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 .
Status of Claims
Receipt of Arguments/Remarks filed on 3/9/2026 is acknowledged. Claims 1, 8, and 9 were amended. Claim 7 was canceled. New claims 11-21 were added. Claims 1-6 and 8-21 are pending.
Withdrawn Rejections
The rejection of claim 9 under 35 U.S.C. § 112(b) is withdrawn.
New and modified objections/rejections necessitated by amendment
Claim Objections
Claim 1 is objected to because of the following informalities: line 22 recites “protease cleave site”. This should instead be “protease cleavage site”. Appropriate correction is required.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-2, 8-11, and 14-17 are rejected under 35 U.S.C. 103 as being unpatentable over Ichtchenko et al., US 2011/0206616 A1.
Regarding claim 1, Ichtchenko teaches isolated nucleic acid molecules encoding Clostridium botulinum propeptides and neurotoxins and methods of expression (Ichtchenko “Abstract”).
Ichtchenko teaches a method for preparing the toxin polypeptide comprising step 1, expressing a modified toxin polypeptide precursor, BoNT/Aadek (Ichtchenko p. 45 Example 10 para. 179).
Ichtchenko teaches step 2, enriching the modified toxin polypeptide precursor, BoNT/Aadek using an affinity resin and affinity chromatography (Ichtchenko p. 46 para. 180).
Ichtchenko teaches step 3, activating the toxin polypeptide precursor to obtain the
modified toxin polypeptide by proteolytic cleavage (Ichtchenko p. 1 para. 4; p. 28 para. 100; p. 46 para. 181). Ichtchenko teaches that activating the polypeptide precursor comprises digesting using a first protease to cleave the first protease cleavage site (TEV protease site, p. 46 para. 182) and a second protease to cleave the second protease cleavage site (enterokinase, p. 46 para. 181), wherein the first and second protease cleavage sites are different.
Ichtchenko teaches that the toxin propeptide produced by the above method comprises a polypeptide fragment including a tag protein (p. 8 para. 48), a protease cleavage site (p. 8 para. 49), and a linker sequence (p. 10 para. 62; see Fig. 1). The tag, protease cleavage site, and linker are at the N-terminus of a second polypeptide fragment, which comprises the neurotoxin light and heavy chain regions (p. 5 para. 18; see Fig. 1). The C. botulinum neurotoxin light region comprises a metalloprotease activity domain (p. 1 para. 5-7) and the heavy chain comprises receptor binding and translocation activity (p. 1 para. 5; p. 2 para. 8). Ichtchenko teaches that the light and heavy chains are connected by an intermediate region comprising a highly specific protease cleavage site (p. 5 para. 18, see Fig. 18). Thus, as seen in Fig. 1 BoNT/Aadek, Ichtchenko teaches a single-chain neurotoxin polypeptide comprising a first fragment with a tag, protease cleavage site, and short linker peptide; and a second fragment comprising a metal-ion dependent protease activity domain (neurotoxin light chain), a second protease cleavage site, and a receptor binding/translocation domain (neurotoxin heavy chain).
Regarding the short linker sequence, Ichtchenko teaches that the linker (spacer) positioned upstream of the light chain region may comprise 2-40 amino acid residues (p. 10 para. 62). When claimed ranges "overlap or lie inside the ranges disclosed by the prior art", a prima facie case of obviousness exists. See MPEP 2144.05(I). Therefore, a linker peptide of no more than 5 amino acid residues is considered obvious in view of Ichtchenko, which teaches linkers with 5 or fewer amino acid residues. Thus, the claimed method is obvious in view of the method of Ichtchenko.
Regarding claim 2, Ichtchenko teaches:
Designing a nucleic acid molecule encoding the toxin polypeptide precursor by synthesizing the synthetic gene for BoNT/Aadek (Ichtchenko p. 27 para. 80; p. 38 para. 137)
Constructing a vector comprising the nucleic acid molecule (Ichtchenko p. 27 para. 80; p. 41 para. 142 “Creation of Vectors for Protein Expression in Baculovirus”)
Transferring the nucleic acid vector into a suitable host cell (Ichtchenko p. 27 para. 80; p. 43 Ex. 5)
Culturing the host cell and allowing the host cell to express the polypeptide (Ichtchenko p. 27 para. 80; p. 43 para. 157 Ex 7).
Regarding claim 8, Ichtchenko teaches that the protein is processed to a heterodimer by cleavage with a protease, enterokinase (Ichtchenko p. 46 para. 181).
