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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 October 30, 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 .
The examiner of your application in the USPTO has changed. To aid in correlating any papers for this application, all further correspondence regarding this application should be directed to David Steadman whose contact information is listed below.
Applicant’s amendment to the claims, filed October 30, 2025, is acknowledged. This listing of the claims replaces all prior versions and listings of the claims.
Claims 1, 3, 4, 8-16, 18, and 20 are pending in the application.
Applicant’s amendment to the specification, filed October 30, 2025, is acknowledged.
Applicant’s amendment to the drawings, filed October 30, 2025, is acknowledged.
Applicant’s remarks filed October 30, 2025 in response to the final rejection filed October 2, 2025 are acknowledged and have been fully considered.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Restriction/Election
In response to a requirement for restriction/election filed January 28, 2025, applicant elected without traverse the invention of Group I, corresponding to pending claims 1, 18, and 20, in the reply filed January 30, 2025.
Claims 3, 4, and 8-16 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Drawing Figures
The objection to the drawings is withdrawn in view of applicant’s submission of a replacement drawing figure.
Specification/Informalities
The objection to the specification for the use of trademarks is withdrawn in view of applicant’s submission of an amendment to the specification to identify trademarks with the symbol ™.
Claim Objections
The objection to claim 1 for reciting “the following the amino acid mutations” is withdrawn in view of applicant’s amendment to claim 1 to delete the phrase at issue.
Claim 1 is newly objected to for reciting “wherein the amino acid K is substituted for the amino acid E at position 23 in the sequence of wild type T4 DNA ligase.” The amino acid sequence of SEQ ID NO: 4 has K at position 23 and thus, the recitation of “wherein the amino acid K is substituted for the amino acid E at position 23 in the sequence of wild type T4 DNA ligase” is redundant. In the interest of improving claim form, it is suggested that claim 1 be amended to delete the phrase “wherein the amino acid K…in the sequence of wild type T4 DNA ligase.”
Claim Rejections - 35 USC § 102
The rejection of claims 1, 18, and 20 under 35 U.S.C. 102(a)(1) or 35 U.S.C. 102(a)(2) as being anticipated by Ong et al. (US Patent No. 10,837,009 B1; cited on Form PTO-892 filed May 20, 2025; hereafter “Ong”) is withdrawn in view of applicant’s amendment to claim 1 to recite “A mutant T4 DNA ligase comprising the amino acid sequence of SEQ ID NO: 4.” Ong does not teach the amino acid sequence of instant SEQ ID NO: 4.
Claim Rejections - 35 USC § 103
The rejection of claim 1 under 35 U.S.C. 103 as being unpatentable over Ong in view of Chen et al. (Adv. Drug Delivery Rev. 65:1357-1369, 2013; cited on Form PTO-892 filed October 2, 2025; hereafter “Chen”) and as evidenced by New England Biolabs, Inc. (PURExpress® In Vitro Protein Synthesis Instruction Manual, NEB catalog # E6800; published 2025; cited on Form PTO-892 filed October 2, 2025) is withdrawn in favor of the new rejection set forth below.
Claims 1, 18, and 20 are newly rejected under 35 U.S.C. 103 as being unpatentable over Ong in view of Chen, Ford et al. (Protein Expression and Purification 2:95-107, 1991; cited on the attached Form PTO-892; hereafter “Ford”), and Zhu et al. (US 2020/0291455 A1; cited on the IDS filed August 16, 2025; hereafter “Zhu”).
As amended, claim 1 is drawn to a mutant T4 DNA ligase comprising the amino acid sequence of SEQ ID NO: 4, wherein the amino acid K is substituted for the amino acid E at position 23 in the sequence of wild type T4 DNA ligase.
Regarding instant claim 1, Ong teaches SEQ ID NO: 1 is the sequence of wild-type T4 DNA ligase (column 7, lines 31-32). SEQ ID NO: 1 of Ong is identical to amino acids 1-487 of instant SEQ ID NO: 4 with the exception of an E23K mutation (see attached Appendix for sequence alignment).
Ong teaches 74 different T4 DNA ligase variants of wild-type T4 DNA ligase that exhibit greater salt tolerance than the wild-type (column 4, lines 34-50). Ong teaches each T4 DNA ligase variant has a single mutation including the mutation E23K (column 4, lines 34-50).
The sequence of the T4 DNA ligase variant with the E23K mutation of Ong is identical to amino acids 1-487 of instant SEQ ID NO: 4.
