DETAILED ACTION
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 25 March 2026 has been entered.
Status of Application
The amendments and response filed 25 March 2026 are acknowledged and have been considered in their entireties. Claims 1-15 remain pending and subject to examination on the merits.
Withdrawal of Previous Objections/Rejection
The rejection of claims 1-6 and 9-14 under 35 U.S.C. 101 because the claimed invention is directed to a judicial exception (i.e., a natural phenomenon) without additional elements that integrate the judicial exception into a practical application is withdrawn in view of the amendments to recite the removal/deletion of amino acids 215-218 of SEQ ID NO: 1.
The rejection of claims 1-6 and 9-14 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by UniProt deposit A0A444R917 (deposited by Potter et al., 2019, cited herein) is withdrawn in view of the amendments to recite the removal/deletion of amino acids 215-218 of SEQ ID NO: 1. Potter/UniProt disclose a hemolysin fragment which does not have TTSA at the end but still possesses effectively amino acids 215-218 of SEQ ID NO: 1.
The rejection of claim(s) 1-15 are rejected under 35 U.S.C. 103 as being unpatentable over by UniProt deposit A0A444R917 (deposited by Potter et al. 2019, cited herein) and Schwarz et al. (EP 2583975 – cited on IDS) is withdrawn for the same reasons recited above in Section 4.
New Objections/Rejections
Claim Objections
Claim 3 is objected to because of the following informalities: the claim refers to claim I and SEQ ID NO: I, rather than claim 1 and SEQ ID NO: 1 (e.g. the current claim utilizes the letter I rather than the number 1). Appropriate correction is required.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim(s) 1-3, 5-8 and 11-15 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Benabdelhak et al. (JMB, 2003 – cited herein) as evidenced by Lecher et al. (Structure, 2012 – cited herein) and UniProt Q1R2T5 (Hemolysin A from E. coli – cited herein).
Benabdelhak et al. teach:
Regarding claims 1-2, a HlyA fragment, termed HlyA2 which lacks the last 57 amino acids of HlyA1. HlyA1 comprises amino acids 807-1023 of full length hemolysin; whereas said HlyA2 fragment comprises amino acids 807-966 (See Materials and Methods). The HlyA1 comprising amino acids 807-1023 of full length hemolysin corresponds to instant SEQ ID NO: 1. The HlyA2 lacking the last 57 amino acids, therefore, would have amino acids 1-161, as can be seen below.
Lecher et al. evidences this construct from Benabdelhak et al. possesses an N-terminal His-tag, three RTX repeats which possess GG repeats; and is devoid of the last 57 amino acids of the HylA1 fragment (e.g. instant SEQ ID NO: 1) as exemplified by Figure 1 reproduced here (also See Experimental Procedures; Expression and Purification of HlyA1 and HlyA2, p. 1784):
PNG
media_image1.png
672
592
media_image1.png
Greyscale
UniProt Q1R2T5 evidences the specific sequence utilized by both Benabdelhak et al. and Leher et al. and the sequence alignment between instant SEQ ID NO: 1 and HlyA2 is below.
Qy=HlyA2 and Db = instant SEQ ID NO: 1
GenCore version 6.5.2
Copyright (c) 1993 - 2026 Biocceleration Ltd.
OM protein - protein search, using sw model
Run on: March 27, 2026, 09:45:13 ; Search time 1 Seconds
(without alignments)
0.035 Million cell updates/sec
Title: AASEQ1_03272026_094512
Perfect score: 858
Sequence: 1 GNSLAKNVLSGGKGNDKLYG..........EYQQSNNKVSYVYGHDASTY 161
Scoring table: BLOSUM62
Gapop 10.0 , Gapext 0.5
Searched: 1 seqs, 218 residues
Total number of hits satisfying chosen parameters: 1
Minimum DB seq length: 0
Maximum DB seq length: inf
Post-processing: Minimum Match 0%
Maximum Match 100%
Listing first 1 summaries
Database : US-17-912-066-1.pep:*
SUMMARIES
%
Result Query
No. Score Match Length DB ID Description
----------------------------------------------------------------------------
1 788 91.8 218 1 US-17-912-066-1 FRAGMENTS OF HLYA
ALIGNMENTS
RESULT 1
US-17-912-066-1
Query Match 91.8%; Score 788; DB 1; Length 218;
Best Local Similarity 90.7%;
Matches 146; Conservative 7; Mismatches 8; Indels 0; Gaps 0;
Qy 1 GNSLAKNVLSGGKGNDKLYGSEGADLLDGGEGNDLLKGGYGNDIYRYLSGYGHHIIDDEG 60
||||||||| ||||||||||||||||||||||:|||||||||||||||||||||||||:|
Db 1 GNSLAKNVLFGGKGNDKLYGSEGADLLDGGEGDDLLKGGYGNDIYRYLSGYGHHIIDDDG 60
Qy 61 GKDDKLSLADIDFRDVAFKREGNDLIMYKAEGNVLSIGHKNGITFKNWFEKESDDLSNHQ 120
||:||||||||||||||||||||||||||||||||||||||||||:||||||| |:||||
Db 61 GKEDKLSLADIDFRDVAFKREGNDLIMYKAEGNVLSIGHKNGITFRNWFEKESGDISNHQ 120
Qy 121 IEQIFDKDGRVITPDSLKKAFEYQQSNNKVSYVYGHDASTY 161
||||||| ||:||||||||| |||| ||| |||||:|| |
Db 121 IEQIFDKSGRIITPDSLKKALEYQQRNNKASYVYGNDALAY 161
Search completed: March 27, 2026, 09:45:13
Job time : 1 secs
Thus, said fragment possess the following instant motifs: SEQ ID NOs: 2, 3, 38, 40.