Regarding claim 9, according to the instant specification, "low toxicity" refers to lower
polypeptide activity (see instant specification p. 6). Ichtchenko teaches that the physiologically active C. botulinum is produced by cleaving the neurotoxin propeptide (Ichtchenko p. 28 para. 98). Thus, Ichtchenko teaches that the toxin precursor/propeptide has lower activity, and therefore lower toxicity, than the active polypeptide.
Regarding claim 10, Ichtchenko teaches that the toxin polypeptide is purified (Ichtchenko p. 45 Ex. 10 “Expression, Purification, and Processing of BoNT/Aadek”; p. 46 para. 181).
Regarding claim 11, Ichtchenko teaches that the expressed neurotoxin propeptide is contacted with a highly specific protease (e.g., enterokinase or TEV sequence) and the Clostridium botulinum neurotoxin propeptide is not cleaved by proteases endogenous to the expression system or the host cell (Ichtchenko p. 27 para. 80; p. 28 para. 97).
Regarding claim 14, Ichtchenko teaches that a protease recognition site is inserted into the loop between the LC and HC, or first and second functional regions (Ichtchenko p. 48 para. 196-197; Fig. 1; Fig. 6).
Regarding claim 15, Ichtchenko teaches that the first functional amino acid structural region is derived from neurotoxin light chain, which comprises a Zn2-+ protease activity domain (p. 1 para. 5-7) and the second functional amino acid structural domain is derived from neurotoxin heavy chain, which comprises antigen receptor binding (neuron binding) activity and translocation activity across the endocytic vesicle membrane (p. 1 para. 5-7; p. 2 para. 8; p. 29 para. 104).
Regarding claims 16 and 17, Ichtchenko teaches that the clostridial neurotoxin is a botulinum neurotoxin (Ichtchenko “Abstract”).
Claims 3-6 are rejected under 35 U.S.C. 103 as being unpatentable over Ichtchenko in view of Ruegg et al., US 9,469,849 B2, as evidenced by Tecnic, "What is a TFF system and how does it work?").
Ichtchenko teaches the method according to claim 1, as set forth above.
Ichtchenko does not teach that step 2 comprises enriching the toxin polypeptide precursor by conducting a multiplex filtration procedure as set forth in claims 3-6.
Regarding claim 3, Ruegg teaches purifying free botulinum neurotoxin from cell
cultures to produce a high purity, high potency product (Ruegg col. 1 lines 19-22). The Botulinum toxin refers to a neurotoxin protein molecule that can be produced by a Clostridial
bacterium, as well as recombinantly produced forms thereof (Ruegg col. 5 lines 26-34) and the
precursor is cleaved to form a modified toxin polypeptide (Ruegg col. 14 lines 24-31).
Ruegg teaches expressing a modified toxin polypeptide precursor, i.e. a recombinant neurotoxin produced by a bacterium (Ruegg col. 5 lines 24-38) and obtaining the toxin from a fermentation culture (Ruegg col. 6 lines 37-40). Ruegg teaches enriching the polypeptide precursor, i.e. pre-chromatography purifications of the fermentation sample which include at least one of tangential flow filtration, nuclease digest, and clarifying centrifugation and/orfiltration (Ruegg col. 6 lines 10-15) used to clarify, concentrate, and purify proteins (Ruegg col. 6 lines 27-32). Ruegg teaches that enriching the polypeptide comprises a multiplex filtration procedure. As understood based on the disclosure, multiplex filtration refers to a process in which multiple filtrations are conducted, i.e. crude liquid filtration and feed liquid circulation filtration (see instant specification p. 5; p. 15 Ex. 2). Ruegg teaches that the sample comprising the polypeptide is subjected to pre-chromatography purification, which may include multiple filtration steps and multiple types of filtration (Ruegg col. 9 lines 10-15).
Regarding claim 4, Ruegg teaches a method comprising purifying a mixture comprising
crude non-complexed botulinum toxin (Ruegg col. 4 lines 20-28). The instant specification does
not specifically define "crude liquid", and a mixture comprising crude botulinum toxin as taught
by Ruegg is considered a crude liquid.
Ruegg teaches that the sample obtained from the fermentation medium, i.e. the crude liquid comprising the crude botulinum toxin, is subjected to one or more pre-chromatography purifications, which include at least one of tangential flow filtration, nuclease digest, and clarifying centrifugation and/or filtration (Ruegg col. 9 lines 10-15). Therefore, Ruegg teaches crude liquid filtration, i.e. filtration of the crude toxin mixture. Ruegg teaches the use of tangential flow filtration, which forces a portion of the fluid to pass through the filter membrane, while the retentate is continuously swept along by the tangentially flowing fluid, and parameters such as pore size, feed flow, and applied pressure may be optimized by a skilled artisan (Ruegg
col. 9 lines 27-48). It is established in the prior art that tangential flow filtration involves circulating a feed liquid through multiple rounds of filtration (see Tecnic reference, first para.