The difference between the T4 DNA ligase variant of Ong and instant SEQ ID NO: 4 is that instant SEQ ID NO: 4 includes a C-terminal six histidine fusion tail with an intervening GSGSSG linker sequence, i.e., SEQ ID NO: 4 includes a C-terminal GSGSSGHHHHHH sequence.
Regarding a C-terminal six histidine tail, Ong teaches nickel resin-purified His-tagged T4 DNA ligase mutants (column 5, line 6) and Ford teaches a poly-histidine fusion tail for purification using immobilized metal affinity chromatography including a six histidine tail fused to the C-terminus of a polypeptide (p. 100, column 1, middle to bottom).
Regarding an intervening GSGSSG linker sequence, Chen teaches that the direct fusion of functional domains without a linker may lead to many undesirable outcomes, including mis-folding of the fusion proteins and impaired bioactivity (p. 2, first full paragraph). Chen teaches the linker is an indispensable element of the successful construction of a recombinant fusion protein (p. 2, first full paragraph). Chen teaches three types of linkers including flexible linkers (p. 4, first full paragraph). Chen teaches flexible linkers are usually applied when the joined domains require a certain degree of movement or interaction. They are generally composed of small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids The incorporation of Ser or Thr can maintain the stability of the linker in aqueous solutions and therefore reduces the unfavorable interaction between the linker and the protein moieties. The most commonly used flexible linkers have sequences consisting primarily of stretches of Gly and Ser residues ("GS" linker)." (p. 4, section 3.1, paragraphs 1-2).
Regarding a C-terminal GSGSSGHHHHHH sequence, Zhu teaches wild-type Taq polymerase with a C-terminal His tag with the sequence of SEQ ID NO: 4 (paragraphs [0006] and [0018]). The C-terminal end of SEQ ID NO: 4 of Zhu has the sequence GSGSSGHHHHHH.
In view of the combined teachings of Ong, Ford, Chen, and Zhu, it would have been obvious to one of ordinary skill in the art before the effective filing date to modify the T4 DNA ligase variant of Ong with a C-terminal GSGSSGHHHHHH sequence.
One would have been motivated to modify the T4 DNA ligase variant of Ong with a C-terminal HHHHHH fusion tail because Ong taught T4 DNA ligase variants having a His-tag for purification and Ford taught a six histidine tail fused to the C-terminus of a polypeptide for purification.
One would have been motivated to include an intervening GSGSSG linker sequence between the T4 DNA ligase variant of Ong and a C-terminal HHHHHH fusion tail because Chen taught the direct fusion of functional domains without a linker may lead to many undesirable outcomes, taught the linker is an indispensable element of the successful construction of a recombinant fusion protein, and taught linker sequences comprising glycine and serine.
One would have expected success to modify the T4 DNA ligase variant of Ong with a C-terminal GSGSSGHHHHHH sequence because Ong taught T4 DNA ligase variants having a His-tag for purification and Zhu taught a C-terminal His-tag with the sequence GSGSSGHHHHHH.
Claim 18 is drawn to a process of conducting polynucleotide ligation between different polynucleotides or by ligating the 5' and 3' ends of polynucleotides to generate circular polynucleotide, wherein the polynucleotides have blunt ends or cohesive ends, comprising:
providing a ligation mixture including the polynucleotides to be ligated and a mutant T4 DNA ligase or a biologically active fragment of claim 1; and
placing said ligation mixture at temperatures wherein ligation takes place.
Regarding instant claim 18, Ong teaches a method for ligating a nucleic acid molecule, that includes: combining a variant bacteriophage DNA ligase with a first polynucleotide and a second polynucleotide to produce a reaction mix; and incubating the reaction mix to permit the variant DNA ligase to ligate the 5' end of the first polynucleotide to the 3' end of the second polynucleotide" (column 2, lines 38-44). Ong teaches that in one example, the incubating is done at a temperature of at least 45° C (column 2, lines 45-46).
Claim 20 is drawn to the process of claim 18, wherein the ligation reaction mixture includes Tris-HCl, MgCl2, ATP, dithiothreitol and water.
Regarding instant claim 20, Ong teaches a standard ligation reaction containing standard ligase reaction buffer (1×T4 DNA Ligase Reaction Buffer: 50 mM Tris-HCl, pH 7.5 @ 25° C, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, i.e., dithiothreitol) (column 13, lines 61-63).
Therefore, claims 1, 18, and 20 would have been obvious to one of ordinary skill in the art before the effective filing date.
