paragraph [00107]).
Regarding CstII of Figure 1, Wetter teaches using one or more additional glycosyltransferases to generate an oligosaccharide or polysaccharide on the glucose-monosaccharide primer (paragraph [00125]; Figure 1). Wetter teaches a sialyltransferase capable of adding one or more sialic acid residues to the monosaccharide (e.g., galactose) linked to the glucose that is linked to the Asn residue can be used in accordance with the methods described herein (paragraph [00109]). Wetter teaches the sialyltransferase is CstI or CstII of Campylobacter jejuni (paragraph [00111]). Although CstI or CstII of Campylobacter jejuni correspond to non-elected species (4I) and (4J), the non-elected species of (4I) and (4J) have yet to be searched and examined on the merits because the reference of Wetter was identified during a search and examination of the elected species of (4G).
Wetter acknowledges the methods for producing glycosylated proteins can be practiced in vitro (paragraph [00124]).
In view of the combined teachings of Natarajan, Wetter, and CN105505959A, it would have been obvious to one of ordinary skill in the art before the effective filing date to apply Natarajan’s method for glycoprotein biosynthesis to the polysialylation pathway of Wetter, i.e., prepare individual E. coli cell extracts each enriched with one of the enzymes of the polysialylation reaction mixture of Figure 1 of Wetter, and combine the cell extracts and appropriate donor substrates to assemble the polysialylation reaction mixture of pathway of Figure 1 of Wetter. One would have been motivated to do this because of the benefits taught by Natarajan including bypassing protein purification and allowing real-time monitoring and fine-tuning of conditions. One would have expected success because Wetter teaches an engineered pathway for N-linked polysialylation of proteins and acknowledges the methods for producing glycosylated proteins can be practiced in vitro, and Natarajan teaches an in vitro method for producing glycosylated proteins.
Therefore, the cell-free system of claims 1-5 and 12 would have been obvious to one of skill in the art before the effective filing date.
Claim 13 is rejected under 35 U.S.C. 103 as being unpatentable over Natarajan in view of Wetter and CN105505959A as applied to claims 1-5 and 12 above, and further in view of Advantages.
The relevant teachings of Natarajan, Wetter, and CN105505959A as applied to claims 1-5 and 12 are set forth above. The combination of Natarajan, Wetter, and CN105505959A does not teach or suggest the polysialylation reaction mixture of the combined teachings of Natarajan, Wetter, and CN105505959A is in a freeze-dried form (i.e., lyophilized).
Advantages teaches a variety of industries including biotechnology and pharmaceutical industries use lyophilization for long-term storage because it helps with sample stability and purity (sentence bridging pp. 1-2). According to Advantages, despite the involved process, pharmaceutical and biotechnology industries employ lyophilization as a standard practice because heat can adversely affect samples (p. 2, top). Advantages teaches that given the adverse effects of heat and water on long-term storage of sample, lyophilization is often the best solution for long-term sample storage, noting that from sample stability to increased shelf life and reduced costs, lyophilization is a convenient, safe and efficient method for long-term storage of lab samples (p. 3, middle).
In view of the combined teachings of Natarajan, Wetter, and CN105505959A, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to lyophilize the polysialylation reaction mixture of the combined teachings of Natarajan, Wetter, and CN105505959A. One would have been motivated and would have expected success to lyophilize the polysialylation reaction mixture of the combined teachings of Natarajan, Wetter, and CN105505959A because Advantages acknowledges the use of lyophilization of samples in biotechnology, and Advantages taught benefits of lyophilization such as sample stability.
Therefore, the cell-free system of claim 13 would have been obvious to one of skill in the art before the effective filing date.
Claim Rejections - 35 USC § 101
The rejection of claims 1-5, 12, and 13 under 35 U.S.C. 101 is withdrawn in view of applicant’s amendment to claim 1 to recite “a first cell-free protein synthesis reaction mixture comprising a lysate prepared from E. coli” and “a second cell-free protein synthesis reaction mixture comprising a lysate prepared from E. coli.” In view of the applicant’s amendment to claim 1, the claimed cell-free system is markedly different from a combination of naturally-occurring components.
Conclusion
Status of the claims:
Claims 1-5, 12, 13, 27, 28, and 30-33 are pending in the application.
Claims 27, 28, and 30-33 are withdrawn from consideration.
Claims 1-5, 12, and 13 are rejected.
No claim is in condition for allowance.
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/David Steadman/Primary Examiner, Art Unit 1656