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 .
Claims 1, 3, 5-8, 15, 28-29, 35, 38, 44, 46, 51-53, 72, 75-76 and 85-89 are pending.
Claims 44, 46, 51-53, 75 and 76 are withdrawn from further consideration by the examiner, 37 C.F.R. 1.142(b) as being drawn to non-elected inventions.
Claims 1, 3, 5-8, 15, 28-29, 35, 38, 72, and 85-89, drawn to a protein conjugate that read on group (e)
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as the masking agent, (B) polyethyleneimine (PEI) as the species of cell penetrating moiety, (C) single domain antibody as the species of biological payload, are being acted upon in this Office Action.
Priority
Applicant’ claim priority to provisional application 63/275,549, filed November 3, 2021, and 63/348,114, filed June 2, 2022 is acknowledged.
New ground of rejection is necessitated by the amendment filed December 2, 2025
Claim rejections under - 35 U.S.C. 112
The following is a quotation of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), first paragraph:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claim 89 is rejected under 35 U.S.C. 112, first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor(s), at the time the application was filed, had possession of the claimed invention. This is New Matter.
The recitation of “does not undergo deprotection at a pH value of 7 or greater” has no support in the specification and the claims as originally filed. Applicant is required to remove the New Matter in response to this Office Action.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102 of this title, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negatived by the manner in which the invention was made.
The factual inquiries set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied for establishing a background for determining obviousness under 35 U.S.C. 103(a) 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 under pre-AIA 35 U.S.C. 103(a), the examiner presumes that the subject matter of the various claims was commonly owned at the time any inventions covered therein were made absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and invention dates of each claim that was not commonly owned at the time a later invention was made in order for the examiner to consider the applicability of pre-AIA 35 U.S.C. 103(c) and potential pre-AIA 35 U.S.C. 102(e), (f) or (g) prior art under pre-AIA 35 U.S.C. 103(a).
Claims 1, 3, 5-8, 15, 35, 72, 85, 86, 87 and 89 are rejected under 35 U.S.C. 103 as being unpatentable over WO2013177421 publication (of record, published November 28, 2013; PTO 892) in view of US Patent 7,019,113 (newly cited, issued March 28, 2006; PTO 892) and Huang et al (Materials Science & Engineering C 102: 558-568, 2019; PTO 892).
Regarding claims 1, 6, 8, the WO2013177421 publication teaches pH-responsive protein conjugated nanoparticle, e.g., HSA-PEI conjugate comprising human serum albumin (HSA) covalently linked to positively-charged cell penetrating moiety, e.g., polyethyleneimine (PEI) via a linker using suitable crosslinking reagent, e.g., l-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC), see para. [0008], [0062]. The reference conjugate may be crosslinked to payload, e.g., targeting agents, proteins, or other molecules, see para. [0062]. The reference polyethyleneimine has a molecular weight of less than 2000 Da, e.g., PEI600 Da, see para. [0012], [0075], [0077], [00116] to [00121]. The polyethyleneimine can be branched or linear type, see para. [0010]. The EDC enhances the link between carboxyl group in HSA and amine group on PEI. The low molecular weight pH-sensitive polymer (polyethyleneimine MW 600, PEI600) is conjugated to human serum albumin (HSA) via cross linking agents, resulting in a hyper-cationized pH-responsive APC, see para. [0077]. The WO2013177421 publication teaches that high molecular weight polymers such as PEI (MW 25,000) are useful as transfection agents, but suffer from cytotoxicity. Low molecular weight PEI (MW 600) does not cause cytotoxicity, but is limited due to its inability to facilitate stable condensation with NAs. The conjugation of low molecular weight polymers to a larger molecule such as albumin is thus a useful method of increasing activity of electrostatic complexation with NA condensation while lowing cytotoxicity of LN formulations, see para. [0060]. The WO2013177421 publication teaches albumin also possesses endosomal lytic activity due to its hydrophobic core, which upon conformational change can be exposed and can induce bilayer disruption or membrane fusion. In some embodiments, such as the HSA-PEI600 conjugate, the APC has an ionization profile that is responsive to pH change. The charge density is increased at endosomal pH, which is acidic. See para. [0075]. A low molecular weight pH-sensitive polymer (polyethyleneimine MW 600, PEI600) is conjugated to human serum albumin (HSA) via cross linking agents, resulting in a hyper-cationized pH-responsive APC. The addition of HSA-PEI600 conjugates to LNs significantly increases downregulation of RRM1 (aka RNR Rl) with ASO LOR-2501 (purchased from Alpha DNA) in the presence of serum without substantial cytotoxicity in KB cells (a subline of HeLa), see para. [0077]. The term “comprising” in claim 1 is open ended. It expands to include lipid. It is desired that the zeta potential of LNs not be excessively positive or negative for systemic delivery. LNs with a highly positive charge tend to interact non-specifically with non-target cells, tissues, and circulating plasma proteins, and may cause cytotoxicity, see para. [0047]. Alternatively, LNs with a highly negative charge cannot effectively incorporate nuclei acids (NAs), which are themselves negatively charged, and may trigger rapid RES-mediated clearance, reducing therapeutic efficacy. LNs with a neutral to moderate charge are best suited for in vivo drug and gene delivery, see para. [0047].