RESPONSE TO REMARKS: Applicant argues SEQ ID NO: 4 is shown by Table 1 of the specification to have two-fold greater activity as compared to wild type T4 ligase. Applicant contends that the high activity of SEQ ID NO: 4, having a linker and a His tag, is an unexpected result over Ong’s T4 ligase with the E23K mutation.
Applicant’s arguments are not found persuasive. According to MPEP 716.02(e), applicant’s results must compare the claimed subject matter with the closest prior art to be effective to rebut a prima facie case of obviousness. In this case, applicant’s alleged unexpected results are a comparison between SEQ ID NO: 4 and wild type T4 ligase. Applicant’s results do not compare SEQ ID NO: 4 with the closest prior art, which is Ong’s T4 ligase with the E23K mutation. As such, applicant fails to satisfy the burden of establishing an unexpected result as required by MPEP 716.02(b).
Conclusion
Status of the claims:
Claims 1, 3, 4, 8-16, 18, and 20 are pending.
Claims 3, 4, and 8-16 are withdrawn from consideration.
Claims 1, 18, and 20 are rejected.
No claim is in condition for allowance.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to DAVID J STEADMAN whose telephone number is (571)272-0942. The examiner can normally be reached Monday to Friday, 7:30 AM to 4:00 PM.
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/David Steadman/Primary Examiner, Art Unit 1656
APPENDIX
Sequence 1, US/16227533
Patent No. 10837009
GENERAL INFORMATION
APPLICANT: New England Biolabs, Inc.
TITLE OF INVENTION: DNA Ligase Variants
FILE REFERENCE: NEB-406-US
CURRENT APPLICATION NUMBER: US/16/227,533
CURRENT FILING DATE: 2018-12-20
PRIOR APPLICATION NUMBER: 62/609,577
PRIOR FILING DATE: 2017-12-22
NUMBER OF SEQ ID NOS: 53
SEQ ID NO 1
LENGTH: 487
TYPE: PRT
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic construct
Query Match 96.8%; Score 2505; Length 487;
Best Local Similarity 99.8%;
Matches 486; Conservative 1; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MILKILNEIASIGSTKQKQAILKKNKDNELLKRVYRLTYSRGLQYYIKKWPKPGIATQSF 60
||||||||||||||||||||||:|||||||||||||||||||||||||||||||||||||
Db 1 MILKILNEIASIGSTKQKQAILEKNKDNELLKRVYRLTYSRGLQYYIKKWPKPGIATQSF 60
Qy 61 GMLTLTDMLDFIEFTLATRKLTGNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGASVS 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 GMLTLTDMLDFIEFTLATRKLTGNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGASVS 120
Qy 121 IANKVWPGLIPEQPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLL 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 IANKVWPGLIPEQPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLL 180
Qy 181 SRAGNEYLGLDLLKEELIKMTAEARQIHPEGVLIDGELVYHEQVKKEPEGLDFLFDAYPE 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 SRAGNEYLGLDLLKEELIKMTAEARQIHPEGVLIDGELVYHEQVKKEPEGLDFLFDAYPE 240
Qy 241 NSKAKEFAEVAESRTASNGIANKSLKGTISEKEAQCMKFQVWDYVPLVEIYSLPAFRLKY 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 NSKAKEFAEVAESRTASNGIANKSLKGTISEKEAQCMKFQVWDYVPLVEIYSLPAFRLKY 300
Qy 301 DVRFSKLEQMTSGYDKVILIENQVVNNLDEAKVIYKKYIDQGLEGIILKNIDGLWENARS 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 DVRFSKLEQMTSGYDKVILIENQVVNNLDEAKVIYKKYIDQGLEGIILKNIDGLWENARS 360
Qy 361 KNLYKFKEVIDVDLKIVGIYPHRKDPTKAGGFILESECGKIKVNAGSGLKDKAGVKSHEL 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 KNLYKFKEVIDVDLKIVGIYPHRKDPTKAGGFILESECGKIKVNAGSGLKDKAGVKSHEL 420
Qy 421 DRTRIMENQNYYIGKILECECNGWLKSDGRTDYVKLFLPIAIRLREDKTKANTFEDVFGD 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 DRTRIMENQNYYIGKILECECNGWLKSDGRTDYVKLFLPIAIRLREDKTKANTFEDVFGD 480
Qy 481 FHEVTGL 487
|||||||
Db 481 FHEVTGL 487
