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 .
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 6/18/25 has been entered.
Election/Restrictions – Retained for the Record
Applicant’s election without traverse of the species of a purified metallothionein protein having an amino acid identified by SEQ ID NO:6, in the reply filed on 3/11/24 is acknowledged.
Claim 2 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 3/11/24. This is consistent with applicant’s specific request, page 2 of 3/11/24 Remarks.
The Examiner notes that on further consideration and analysis that the sequence of SEQ ID NO:6 excluding the C-terminus histidine tag is a species of the genus of the claim 2 formula.
Claim Status
Claims 1 and 3-20 are pending.
Claim 2 is cancelled.
Claims 1, 3-20 are under examination.
Claims 1, 3-20 are rejected.
Priority
The instant application, filed 01/12/2021 is a Continuation in Part of 14937142 , filed 11/10/2015, now abandoned, claims foreign priority to 103139135, filed 11/11/2014.
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C 120 as follows:
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
The disclosure of the prior-filed application, Application No. 14937142, fails to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application. SEQ ID NO:6, explicitly recited in instant claim 1, was not taught in the prior-filed application, Application No. 14937142, nor, from the examiner’s understanding, in the foreign priority document from which Application No. 14937142 claims priority. The first recitation of SEQ ID NO:6 is in the instant application, which is identified as a Continuation in Part. This sequence, as well as the instant claim 1 step of purifying the metallothionein protein by utilizing cleavage of histidine tag and metal affinity chromatography, are found in instant claim 1, but not in prior applications.
The earliest priority for what is instantly claimed in claim 1 and all claims depending from it is 01/12/2021.
Claim Interpretation
The claim limitations are given their broadest reasonable interpretation (BRI) consistent with the specification, MPEP 2111, and under the BRI, words of the claim must be given their plain meaning, unless such meaning is inconsistent with the specification, MPEP 2111.01.
The transitional term “comprising” is inclusive or open-ended and does not exclude additional, unrecited elements. See MPEP 2111.03.
Claim Rejections - 35 USC § 103
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 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.
Response to Arguments and Reconsideration of Declaration
Applicant's arguments filed 4/16/26 have been fully considered but they are not persuasive.
As to applicant’s arguments against Melcher, beginning on page 5, that is, in response to applicant's arguments against the references individually, 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).
Regarding Applicant’s assertions that Melcher’s SEQ ID Number 2 and 4 “could not be successfully expressed” page 6, and upon reconsideration of the Declaration of Dr. Resina, and including assertion of production of MT2a of SEQ ID NO:6 being “completely unexpected,” this is not found persuasive given the bases for using a human sequence that is responsible for metallothionein activity, and also given the known approaches for optimizing production of the widely used P. pastoris yeast, see Karbalaei reference applied below. Please also note that prior art other than Melcher alone was applied to establish a rationale to arrive at SEQ ID NO:6 rather than a closely related sequence set forth in Melcher.
The examiner does not understand the line of arguments beginning on page 7 with “Thus, the Examiner's suggested substitution of SEQ ID NO: 6 in view of the prior art Melcher's SEQ ID NO: 4 would destroy the present method and would prevent obtaining the aimed for results” extending to the end of that paragraph, so finds this unpersuasive. How would such “substitution” “destroy the present method”? Again, please also note that prior art other than Melcher alone was applied to establish a rationale to arrive at SEQ ID NO:6 rather than a closely related sequence set forth in Melcher.
As to arguments in B and C on pages 8-9, and D on page 9, and also later arguments directed against references secondary to Melcher, including GE on page 17, and Rice on page 18, in response to applicant's arguments against the references individually, 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).
As to claim 18, given the new inclusion of use of P. pastoris, a new reference was required, so arguments against previously applied references not teaching this are not relevant to the instant rejections that needed to be modified to include new limitations.
Please note that the first full sentence on page 16 is not clear and seems to be missing a subject (the subject of “does not provide”), so the examiner cannot properly respond to this unclear argument. The rationale regarding the common core is retained in the rejection below.
Regarding arguments on page 19, these are not considered persuasive because there is reasoning to use human MT protein having the amino acid sequence of SEQ ID NO:6, with an affinity tag, as set forth in the rejection below, modified to include use of P. pistoris yeast.
Claim(s) 1, 3-12 and 14-16 are rejected under 35 U.S.C. 103 as being unpatentable over US 20160130325, published 5/12/16, inventors Melcher et al. (Melcher, parent of instant CIP), in view of Honda et al, “One-step purification of metallothionein extracted from two different sources,” Journal of Chromatography B, 820 (2005) 205–210 (Honda), Abdullah and Chase, BIOTECHNOLOGY AND BIOENGINEERING, VOL. 92, NO. 4, NOVEMBER 20, 2005, 501-513 (Abdullah), and GenBank AAP36553.1 (2003), these references previously applied and prior art references provided, and Karbalaei et al., J Cell Physiol. 2020;235:5867–5881(Karbalaei), as evidenced by Metallothionein Pfam downloads, two screen shots combined to one page, downloaded 10/15/25 (“Pfam”, previously provided).