Regarding claim 6, the WO2013177421 publication teaches that up to 20 polycations can be linked to each albumin molecule, which is between 2 and 30 PEI, p. 6, para. [0048]. The term “or” does not require all of a, b, d to j.
Regarding claim 15, the WO2013177421 publication teaches that the payload, e.g., oligonucleotides, such as antisense ODNs, pDNAs, siRNAs, shRNAs, miRs, and anti-miRs, (see para. [0075], [0078]), alpha DNA and Era, see [00126] to [00127] form by ionic complex (aka devoid of a disulfide bond) with the positively charged polyethyleneimine.
Regarding claim 72, the WO2013177421 publication teaches a pharmaceutical composition and pharmaceutically acceptable excipient, see p. 15-16.
Regarding claim 85, the WO2013177421 publication teaches that the HSA-PEI has zeta potential of about 0 to about + 40 mV, which is at least 8 mV, see reference claim 3. Claim 35 is included as it is within the purview of one of ordinary skill in the art to optimize the negative zeta potential to less than -1 mV.
Regarding claim 87, the WO2013177421 publication teaches that the payload is antineoplastic agents (aka devoid of DNA), see reference claim 19, in particular.
The WO2013177421 publication teaches that the albumin-PEI conjugate via disulfide linkages, see p. 20, para. [00118].
The WO2013177421 publication does not teach that the protein conjugate has a negative zeta potential and the PEI is bound to a protecting group by a covalent bond, wherein said protecting group undergoes deprotection at a pH value of less than 7 and is represented by Formula 1:
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However, the ‘113 patent teaches a process to modify an amine-containing molecule with a substituted maleic anhydride or derivative thereof wherein cleavage of the modification is accelerated upon exposure to pH less than 7 (aka protecting group undergoes deprotection at a pH value of less than 7) as per claim 89, see entire document, claims, in particular. Upon cleavage of the anhydride, the amine on the molecule is regenerated, see entire document, Summary in particular. Example of pH-sensitive amines present on polyamines such as PEI may also play a role in endosomal release although it is unclear what role protonation plays. To protect or mask or inactivating the amine-containing molecule, the amine-containing molecule is reacted with a substituted maleic anhydrides having the general structure shown in FIG. 1A wherein the R to anhydride bonds can be carbon-hydrogen or carbon-carbon bonds and either R.sup.1 or R.sup.2 but not both, can be a hydrogen atom.
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When reacting amine containing compound with anhydride, the anhydrides form covalent bonds with primary amines as shown
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see Fig 2. For maleic anhydrides (FIG. 1A), the resultant covalent bond can be cleaved under physiological conditions, see col. 5, lines 57-61. Citraconic and cis-aconitic-derived maleamic acids each have one substitution and are more pH-labile. Maleamic acids derived from disubstituted dimethyl maleic anhydride are the most pH-labile, see col. 5, line 64-67. pH-labile refers to the selective breakage of a covalent bond under acidic conditions (pH <7), see col. 10, lines 58-67, in particular. A preferred substituted maleic anhydride is a disubstituted maleic anhydride wherein neither R.sup.1 or R.sup.2 is a hydrogen atom. The ‘113 patent teaches that disubstitution increases the pH-lability of the covalent bond formed between the anhydride and an amine. A preferred disubstituted maleic anhydride is carboxy dimethylmaleic anhydride (CDM), or 2-propionic-3-methylmaleic anhydride, wherein R.sup.1 is --CH.sub.3 and R.sup.2 is --(CH.sub.2).sub.2COOH or R.sup.2 is --CH.sub.3 and R.sup.1 is --(CH.sub.2).sub.2COOH. The addition of the carboxyl group increases the charge and water solubility of the anhydride and enhances inactivation of membrane active and cell permeable compounds. Other functional groups may be added to either R.sup.1, R.sup.2, or both. The reversible inactivation of a cationic import molecule comprising: modifying the molecule by reaction with a substituted maleic anhydride. The reversibly inactivated import molecule can then be associated with a cell and the activity of the import molecule is restored. The reactivation of the import molecule by cleavage of the anhydride modification is enhanced in an acidic environment such as in an endosome/lysosomes or tumor region. The modified cationic import molecule may be delivered to a cell that is in vitro or in vivo, see col. 4, in particular.