Regarding claim 3, said fragment clearly has a deletion of 57 amino acids at the C-terminus.
Regarding claim 5, said fragment meets limitations of iv, vi, xiii, xv, xix, xxi.
With regard to claims 6-8, said sequence comprises an N-terminal His6x tag. This is deemed a “second amino acid sequence”. Further, the linker in claims 7 and 8 is entirely optional and not required to be present.
With regard to claim 11, “Products of identical chemical composition can not have mutually exclusive properties.” In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655, 1658 (Fed. Cir. 1990). – See MPEP 2112.01(II). Thus, while silent to the aspect of increased expression, the claimed product as taught falls within the scope of the claims and must therefore possess said increased expression property.
Regarding claims 12-15, said variant is encoded by nucleic acids in specified vectors and expressed in E. coli and purified (See pp1776-1777, Materials and Methods).
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claim(s) 9-10 are rejected under 35 U.S.C. 103 as being unpatentable over Benabdelhak et al. (JMB, 2003 – cited herein) as evidenced by Lecher et al. (Structure, 2012 – cited herein) and UniProt Q1R2T5 (Hemolysin A from E. coli – cited herein) as applied to claims 1-3, 5-8 and 11-15 above, and further in view of Schwarz et al. (EP 2583975 – cited on IDS).
The teachings of Benabdelhak et al. as evidenced by Lecher et al. and UniProt Q1R2T5 are discussed above and incorporated into the instant rejection.
The Benabdelhak et al., however, does not teach fusion of the His-tagged HylA2 fragment to a protein of interest.
Schwarz et al. teach recombinant expression and secretion of proteins of interest by fusing them to C-terminal fragments made from the full-length HlyA toxin proteins (SEQ ID NO: 33; wherein full-length is 1023/1024 amino acids), wherein said fusion proteins results in the enhanced expression/secretion of a recombinant peptide or protein of interest, further wherein the method comprises: (a) transforming a suitable host cell with a vector as disclosed expressing a fusion of the HlyA fragment and protein of interest, wherein the vector encodes the recombinant peptide or protein; (b) cultivating the host cell in a culture medium under conditions that allow growth of the host cell; (c) cultivating the host cell in a culture medium under conditions that allow expression/secretion of the recombinant peptide or protein; and optionally (d) purifying the protein. – See paragraphs 0188. It is stipulated for HlyA C-terminal fragments to be successful in aiding secretion efficiency of the fusion protein (fused to a peptide or polypeptide of interest), then the C-terminal fragment should comprise one or more GG repeats (See paragraph 0158). It is specifically stipulated that said HlyA C-terminal fragment should comprise a GGXGXDXXX motif, wherein X can be any amino acid (See paragraph 0047).
The sequence alignment of instant the Benabdelhak et al. as evidenced by Lecher et al. and UniProt Q1R2T5 and instant SEQ ID NO: 1 is described above and reiterated herein. It demonstrates at least two GG containing motifs comprising the GGXGXDXXX motif (Bold and underlined):
Qy 1 GNSLAKNVLSGGKGNDKLYGSEGADLLDGGEGNDLLKGGYGNDIYRYLSGYGHHIIDDEG 60
||||||||| ||||||||||||||||||||||:|||||||||||||||||||||||||:|
Db 1 GNSLAKNVLFGGKGNDKLYGSEGADLLDGGEGDDLLKGGYGNDIYRYLSGYGHHIIDDDG 60
Qy 61 GKDDKLSLADIDFRDVAFKREGNDLIMYKAEGNVLSIGHKNGITFKNWFEKESDDLSNHQ 120
||:||||||||||||||||||||||||||||||||||||||||||:||||||| |:||||
Db 61 GKEDKLSLADIDFRDVAFKREGNDLIMYKAEGNVLSIGHKNGITFRNWFEKESGDISNHQ 120
Qy 121 IEQIFDKDGRVITPDSLKKAFEYQQSNNKVSYVYGHDASTY 161
||||||| ||:||||||||| |||| ||| |||||:|| |
Db 121 IEQIFDKSGRIITPDSLKKALEYQQRNNKASYVYGNDALAY 161
Therefore, it would have been obvious to one of ordinary skill prior to the effective filing date of the claimed invention to utilize any or all known HlyA fragments comprising the GGXGXDXXX motifs as taught by Schwartz et al., including that disclosed as the naturally occurring one deposited as Benabdelhak et al. as evidenced by Lecher et al. and UniProt Q1R2T5, for use in the methods of making fusion proteins of HlyA C-terminal proteins/peptides having the requisite GG containing motifs, specifically GGXGXDXXX motifs, in order to enhance the secretion of peptides of polypeptides of interest in a prokaryotic host cell by fusing said HlyA fragments thereto, cultivating the host cell, expressing the fusion and recovering the fusion because Schwarz et al. teach these C-terminal fragments enhance secretion of the proteins of interest. This would be motivation in and of itself to utilize any known C-terminal HlyA fragment possessing the requisite motifs to utilize in the materials and methods of enhanced secretion of polypeptides of interest. There would be a reasonable expectation of success in utilizing the known HlyA2 fragment as a fusion partner because it possesses all of the requisite motifs required for the enhanced secretion function as taught by Schwarz et al.
Conclusion
No claim is allowed. Claim 4 is objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to SUZANNE M NOAKES whose telephone number is (571)272-2924. The examiner can normally be reached M-F (7-4).
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/SUZANNE M NOAKES/Primary Examiner, Art Unit 1656 27 March 2026