Claim 1 as amended 4/16/26 is directed to a method for producing a hypo-metallated redox
active metallothionein protein, the hypo-metallated redox-active
metallothionein protein having 20 cysteine sulfhydryl groups and 7 binding
pockets, 2 to 16 of the 20 cysteine sulfhydryl groups being free and reduced,
and 1 to 6 of the 7 binding pockets being occupied by metal ions, comprising:
producing a metallothionein protein in a Pichia yeast host, said
metallothionein protein having the amino acid sequence of SEQ ID NO: 6 and
comprising at least one peptide extension in the form of an affinity tag, said
affinity tag being a histidine tag (His Tag);
subsequent to producing said metallothionein protein, exposing said
metallothionein protein to His Tag cleavage and metal affinity chromatography,
thus purifying [[the]] said metallothionein protein by utilizing a from said
histidine tag and metal affinity chromatography and obtaining His Tag purified
metallothionein protein;
subsequent to obtaining said His Tag purified metallothionein protein, de-metallating the His Tag purified metallothionein protein, thus obtaining de-metalled metallothionein protein;
chemically reducing the de-metallated metallothionein protein, thus obtaining reduced de-metallated metallothionein protein; and
partially metallating the reduced de-metallated metallothionein protein with 4 molar equivalents of zinc ions, thus obtaining the hypo-metallated redox active metallothionein protein.
s
The elected sequence is SEQ ID NO:6, which includes a six histidine “his tag” at its C-terminus.
Melcher teaches an invention that relates to method for producing hypo-metallated redox-active metallothionein (MT) proteins, pharmaceutical compositions containing the proteins, and uses the pharmaceutical compositions for treatment of conditions originating from elevated intracellular oxidative stress and/or dis-balanced intracellular redox-potential and/or redox-potential-dependent imbalance of metal ions, Abstract.
In some embodiments Melcher’s method steps are as follows:
[0053] The first aspect of the present invention relates to a method for producing a hypo-metallated redox-active metallothionein protein. The hypo-metallated redox-active metallothionein protein has 20 cysteine sulfhydryl groups, thus forming 7 metal ion binding pockets. Two (2) to 16 of the 20 cysteine sulfhydryl groups are free and reduced, and 1 to 6 of the 7 binding pockets are occupied by metal ions. The method comprises the following steps:
[0054] providing a metallothionein protein;
[0055] de-metallating the metallothionein protein;
[0056] chemically reducing all the 20 cysteine sulfhydryl groups of the de-metallated metallothionein protein; and
[0057] partially metallating the reduced de-metallated metallothionein protein by providing 1, 2, 3, 4, 5, or 6 metal ions to the 7 binding pockets.
Melcher teaches that
[0083] The metallothione in apo-proteins of the present invention, in their native form with undefined metallation status and undefined redox-status of their cysteine sulfur residues, may be produced by any conventional method of recombinant DNA technology, protein synthesis, enzymatic cleavage of pre-pro-proteins, or isolation of naturally occurring variants of mammalian metallothionein proteins.
[0084] Thus, in one preferred embodiment of the invention, the metallothionein apo-proteins are produced by established recombinant DNA technology (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, 1989, and references therein). The DNA sequence encoding the respective full-length metallothionein protein isoform may either be synthesized by standard nucleic acid synthesis methods, or may be obtained by established laboratory methods of directly isolating it from appropriate genomic or cDNA libraries, or indirectly by copying and amplifying it from said libraries by standard polymerase chain reaction (PCR) method.
[0085] The DNA sequence, after appropriate preparation and purification, may then be introduced into a recombinant expression vector of choice, using, again, established standard methods. The choice of an appropriate vector may depend on the choice of protein expression system to be used. The expression systems, as established and extensively described in scientific literature well known to the person skilled in the art, include prokaryotic bacterial cells, eukaryotic yeast and fungal cells, as well as cell cultures derived from higher animals such as insects, arthropods or mammals. Introduction of the protein expression vector, equipped with the coding sequence for the chosen MT protein variant, into appropriate host cells, may be facilitated by established protocols.
As to instant claim 1’s step 5, in addition to para 57 above, that statement from the first Example, from para 143, “As representative examples for one preferred embodiment of the invention, the main protein fractions were then partially metallated with 4 molar equivalents of zinc ions (“hypo-MT1a” and “hypo-MT2a”), by adding respective amounts of zinc chloride, based on Bradford protein quantification,” see also para 145, make obvious step 5’s “partially metallating the reduced de-metallated metallothionein protein with 4 molar equivalents of zinc ions,” based on applying a specifically taught amount in the same step.
Melcher paras 55 and 56 make obvious instant claim 1’s steps involving de-metallating and chemically reducing, and per any of the approaches of paras 83-85 instant claim 1’s first step of producing a metallothionein protein would have been obvious except for the now-specified use of a Pichia yeast host and SEQ ID NO:6 comprising at least one peptide extension in the form of a histidine affinity tag.
The level of ordinary skill in the art of yeast production and also purification of polypeptides and proteins is high.