Claim 89 is included because the ‘113 patent teaches substituted maleic anhydride or derivative thereof wherein cleavage of the modification is accelerated upon exposure to pH less than 7 (aka protecting group undergoes deprotection at a pH value of less than 7) as per claim 89, see entire document, claims, in particular. Upon cleavage of the anhydride, the amine on the molecule, e.g., PEI is regenerated, see entire document, Summary in particular. Given the claimed anhydride represented by Formula 1 encompassed the reference’s anhydrides, e.g., citraconic anhydride, if the claimed anhydride undergoes deprotection (cleaved) at a pH value of less than 7 and does not undergo deprotection at a pH value of 7 or greater, so is the reference’s. Since the Patent Office does not have the facilities for examining and comparing the anhydride of the instant invention to those of the prior art. The burden is on applicant to show that the prior art anhydride is different from the claimed anhydride. See In re Best. 562 F2d 1252. 195 USPQ 430(CCPA 1977).
Likewise, Huang teaches that the branched polyethyleneimine (bPEI) has a much higher amine group density than most, if not all, of other amine group-containing polymer, see p. 559, left col. Huang teaches that the bPEI was modified with 2,3-dimethylmaleic anhydride to form pH-triggered charge-reversal nanoparticle, see p. 559, left col. Huang shows that reversible blocking of branched polyethyleneimine (bPEI) with a protecting group, e.g., 2,3-dimethylmaleic anhydride (DMA) temporary shield the positive charge of the polymer in bloodstream environment (pH 7.4), as shown in Fig. 4.
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Claim 5 is included as the branched PEI has three kinds of amine groups, primary, secondary and tertiary amines and the primary amines of bPEI are amenable to chemical modification by DMA above. It is within the purview of one of ordinary skill in the art to optimize the % of amine groups of the PEI bound by the protecting group DMA. The claims would have been obvious because "a person of ordinary skill has good reason to pursue the known options within his or her technical grasp. If this leads to the anticipated success, it is likely the product not of innovation but of ordinary skill and common sense". See KSR International Co. v. Teleflex Inc., 82 USPQ2d 1385 (U.S. 2007).
The reference DMA has the claimed formula I wherein n is 0, R and R1 are methyl (aka alkyl). Once located in tumor, the acidic pH-sensitive β-carboxylate amide of DMA-modified bPEI was supposed to be hydrolyzed because of the acidic extracellular environment of solid tumor, regenerating b-PEI-10k and subsequently causing an increase in the permeability of more anticancer drug molecules that entered in tumor tissue, see p. 561, left col. Huang teaches that the pH-triggered zeta potential changes of these modified bPEI-10k and shown in Fig. 5. The zeta (ζ) potentials of bPEI-DMA-1 at pH 7.4 and 6.8 were always negative values during the incubation time period. Only at more acidic pH (6.5) bPEI-DMA-1 showed charge reversal with incubation time, see p. 562, left coll. bPEI-DMA-4 contained zwitterions with a slightly negative charge in bloodstream environment, (pH 7.4). And in tumor acidic environment (pH 6.5 and 6.8), it took a short time to change from negative ζ potentials to positive ones during incubation, see p. 562, left, in particular.
Claims 3 and 86 are included as the conjugate increased accumulation within a biological tissue, e.g., tumor having a pH of less than 7, see p. 562, left, in particular.