"What is tangential flow filtration?"). Thus, it is considered that the tangential flow filtration as
taught by Ruegg is a feed liquid circulation filtration.
Regarding claim 5, Ruegg teaches that the crude liquid mixture may be filtered using a
filter with a pore size of 0.1 μm to about 0.3 μm (Ruegg col. 9 lines 50-52; col. 10 lines 30-40).
Regarding claim 6, Ruegg teaches that the tangential flow filtration, or liquid circulation
filtration, comprises retaining some of the fluid, rather than building up the retained components
on the membrane surface like in normal flow filtration (Ruegg col. 9 lines 26-45). Further, as
evidenced by the Tecnic reference, is established that tangential flow filtration involves multiple
rounds of filtration via recirculation of the feed liquid (see Tecnic reference "How does a TFF
system work?"). Thus, it is considered that this type of filtration involves multiple rounds of feed
liquid filtration. Ruegg teaches that tangential flow filtration with filters having a pore size of
about 0.1 μm (i.e. less than 0.2 μm) may be used (Ruegg col. 9 lines 50-51).
It would have been obvious for a skilled artisan, before the effective filing date, to modify the method of Ichtchenko and incorporate a multiplex filtration procedure as taught by Ruegg. Both Ichtchenko and Ruegg are directed to purification of clostridial neurotoxin polypeptides by expressing the polypeptide in a host microorganism, enriching the polypeptide, and activating via proteolytic cleavage. It would have been obvious to a skilled artisan that the multiplex filtration procedure for enrichment taught by Ruegg could be used in the method of Ichtchenko, both of which utilize similar steps to achieve the same purpose of toxin polypeptide preparation.
A person of ordinary skill in the art would have been motivated, with a reasonable expectation of success, to utilize the multiplex filtration technique taught by Ruegg because this technique is effective for the production of a toxin polypeptide. Additionally, in tangential flow filtration the feed solution is continuously recirculated across the membrane, and retained molecules remain in the flowing stream and can be recirculated for further filtration, which keeps the filtration performance more consistent over time and allows processing to continue until the desired concentration or separation is achieved (see Tecnic reference “What is tangential flow filtration?”; “How does a TFF system work?”). Thus, it would be considered beneficial to incorporate this technique, which is known to be an efficient and valuable method, in the process of Ichtchenko, with reasonable expectation of success given the successful use of this technique in a similar method of preparing toxin polypeptides as taught by Ruegg,
Claims 12-13 and 19-21 are rejected under 35 U.S.C. 103 as being unpatentable over Ichtchenko in view of Dolly et al., US 8,119,370 B2.
Ichtchenko teaches the method according to claim 1, as set forth above. Ichtchenko does not teach that the first and second proteases are both proteases of rhinoviruses (claim 12), the proteases are LEVLFQGP (claim 13), SEQ ID NO: 7 (claim 19), that the tag protein is glutathione-S-transferase (claim 20), or SEQ ID NO: 11 (claim 21).
Regarding claims 12 and 13, Dolly teaches recombinant clostridial neurotoxin polypeptides (Dolly col. 1 lines 15-22). Dolly teaches that cleavage sites can be engineered in the neurotoxin loop region between the heavy and light chains, and that the protease cleavage site is designed to reduce cleavage by human proteases (Dolly col. 5 lines 30-32). Dolly teaches that the protease may be the protease from human rhinovirus 3C, which cleaves the amino acid sequence LEVLFQGP (Dolly col. 5 lines 40-45).
Regarding claim 20, Dolly teaches that the neurotoxin proteins comprise a binding tag, and that the binding tag may be glutathione-S-transferase (Dolly col. 5 lines 5-10).
It would have been obvious for a skilled artisan to substitute the protease cleavage site and tag protein of Ichtchenko for the rhinovirus cleavage site and GST tag of Dolly. Both Ichtchenko and Dolly are directed to preparation of clostridial neurotoxin polypeptides, wherein the polypeptides are modified to include protease sites and tag proteins. It would have been obvious to a skilled artisan that the protease site of Ichtchenko (enterokinase or TEV) could be substituted with the rhinovirus protease site taught by Dolly. Dolly teaches that the cleavage sites are introduced in the loop region between the light and heavy chains to reduce unintentional activation by human proteases, and can include enterokinase, TEV, or rhinovirus protease sites (Dolly col. 5 lines 30-45). It would therefore be considered a simple substitution of one known element for another having the same function, with a reasonable expectation of predictable results, given the teachings of Dolly and Ichtchenko that these protease sites may be substituted for one another in the neurotoxin polypeptide. Similarly, Ichtchenko teaches that the tag is a His tag, and Dolly teaches that the tag may be a GST tag or His tag, among others (Ichtchenko col. 5 lines 8-12). Therefore, it would be considered a simple substitution of one known element for another having the same function, with a reasonable expectation of predictable results, given the teachings of Dolly that these tag proteins may be substituted for one another in a neurotoxin polypeptide.