Karbalaei clearly teaches that use of Pichia pastoris yeast, this by its biological genus and species encompassed by the claim’s “Pichia yeast host”, as a host for production of a wide range of proteins is very common and successful, and affords a number of advantages over other protein production hosts, see entire document, noting from the Abstract, “P. pastoris expression system is one of the most popular and standard tools for the production of recombinant protein in molecular biology.” Selecting and claiming one of the most popular and standard tools for the production of recombinant protein in molecular biology would have been obvious based on the expected advantages set forth in Karbalaei, and would have had a reasonable expectation of success given the wide and popular use of this host in the art for diverse polypeptides.
Karbalaei does not teach SEQ ID NO:6 comprising at least one peptide extension in the form of a histidine affinity tag, nor the metal affinity chromatography.
Honda teaches a “one-step purification of hepatic metallothionein from the Amazon fish Colossoma macropomum injected with cadmium and from the copper-loaded metallothionein from the yeast Saccharomyces cerevisiae, performed by affinity chromatography through metal chelating columns. … hepatic metallothionein extracted from fishes was purified by Ni2+-loaded resin and eluted by a continuous imidazol gradient. Purified metallothioneins were evaluated by SDS–PAGE and characterized by UV spectra of the apo- and Cd2+-loaded protein. This method allowed high purity and yield as well as rapid one-step extraction of both metal-loaded and apoprotein.” Abstract.
Honda teaches that other methods for metallothionein purification generally include several steps, take time, and have a high loss of protein mass, page 205, whereas its method is simpler and only requires a single column, Abstract and page 206, section 2.4.
Based on Honda’s teachings and results, one of ordinary skill in the art would have been motivated to further improve the methods of Melcher by purification that includes metal affinity chromatography. The motivation is to purify metallothioneins with a simpler, faster method that provides greater yield by having less protein losses. There would have been a reasonable expectation of success given the teachings and results of Honda, including for metallothioneins from a fish species.
Honda teaches that affinity chromatography “has been a widespread and powerful technique for purification of mainly recombinant proteins modified with specific sequences, e.g., polyhistidine tails, enzymes, monoclonal antibodies, DNA-binding proteins and so on,” page 209, but does not extensively teach the advantages of utilizing a poly-histidine tag for purification procedures.
Abdullah teaches that poly-histidine tags, “fused to either the N- or C-terminal end of the target protein and hence provides a strong selective affinity for binding to metal ions and allows histidine tagged proteins to be separated using the immobilized metal affinity chromatography (IMAC) strategy. IMAC separation works efficiently under mild operating condition is low in cost, robust, and has high ligand selectivity and capacity, thus making it suitable for large-scale applications,” (citation omitted), page 502.
Thus Abdullah expands on the single statement in Honda regarding “polyhistidine tails”, and teaches that this provides a strong selective affinity for binding to metal ions and allows histidine tagged proteins to be separated using the immobilized metal affinity chromatography. This clearly supports that metal affinity chromatography utilizing histidine tags was commonly practiced and common in the art, so that one of ordinary skill in the art wanting to improve purification and separation of a desired protein would reasonably consider applying his-tag separation and purification. The rationale is to improve the process of Melcher to obtain a desired purified metallothionein by incorporating a Histidine tag, as routinely used in the art to obtain a simpler purification with improved yield. There would have been a reasonable expectation of success given the teachings and results of Abdullah, also considering the teachings of Honda.
None of Melcher, Karbalaei, Honda or Abdullah explicitly teach the specific sequence of SEQ ID NO:6’s first 61 amino acids.
However, Melcher clearly teaches producing and purifying and using metallothionein 2a (MT2a), see paras 69-72, 106 with SEQ ID NO:4 referred to as bovine metallothionein 2a protein (MT2a), 143, 145-150.
Melcher’s SEQ ID NO:4 compared with instant SEQ ID NO:6 is as follows:
Query Match 95.5%; Score 364; DB 1; Length 67;
Best Local Similarity 91.8%;
Matches 56; Conservative 3; Mismatches 2; Indels 0; Gaps 0;
Qy 1 MDPNCSCTAGESCTCAGSCKCKDCKCASCKKSCCSCCPVGCAKCAQGCVCKGASDKCSCC 60
||||||| ||:|||||||||||:||| |||||||||||||||||||||:|||||||||||
Db 1 MDPNCSCAAGDSCTCAGSCKCKECKCTSCKKSCCSCCPVGCAKCAQGCICKGASDKCSCC 60
Qy 61 A 61
|
Db 61 A 61
Melcher teaches multiple uses of its processed metallothioneins for treating human subjects having a variety of diseases/conditions, see paras 5, 7-9 (providing background), 92, teaching alternatives of bovine and human MT isoforms, and 120-132, see also claims 10-15, so one of ordinary skill in the art would have considered substituting a similar human MT2a for Melcher’s bovine MT2a at least because a human MT2a would reasonably have greater chance of greater tolerance and effectiveness than a bovine MT2a when administering to a human subject.
Among known human metallothionein 2A sequences is a 62 amino acid sequence, identified as a partial synthetic construct, that is 100% identical with SEQ ID NO:2’s first 61 amino acids (i.e., without the His tag), this provided by Kalnine et al., submitted to GenBank 5/13/2003, having the following sequence:
mdpncscaag dsctcagsck ckeckctsck ksccsccpvg cakcaqgcic kgasdkcscc al (GenBank AAP36553.1, provided).