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filling date of the claimed invention to combine the teachings of WO2013177421 publication in view of the ‘113 patent and/or Huang by reversibly to modify an amine-containing PEI in the conjugate of the WO2013177421 publication using any one of the ‘113 patent’s maleic anhydride wherein R1 and R2 are H or derivative thereof, e.g., dimethyl maleic anhydride wherein R1 and R2 are CH3 or citraconic anhydride wherein R1 or R2 is H and R2 or R1 is CH3, or cis-aconitic anhydride wherein R1 or R2 is H and R2 and R1 are CH2CO2H or 2-propionic-3-methylmaleic anhydride (CDM) wherein R1 or R2 is CH3 and R2 or R1 is (CH2)2CO2H or Huang’s 2,3-dimethylmaleic anhydride (DMA) in order to enabling reversible conversion of any positively charged amine groups on PEI to negatively charged carboxyl groups to lower the ζ potentials of HSA-PEI conjugate from positive to negative charge and the regeneration of the amine by cleavage of the negatively charged anhydride when exposed to pH < 7 in the endosome.
One of ordinary skill in the art would have had an expectation of success at the time the invention was made to modify the conjugate of the WO2013177421 publication in view of the ‘113 patent because the ‘113 patent teaches that polyamine can be partially or completely modified with anhydride is straightforward and facile. Furthermore, the reversal of the modification in vivo or in vitro is essentially unidirectional. Once the anhydride is cleaved in the acidic endosome, the compound retains activity even if the local pH does not stay acidic, see col. 5, in particular.
One of ordinary skill in the art would have been motivated to do so because the WO2013177421 publication teaches that highly positive charge of the conjugate tend to interact non-specifically with non-target cells, tissues, and circulating plasma proteins, and may cause cytotoxicity and Huang teaches the advantage of using the protecting group 2,3-dimethylmaleic anhydride (DMA) to reversibly blocking the amino groups of HSA-PEI in bloodstream environment (pH 7.4) in order to reduce systemic toxicity (lytic activity); the blocking group DMA could be removed in the endosome at a more acidic pH (6.5). The DMA group could be removed because of the protonic forms of the free carboxylic groups which catalyze the hydrolysis of the amide bonds, see p. 562, left col.
One of ordinary skill in the art would have been motivated to do so because the ‘113 patent teaches that the addition of the carboxyl group using anhydride increases the charge and water solubility of the conjugate molecule and the negatively charge anhydride shields or masks or protects the positive charge amine groups of PEI of the nanoparticles therefore reduce membrane irritation or damage as higher amount of PEI were toxic to cells.
One of ordinary skill in the art would have had an expectation of success before the effective filling date of the claimed invention to modify the HSA-PEI of WO2013177421 publication in view of Huang because Huang each teaches that the dimethylmaleic anhydride (DMMAn or DMA) can be used as a protecting group to mask the positive amine groups on PEI at neutral pH and then undergoes deprotection by hydrolysis of the amide bonds at acidic pH in the endosome of the target cells.
“The combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results.” KSR Int’l Co. v. Teleflex Inc., 550 U.S. 398, 416 (2007).
“The test of obviousness is not express suggestion of the claimed invention in any or all of the references but rather what the references taken collectively would suggest to those of ordinary skill in the art presumed to be familiar with them.” See In re Rosselet 146 USPQ 183, 186 (CCPA 1965).
“There is no requirement (under 35 USC 103(a)) that the prior art contain an express suggestion to combine known elements to achieve the claimed invention. Rather, the suggestion to combine may come from the prior art, as filtered through the knowledge of one skilled in the art.,” Motorola, Inc, v. Interdigital Tech. Corn., 43 USPQ2d 1481, 1489 (Fed. Cir. 1997).
Accordingly, the claimed invention as a whole was prima facie obvious to one of ordinary skill in the art before the effective filling date of the claimed invention especially in the absence of evidence to the contrary.
Applicants’ arguments filed December 2, 2025 have been fully considered but are not found persuasive.
Applicants’ position is that Claim 1 is currently amended. Claim 88 is cancelled without prejudice. Claim 89 is added as a new claim.
All of the rejections are based upon Lee. However, Applicant asserts that Lee is not relevant to instant claim 1 and is an inappropriate piece of art for producing an obviousness type rejection. Lee teaches HSA linked to PEI600, but Lee is silent with respect to any "protecting group". The Office Action acknowledges that Lee does not teach: 1) a negative zeta potential, 2) a covalent bond from the PEI to the protecting group and 3) the protecting group being represented by Formula 1. However, the Office Action seems to be stating that Lee does in fact teach a protecting group and that the protecting group of Lee can be swapped for the protecting group of Meyer and Huang.