Regarding claim 19, instant SEQ ID NO: 7 corresponds to the second polypeptide fragment, which is the light and heavy chains of C. botulinum neurotoxin, with a protease cleavage site LEVLFQGP between the light and heavy chains. SEQ ID NO: 3 of Ichtchenko is 98.8% identical to SEQ ID NO: 7, the difference being in the region between LC and HC, where the protease cleavage site is located (see sequence alignment in OA Appendix). As discussed above, it would have been obvious to modify the teachings of Ichtchenko and create a neurotoxin polypeptide with a second fragment comprising a first structural region (light chain), LEVLFQGP cleavage site in the loop region, and a second structural region (heavy chain). Therefore, a polypeptide having SEQ ID NO: 7 is obvious in view of Ichtchenko and Dolly.
Regarding claim 21, instant SEQ ID NO: 11 corresponds to the neurotoxin polypeptide precursor sequence comprising a GST tag (residues 1-218), an LEVLFQGP protease cleavage site (residues 221-228), and the second polypeptide fragment (residues 232-1524; or SEQ ID NO: 7). As discussed above, it would have been obvious to modify the teachings of Ichtchenko and create a neurotoxin polypeptide with a GST tag and LEVLFQGP cleavage site upstream of the second polypeptide fragment with light and heavy neurotoxin chains and an LEVLFQGP site. Therefore, a polypeptide having SEQ ID NO: 11 is obvious in view of Ichtchenko and Dolly.
Claims 18 is rejected under 35 U.S.C. 103 as being unpatentable over Ichtchenko in view of Chen et al., Advanced drug delivery reviews. 2013 Oct 15;65(10):1357-69.
Ichtchenko teaches the method according to claim 1, as set forth above. Ichtchenko
teaches that the linker peptide can be 5 amino acids or less and serves to preserve and protect conformational independence of the cargo attachment peptide and the neurotoxin (Ichtchenko p. 10 para. 62).
Ichtchenko does not teach that the short linker peptide is glycine-serine (claim 18).
Regarding claim 18, Chen teaches peptide linkers used in fusion proteins (Chen “Abstract”). Chen teaches that the most commonly used flexible linkers have sequences primarily consisting of stretches of Gly and Ser residues, an example being (Gly-Gly-Gly-Gly-Ser)n, with “n” being optimized to achieve appropriate separation or flexibility for the fusion protein of interest (Chen p. 1360 para. 1).
It would have been obvious for a skilled artisan to substitute the short linker of Ichtchenko for a short linker that is a glycine-serine peptide as taught by Chen. Chen teaches the Gly-Ser are the most commonly used small amino acids in fusion proteins, with a 5 amino acid peptide GGGGS being an example of a commonly used linker. Ichtchenko teaches that linkers may be 5 or fewer amino acids, and are used for the purpose of maintaining desired conformation. It would have been obvious that the linker of Ichtchenko could be substituted with a Gly-Ser linker of five or fewer amino acids. This would be considered a simple substitution of one known element for another of the same function, as both the linker of Ichtchenko and the Gly-Ser linkers taught by Chen are used to promote proper conformation and flexibility of protein domains. Thus, a skilled artisan could substitute one linker for another, with a reasonable expectation of predictable results.
Response to Arguments
In light of amendments to the claims, the rejections of claims 1-10 under 35 U.S.C. § 102 has been withdrawn. However, upon further consideration, new grounds of rejection of claims 1-6 and 8-21 are made under 35 U.S.C. § 103 as set forth above. Given these new grounds of rejection, the arguments presented regarding claims rejected under 35 U.S.C. § 102 are moot.
Conclusion
Claims 1-6 and 8-21 are rejected. No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, 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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to EMILY F EIX whose telephone number is (571)270-0808. The examiner can normally be reached M-F 8am-5pm ET.
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/EMILY F EIX/Examiner, Art Unit 1653
/JENNIFER M.H. TICHY/Primary Examiner, Art Unit 1653