The only difference between this sequence and instant SEQ ID NO:6 excluding the His tag is that the GenBank sequence comprises a 62nd amino acid, Leucine. However, GenBank AAP36553.1 clearly teaches that the region of 4 to 61, which excludes the leucine at position 62, is the region having the name “Metallothio” that belongs in pfam00131. Pfam00131 comprises a group of sequences of different species having a common core that is identified as having metallothionein function, see evidentiary reference Pfam. At a minimum, this suggests to one of ordinary skill in the art that the C-terminus leucine is not required for metallothionein activity, and a person of ordinary skill in the art would reasonably understand that the leucine at position 62 is not expected to be required for metallothionein function or activity based on this distinction. That is, GenBank AAP36553.1 states that from the sequence between 4 to 61, inclusive of these end amino acids, is that portion of the entire sequence that belongs within the pfam00131 of methallothioneins, and is responsible for metallothionein activity, so the C-terminus leucine would not be required for such metallothionein activity.
One of ordinary skill in the art seeking a human MT2a sequence instead of the bovine MT2a taught by Melcher, to better treat human diseases/conditions, would have identified the human MT2A sequence of GenBank AAP36553.1, and would have considered substitution of this for the bovine sequence of Melcher. Based on the above teachings in GenBank AAP36553.1, and as further evidenced by Pfam, in developing a desired metallothionein for therapeutic use the C-terminal leucine would be understood by one of ordinary skill in the art to not be required for function. The motivation to not include this C-terminus lysine is to provide a human metallothionein-derived sequence that comprises the sequence providing the metallothionein function, without amino acids not required for such function or for other functions (e.g., methionine as the first amino acid in an expressed sequence) and is derived from a human metallothionein so as to afford potentially improved performance when treating human diseases/conditions, including, additionally, with less risk of potential adverse immune response.
As an additional basis, when considering that Melcher SEQ ID NO:4 terminates with SCCA, without a leucine, one of ordinary skill in the art would have found additional support for there being no need for the terminal leucine of GenBank AAP36553.1. That is, this teaching and example of Melcher further supports that the C-terminus leucine of GenBank AAP36553.1 would not be required for metallothionein activity.
There would have been a reasonable expectation of success given the description in GenBank AAP36553.1 of the 4..61 portion being that portion in the metallothio pfam00131 – so indicating desired function, and also the teachings of Melcher including that of SEQ ID NO:4 and as to use of human MT2a. Additionally, it is not inventive to substitute a known human protein for a previously taught bovine protein of the same type and function when considering use of that protein in therapies for humans, and to not include any amino acid of a reported human sequence that is not attributed to the desired function, here the metallothionein function.
Accordingly, claim 1 would have been obvious, as would the elected species SEQ ID NO:6.
Claim 3 would have been obvious based on the teachings in references applied to claim 1, specifically including Melcher’s teachings of fully pre-metallating, see para 59, and Melcher claim 3, and of magnetic force purification including when fully pre-metallated, paras 96 and 97.
Claim 4 would have been obvious based on the additional teaching of using zinc sulfate or zinc chloride, “…in order to allow for full pre-metallation. From this solution, the fully reconstituted Zn.sub.7-MT protein may be recovered using magnetic force,” para 97 of Melcher.
Claim 5 would have been obvious based on the teachings in references applied to claim 1, and the additional Melcher claim 4, having substantially the same language as instant claim 5.
Claim 6 would have been obvious based on the teachings in references applied to claim 1, and specifically Melcher’s para 99 teachings, these including relevant portions of claim 6 limitations.
Claims 7 and 8 would have been obvious based on the teachings in references applied to claim 1, and specifically Melcher’s para 100 teachings combined with Melcher claims 5 and 6.
Claims 9 and 10 would have been obvious based on the teachings in references applied to claim 1, and specifically Melcher’s para 101 teachings combined with Melcher claims 7 and 8.
Claims 11 and 12 would have been obvious based on the teachings in references applied to claim 1, and specifically Melcher’s para 102 teachings.
Claim 14 would have been obvious based on the teachings in references applied to claim 1, and specifically Melcher’s para 82 teachings combined with Melcher claims 11 and 12.
Claim 15 would have been obvious based on the teachings in references applied to claim 1, and specifically Melcher’s paras 128, 148-150 and Fig. 3A and 3B.
Claim 16 would have been obvious based on the teachings in references applied to claim 1, and specifically Melcher’s paras 115, 121-130, and 151-154.
Claim(s) 13 is rejected under 35 U.S.C. 103 as being unpatentable over US 20160130325, published 5/12/16, inventors Melcher et al. (Melcher, parent of instant CIP), in view of Honda et al, “One-step purification of metallothionein extracted from two different sources,” Journal of Chromatography B, 820 (2005) 205–210 (Honda), Abdullah and Chase, BIOTECHNOLOGY AND BIOENGINEERING, VOL. 92, NO. 4, NOVEMBER 20, 2005, 501-513 (Abdullah), GenBank AAP36553.1 (2003), and Karbalaei et al., J Cell Physiol. 2020;235:5867–5881(Karbalaei), as evidenced by Metallothionein Pfam downloads, two screen shots combined to one page, downloaded 10/15/25 (“Pfam”, previously provided), as applied to claim 1 above, and as evidenced by GE Instructions 11-0008-88 AH, 24 pages, 2014 (GE, previously provided).