Applicant, however, is of the opinion that a careful reading of Lee does not indicate that there is any protecting group. Lee merely encapsulates the HSA-PEI in a lipid nanoparticle (LNP). It is true that claim 1 recites "comprising" and so an LNP can be part of the composition, but this does not negate the fact the Lee is silent with respect to any type of "protecting group". The Office Action seems to be arguing that the LNP itself is a kind of pH-responsive protecting group, but this is not so. Lee states that conjugation of PEI600 to HSA results in a "hyper-cationized pH-responsive albumin- polymer conjugate (APC)". Thus, the HSA-PEI is "pH-responsive" not the LNP. PEI600 is well- known in the art to be pH-sensitive on its own, in that it becomes more positive at low pH. This is what Lee is referring to when he states that the APC is "pH-responsive". This has nothing to do whatsoever with the LNP.
The LNP is merely a carrier. The term "protecting group" is well-known in the art of Chemistry. A protecting group is a temporary chemical modification to a functional group in a molecule that renders it unreactive. The LNP does not protect the PEI. This is evident because the LNPs still have a positive zeta potential once the HSA-PEI is incorporated, while they were neutral before. This means that the PEI is not protected and can still react. Indeed, this is why Lee puts the HSA-PEI in the LNP in the first place, so that the PEI can improve the transfection of the LNP. Lee states this explicitly in paragraph [008], "The incorporation of hyper-cationized albumin- polymer conjugates (APC) increases the transfection efficiency of LN formulations." The HSA- PEI is meant to improve LNP transfection, so the PEI is clearly still active and not protected. If the LNP does not protect, it certainly does not deprotect at a pH value of less than 7. Essentially in calling an LNP a "protecting group" the Office Action has given no patentable weight to this well-known term. The protecting group is protecting the positive charge of the PEI so that it cannot react. The claim has previously stated that the protein conjugate has a negative zeta potential, indicated that the full positive charge of the PEI has been blocked (i.e., protected). Blocking/protecting the positive charge of the PEI is what the protecting group does. It is thus clear that a LNP is not actually a protecting group, but merely a carrier.
A skilled artisan reading Lee would learn nothing about protection or pH sensitivity of a protecting group, but merely that LNP encapsulation is a mode of delivery and that the PEI improves the transfection of the LNP. The Office Action argues that a skilled artisan would have been motivated to reversibly protect the HSA-PEI of Lee with the DMMAn of Meyer or the DMA of Huang, but there is no motivation in Lee to protect the HSA-PEI.
Without protection being recited in Lee there is no reason a skilled artisan would even look to Meyer and Huang. In fact, since Lee's whole reason for incorporating the HSA-PEI into LNPs is so that the PEI can actively improve the transfection of the LNPs, protecting (i.e., blocking the PEI) would be counterproductive. The combination with Meyer and Huang is motivated only by hind-sight reasoning and not by anything in Lee that would suggest such a combination. When the term "protecting group" is given is proper meaning Lee becomes irrelevant and the case for prima facia obviousness falls apart.
For the sake of completeness it should be noted that the instant application provides data that blocking with the protecting group of claim 38 (citraconic anhydride) is superior to other blocking groups. A dimethyl maleic anhydride such as is taught by Meyer and Huang was tested in the instant application. In paragraph [0430] it states "dimethyl maleic anhydride gave a smear probably suggesting removal of this masking prior and during this analysis." This indicates that dimethyl maleic anhydride deprotects even at a pH around or above 7 and not only at acidic pH. This early deprotecting was not observed with citraconic anhydride protection. It should be noted that new claim 89 recites that the protecting group does not deprotect "at a pH value of 7 or greater". Thus, Meyer and Huang cannot render this claim obvious.
In response, the amendment to the claims is acknowledged. The argument with respect to Meyer is moot as the reference has been withdrawn.
In response to the argument that Lee is silent with respect to any "protecting group", the use of anhydride to reversibly mask amine containing compound to facilitate delivery of molecules to cells in vitro or in vivo is known in the art as evidenced by the teachings of the ‘113 patent and/or Huang.
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).