The rejection of claim 1 is set forth above.
Claim 13 depends from claim 1 and states “wherein the metal affinity chromatography utilizes a nickel (Ni++) prepacked column.
Honda teaches using a HiTrapTM Chelating HP column that comprises Ni2+, and was previously loaded, page 206 sections 2.2 and 2.4, see also page 207 bottom right column, but does not explicitly state that this was prepacked.
As noted above, the level of ordinary skill in the art of purification of peptides and proteins is high.
GE evidences that “HisTrap FF is a ready to use HiTrap™ column, prepacked with precharged Ni Sepharose™ 6 Fast Flow. This prepacked column is ideal for preparative purification of histidine-tagged recombinant proteins by immobilized metal ion affinity chromatography (IMAC).
HisTrap FF columns provide fast, simple, and easy separations in a convenient format, including an excellent start for scaling up.”
GE evidences that the column used by Honda was prepacked, so claim 13 would have been obvious over the rejection applied to claim 1 above, with the evidentiary reference GE supporting the rejection as to the prepacked limitation.
Claim(s) 17 is rejected under 35 U.S.C. 103 as being unpatentable over US 20160130325, published 5/12/16, inventors Melcher et al. (Melcher, parent of instant CIP), in view of Honda et al, “One-step purification of metallothionein extracted from two different sources,” Journal of Chromatography B, 820 (2005) 205–210 (Honda), Abdullah and Chase, BIOTECHNOLOGY AND BIOENGINEERING, VOL. 92, NO. 4, NOVEMBER 20, 2005, 501-513 (Abdullah), GenBank AAP36553.1 (2003), and Karbalaei et al., J Cell Physiol. 2020;235:5867–5881(Karbalaei), as evidenced by Metallothionein Pfam downloads, two screen shots combined to one page, downloaded 10/15/25 (“Pfam”, previously provided), as applied to claim 1 above, and further in view of Quantifying protein using absorbance at 280 nm, Experimental Biosciences web page from www.rice.edu, 2015, 3 pages, downloaded from the internet 10/14/25 (Rice, previously provided).
The rejection of claim 1 is set forth above.
Claim 17 depends from claim 1 and further comprises determining the concentration of the purified metallothionein protein by ultraviolet (UV) absorbance at 280 nm.
None of the references applied to claim 1 explicitly teach this claim 17 step.
As noted above, the level of ordinary skill in the art of purification of peptides and proteins is high.
Rice teaches that “Quantifying protein by directly measuring absorbance is fast and convenient, since no additional reagents or incubations are required. No protein standard need be prepared and the procedure does not consume the protein,” page 1. Rice also teaches specific steps for determining protein content by measuring ultraviolet (UV) absorbance at 280 nm, page 2, Procedure.
It would have been obvious to apply the method of Rice to measure protein content as set forth in claim 17 because this method is fast, convenient, and does not consume the protein. There would have been a reasonable expectation of success because the method as taught by Rice is commonly used for the same or similar purpose as set forth in Rice, “The most common use for this method is to monitor fractions from chromatography columns, or any time a quick estimation is needed and error in protein concentration is not a concern.”
Accordingly, claim 17 would have been obvious and is rejected under this section.
NEW claims 18 and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Karbalaei et al., J Cell Physiol. 2020;235:5867–5881(Karbalaei) in view of METHODS IN ENZYMOLOGY, VOL. 326, chapter 16, 2000 (“BF”).
Claim 18, broader than claim 1, is as follows:
18. (New) A method for producing a biologically active polypeptide, comprising:
expressing a nucleic acid sequence encoding a polypeptide of choice in a Pichia yeast host cell, thus obtaining an expressed polypeptide of choice, the Pichia yeast host cell comprising a protein expression vector comprising a nucleic acid operably linked to a Pichia compatible promoter and equipped with a coding sequence for said polypeptide of choice; and purifying said expressed polypeptide of choice by affinity chromatography.
Claims 18 and 19 are much broader than just that elected sequence, which is not included in claim 18 or claim 19 limitations, and are rejected herein expanding beyond the elected species to address this breadth.
Karbalaei clearly teaches that use of Pichia pastoris yeast as a host for production of a wide range of proteins is very common and successful, and affords a number of advantages over other protein production hosts, see entire document, noting from the Abstract, “P. pastoris expression system is one of the most popular and standard tools for the production of recombinant protein in molecular biology.” Selecting and claiming one of the most popular and standard tools for the production of recombinant protein in molecular biology would have been obvious based on the expected advantages set forth in Karbalaei, and would have had a reasonable expectation of success given the wide and popular use of this host in the art.
Karbalaei clearly makes obvious the use of Pichia yeast host cell for production to obtain an expressed polypeptide of choice, at least given the multiple examples in that reference of successful production, and teaches use of a Pichia compatible promoter operably linked to a protein expression vector comprising a nucleic acid, equipped with a coding sequence for the polypeptide of choice, see Section 3.2 and Figure 2, however does not teach purifying by affinity chromatography the expressed polypeptide.
The level of ordinary skill in the art of yeast production and also purification of polypeptides and proteins is high.
BF broadly teaches a versatile method “A powerful purification method involves the use of peptide affinity tags, which are fused to the protein of interest and used to expedite protein purification via affinity chromatography. 1'2 A widely employed method utilizes immobilized metal-affinity chromatography (IMAC) to purify recombinant proteins containing a short affinity tag consisting of polyhistidine residues. IMAC is based on the interactions between a transition metal ion (Co 2+, Ni 2÷ , Cu 2÷ , Zn 2+) immobilized on a matrix and specific amino acid side chains. Histidine is the amino acid that exhibits the strongest interaction with immobilized metal ion matrices, as electron donor groups on the histidine imidazole ring readily form coordination bonds with the immobilized transition metal. Peptides containing sequences of consecutive histidine residues are efficiently retained on IMAC column matrices. Following washing of the matrix material, peptides containing polyhistidine sequences can be easily eluted by either adjusting the pH of the column buffer or adding free imidazole to the column buffer.”
Given its versatility and effectiveness, see entire document, one of ordinary skill in the art would have been motivated to apply this purification technique to purify any of a range of polypeptides that could be produced per Karbalaei’s yeast production teachings. This is based on using a known purification technique that could be applied to purify polypeptides produced by a common production method, with a reasonable expectation of success given the respective teachings.
Accordingly, claim 18 would have been obvious and is rejected.
Claim 19 also would have been obvious based on BF’s teaching that “If necessary, the affinity tag can be removed by use of a protease cleavage site inserted between the tag and the protein,” page 246, see entire para that includes this ending sentence. Note also that cleavable can be interpreted broadly to include any method of cleavage, however the teaching in BF is sufficient to reject this limitation.
NEW claims 18-20 are rejected under 35 U.S.C. 103 as being unpatentable over US 20160130325, published 5/12/16, inventors Melcher et al. (Melcher, parent of instant CIP), in view of Honda et al, “One-step purification of metallothionein extracted from two different sources,” Journal of Chromatography B, 820 (2005) 205–210 (Honda), Abdullah and Chase, BIOTECHNOLOGY AND BIOENGINEERING, VOL. 92, NO. 4, NOVEMBER 20, 2005, 501-513 (Abdullah), and GenBank AAP36553.1 (2003), these references previously applied and prior art references provided, and Karbalaei et al., J Cell Physiol. 2020;235:5867–5881(Karbalaei), as evidenced by Metallothionein Pfam downloads, two screen shots combined to one page, downloaded 10/15/25 (“Pfam”). Karbalaei et al., J Cell Physiol. 2020;235:5867–5881.
This rejection considers the elected species of a purified metallothionein protein having an amino acid identified by SEQ ID NO:6 as being the claim 18 polypeptide of choice.
The elected sequence is SEQ ID NO:6, which includes a six histidine “his tag” at its C-terminus.
Melcher teaches an invention that relates to method for producing hypo-metallated redox-active metallothionein (MT) proteins, pharmaceutical compositions containing the proteins, and uses the pharmaceutical compositions for treatment of conditions originating from elevated intracellular oxidative stress and/or dis-balanced intracellular redox-potential and/or redox-potential-dependent imbalance of metal ions, Abstract.
In some embodiments Melcher’s method steps are as follows:
[0053] The first aspect of the present invention relates to a method for producing a hypo-metallated redox-active metallothionein protein. The hypo-metallated redox-active metallothionein protein has 20 cysteine sulfhydryl groups, thus forming 7 metal ion binding pockets. Two (2) to 16 of the 20 cysteine sulfhydryl groups are free and reduced, and 1 to 6 of the 7 binding pockets are occupied by metal ions. The method comprises the following steps:
[0054] providing a metallothionein protein;
[0055] de-metallating the metallothionein protein;
[0056] chemically reducing all the 20 cysteine sulfhydryl groups of the de-metallated metallothionein protein; and
[0057] partially metallating the reduced de-metallated metallothionein protein by providing 1, 2, 3, 4, 5, or 6 metal ions to the 7 binding pockets.
Melcher teaches that
[0083] The metallothione in apo-proteins of the present invention, in their native form with undefined metallation status and undefined redox-status of their cysteine sulfur residues, may be produced by any conventional method of recombinant DNA technology, protein synthesis, enzymatic cleavage of pre-pro-proteins, or isolation of naturally occurring variants of mammalian metallothionein proteins.
[0084] Thus, in one preferred embodiment of the invention, the metallothionein apo-proteins are produced by established recombinant DNA technology (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, 1989, and references therein). The DNA sequence encoding the respective full-length metallothionein protein isoform may either be synthesized by standard nucleic acid synthesis methods, or may be obtained by established laboratory methods of directly isolating it from appropriate genomic or cDNA libraries, or indirectly by copying and amplifying it from said libraries by standard polymerase chain reaction (PCR) method.
[0085] The DNA sequence, after appropriate preparation and purification, may then be introduced into a recombinant expression vector of choice, using, again, established standard methods. The choice of an appropriate vector may depend on the choice of protein expression system to be used. The expression systems, as established and extensively described in scientific literature well known to the person skilled in the art, include prokaryotic bacterial cells, eukaryotic yeast and fungal cells, as well as cell cultures derived from higher animals such as insects, arthropods or mammals. Introduction of the protein expression vector, equipped with the coding sequence for the chosen MT protein variant, into appropriate host cells, may be facilitated by established protocols.
Melcher thus teaches production and purification of polypeptides “of choice” per claim 18 that have a “biological activity characteristic of the polypeptide defined by SEQ ID NO:6” per claim 20 (this interpreted broadly), but does not teach production of same in a Pichia yeast host cell per the limitations of claim 18.
The level of ordinary skill in the art of yeast production and also purification of polypeptides and proteins is high.
Karbalaei clearly teaches that use of Pichia pastoris yeast as a host for production of a wide range of proteins is very common and successful, and affords a number of advantages over other protein production hosts, see entire document, noting from the Abstract, “P. pastoris expression system is one of the most popular and standard tools for the production of recombinant protein in molecular biology.” Selecting and claiming one of the most popular and standard tools for the production of recombinant protein in molecular biology would have been obvious based on the expected advantages set forth in Karbalaei, and would have had a reasonable expectation of success given the wide and popular use of this host in the art.
Karbalaei clearly makes obvious the use of Pichia yeast host cell for production to obtain an expressed polypeptide of choice, at least given the multiple examples in that reference of successful production, and teaches use of a Pichia compatible promoter operably linked to a protein expression vector comprising a nucleic acid, equipped with a coding sequence for the polypeptide of choice, see Section 3.2 and Figure 2, however does not teach purifying by affinity chromatography the expressed polypeptide.
The level of ordinary skill in the art of yeast production and also purification of polypeptides and proteins is high.
Karbalaei does not teach SEQ ID NO:6 comprising at least one peptide extension in the form of a histidine affinity tag, nor the metal affinity chromatography.
Honda teaches a “one-step purification of hepatic metallothionein from the Amazon fish Colossoma macropomum injected with cadmium and from the copper-loaded metallothionein from the yeast Saccharomyces cerevisiae, performed by affinity chromatography through metal chelating columns. … hepatic metallothionein extracted from fishes was purified by Ni2+-loaded resin and eluted by a continuous imidazol gradient. Purified metallothioneins were evaluated by SDS–PAGE and characterized by UV spectra of the apo- and Cd2+-loaded protein. This method allowed high purity and yield as well as rapid one-step extraction of both metal-loaded and apoprotein.” Abstract.
Honda teaches that other methods for metallothionein purification generally include several steps, take time, and have a high loss of protein mass, page 205, whereas its method is simpler and only requires a single column, Abstract and page 206, section 2.4.
Based on Honda’s teachings and results, one of ordinary skill in the art would have been motivated to further improve the methods of Melcher by purification that includes metal affinity chromatography. The motivation is to purify metallothioneins with a simpler, faster method that provides greater yield by having less protein losses. There would have been a reasonable expectation of success given the teachings and results of Honda, including for metallothioneins from a fish species.
Honda teaches that affinity chromatography “has been a widespread and powerful technique for purification of mainly recombinant proteins modified with specific sequences, e.g., polyhistidine tails, enzymes, monoclonal antibodies, DNA-binding proteins and so on,” page 209, but does not extensively teach the advantages of utilizing a poly-histidine tag for purification procedures.
Abdullah teaches that poly-histidine tags, “fused to either the N- or C-terminal end of the target protein and hence provides a strong selective affinity for binding to metal ions and allows histidine tagged proteins to be separated using the immobilized metal affinity chromatography (IMAC) strategy. IMAC separation works efficiently under mild operating condition is low in cost, robust, and has high ligand selectivity and capacity, thus making it suitable for large-scale applications,” (citation omitted), page 502.
Thus Abdullah expands on the single statement in Honda regarding “polyhistidine tails”, and teaches that this provides a strong selective affinity for binding to metal ions and allows histidine tagged proteins to be separated using the immobilized metal affinity chromatography. This clearly supports that metal affinity chromatography utilizing histidine tags was commonly practiced and common in the art, so that one of ordinary skill in the art wanting to improve purification and separation of a desired protein would reasonably consider applying his-tag separation and purification. The rationale is to improve the process of Melcher to obtain a desired purified metallothionein by incorporating a Histidine tag, as routinely used in the art to obtain a simpler purification with improved yield. There would have been a reasonable expectation of success given the teachings and results of Abdullah, also considering the teachings of Honda.
None of Melcher, Karbalaei, Honda or Abdullah explicitly teach the specific sequence of SEQ ID NO:6’s first 61 amino acids.
However, Melcher clearly teaches producing and purifying and using metallothionein 2a (MT2a), see paras 69-72, 106 with SEQ ID NO:4 referred to as bovine metallothionein 2a protein (MT2a), 143, 145-150.
Melcher’s SEQ ID NO:4 compared with instant SEQ ID NO:6 is as follows:
Query Match 95.5%; Score 364; DB 1; Length 67;
Best Local Similarity 91.8%;
Matches 56; Conservative 3; Mismatches 2; Indels 0; Gaps 0;
Qy 1 MDPNCSCTAGESCTCAGSCKCKDCKCASCKKSCCSCCPVGCAKCAQGCVCKGASDKCSCC 60
||||||| ||:|||||||||||:||| |||||||||||||||||||||:|||||||||||
Db 1 MDPNCSCAAGDSCTCAGSCKCKECKCTSCKKSCCSCCPVGCAKCAQGCICKGASDKCSCC 60
Qy 61 A 61
|
Db 61 A 61
Melcher teaches multiple uses of its processed metallothioneins for treating human subjects having a variety of diseases/conditions, see paras 5, 7-9 (providing background), 92, teaching alternatives of bovine and human MT isoforms, and 120-132, see also claims 10-15, so one of ordinary skill in the art would have considered substituting a similar human MT2a for Melcher’s bovine MT2a at least because a human MT2a would reasonably have greater chance of greater tolerance and effectiveness than a bovine MT2a when administering to a human subject.
Among known human metallothionein 2A sequences is a 62 amino acid sequence, identified as a partial synthetic construct, that is 100% identical with SEQ ID NO:2’s first 61 amino acids (i.e., without the His tag), this provided by Kalnine et al., submitted to GenBank 5/13/2003, having the following sequence:
mdpncscaag dsctcagsck ckeckctsck ksccsccpvg cakcaqgcic kgasdkcscc al (GenBank AAP36553.1, provided).
The only difference between this sequence and instant SEQ ID NO:6 excluding the His tag is that the GenBank sequence comprises a 62nd amino acid, Leucine. However, GenBank AAP36553.1 clearly teaches that the region of 4 to 61, which excludes the leucine at position 62, is the region having the name “Metallothio” that belongs in pfam00131. Pfam00131 comprises a group of sequences of different species having a common core that is identified as having metallothionein function, see evidentiary reference Pfam. At a minimum, this suggests to one of ordinary skill in the art that the C-terminus leucine is not required for metallothionein activity, and a person of ordinary skill in the art would reasonably understand that the leucine at position 62 is not expected to be required for metallothionein function or activity based on this distinction. That is, GenBank AAP36553.1 states that from the sequence between 4 to 61, inclusive of these end amino acids, is that portion of the entire sequence that belongs within the pfam00131 of methallothioneins, and is responsible for metallothionein activity, so the C-terminus leucine would not be required for such metallothionein activity.
One of ordinary skill in the art seeking a human MT2a sequence instead of the bovine MT2a taught by Melcher, to better treat human diseases/conditions, would have identified the human MT2A sequence of GenBank AAP36553.1, and would have considered substitution of this for the bovine sequence of Melcher. Based on the above teachings in GenBank AAP36553.1, and as further evidenced by Pfam, in developing a desired metallothionein for therapeutic use the C-terminal leucine would be understood by one of ordinary skill in the art to not be required for function. The motivation to not include this C-terminus lysine is to provide a human metallothionein-derived sequence that comprises the sequence providing the metallothionein function, without amino acids not required for such function or for other functions (e.g., methionine as the first amino acid in an expressed sequence) and is derived from a human metallothionein so as to afford potentially improved performance when treating human diseases/conditions, including, additionally, with less risk of potential adverse immune response.
As an additional basis, when considering that Melcher SEQ ID NO:4 terminates with SCCA, without a leucine, one of ordinary skill in the art would have found additional support for there being no need for the terminal leucine of GenBank AAP36553.1. That is, this teaching and example of Melcher further supports that the C-terminus leucine of GenBank AAP36553.1 would not be required for metallothionein activity.
There would have been a reasonable expectation of success given the description in GenBank AAP36553.1 of the 4..61 portion being that portion in the metallothio pfam00131 – so indicating desired function, and also the teachings of Melcher including that of SEQ ID NO:4 and as to use of human MT2a. Additionally, it is not inventive to substitute a known human protein for a previously taught bovine protein of the same type and function when considering use of that protein in therapies for humans, and to not include any amino acid of a reported human sequence that is not attributed to the desired function, here the metallothionein function.
Accordingly, claim 18 would have been obvious considering the elected species SEQ ID NO:6.
Claim 19 also would have been obvious based on Abdull, see page 502 regarding cleavage of fusion tags using either chemical or enzymatic methods, clearly indicating that such removal is routine and achieved by a number of approaches that include such claimed “at least one peptide extension comprising an affinity tag comprising a histidine tag cleavable from said expressed polypeptide.
Claim 20 would have been obvious on the same bases applied above to claim 18.
Conclusion
No claim is 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 JOSEPH FISCHER whose telephone number is (571)270-7925. The examiner can normally be reached on Monday to Friday, 9:00 AM to 5:00 PM, however noting that the examiner will not normally be working on Wednesday-Friday and on Monday/Tuesday on alternating weeks, but will promptly answer messages upon his return to work.
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/JOSEPH FISCHER/Primary Examiner, Art Unit 1658
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