In this case, the teachings of Lee pertaining to low molecular weight pH-sensitive polymer (polyethyleneimine MW 600, PEI600) conjugated to human serum albumin (HSA) via cross linking agents, resulting in a hyper-cationized pH-responsive APC and LNs with a highly positive charge tend to interact non-specifically with non-target cells, tissues, and circulating plasma proteins, and may cause cytotoxicity (see para. [0047]), the teachings of the ‘113 patent pertaining to reversibly protecting the positively charge amines on polyamines such as PEI with substituted maleic anhydride or derivative thereof and upon cleavage of the anhydride protecting group at pH < 7, the amine on the PEI molecule is regenerated, and the teachings of Huang pertaining to 2,3-dimethylmaleic anhydride to mask the amine groups on branched polyethyleneimine (bPEI) to form pH-triggered charge-reversal nanoparticle (see p. 559, left col) would have lead a person of ordinary skill in the art before the effective filling date of the claimed invention to reversibly block or protect the positively charge amine on the PEI in the conjugate of Lee with any one of the negatively charge carboxylate group of maleic anhydride wherein R1 and R2 are H or derivative thereof, e.g., dimethyl maleic anhydride wherein R1 and R2 are CH3 or citraconic anhydride wherein R1 or R2 is H and R2 or R1 is CH3, or cis-aconitic anhydride wherein R1 or R2 is H and R2 and R1 are CH2CO2H or 2-propionic-3-methylmaleic anhydride (CDM) wherein R1 or R2 is CH3 and R2 or R1 is (CH2)2CO2H or Huang’s 2,3-dimethylmaleic anhydride (DMA) in order to enabling reversible conversion of any positively charged amine groups on PEI to a negatively charged carboxyl to lower the ζ potentials of HSA-PEI conjugate from positive to negative charge and the regeneration of the amine by cleavage of the negatively charged anhydride when exposed to pH < 7.
One of ordinary skill in the art would have had an expectation of success at the time the invention was made to modify the conjugate of the WO2013177421 publication in view of the ‘113 patent because the ‘113 patent teaches that polyamine can be partially or completely modified with anhydride is straightforward and facile. Furthermore, the reversal of the modification in vivo or in vitro is essentially unidirectional. Once the anhydride is cleaved in the acidic endosome, the compound retains activity even if the local pH does not stay acidic, see col. 5, in particular.
One of ordinary skill in the art would have been motivated to do so because the WO2013177421 publication teaches that highly positive charge of the conjugate tend to interact non-specifically with non-target cells, tissues, and circulating plasma proteins, and may cause cytotoxicity and Huang teaches the advantage of using the protecting group 2,3-dimethylmaleic anhydride (DMA) to reversibly blocking the amino groups of HSA-PEI in bloodstream environment (pH 7.4) in order to reduce systemic toxicity (lytic activity); the blocking group DMA could be removed in the endosome at a more acidic pH (6.5). The DMA group could be removed because of the protonic forms of the free carboxylic groups which catalyze the hydrolysis of the amide bonds, see p. 562, left col.
One of ordinary skill in the art would have been motivated to do so because the ‘113 patent teaches that the addition of the carboxyl group using anhydride increases the charge and water solubility of the conjugate molecule and the negatively charge anhydride shields or masks or protects the positive charge amine groups of PEI of the nanoparticles therefore reduce membrane irritation or damage as higher amount of PEI were toxic to cells.
One of ordinary skill in the art would have had an expectation of success before the effective filling date of the claimed invention to modify the HSA-PEI of WO2013177421 publication in view of Huang because Huang each teaches that the dimethylmaleic anhydride (DMMAn or DMA) can be used as a protecting group to mask the positive amine groups on PEI at neutral pH and then undergoes deprotection by hydrolysis of the amide bonds at acidic pH in the endosome of the target cells.
In response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971).
In response to applicant's argument that protecting group citraconic anhydride (claim 38) is superior to other blocking groups such as dimethyl maleic anhydride as taught by Huang as indicated in the paragraph [0430] of the specification, it is noted that the anhydride represented by Formula 1 encompassed dimethyl maleic anhydride (claim 1). Further, the ‘113 patent teaches citraconic anhydride, see Fig 1A wherein the R to anhydride bonds can be carbon-hydrogen or carbon-carbon bonds and either R.sup.1 or R.sup.2 but not both, can be a hydrogen atom.
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When reacting amine containing compound with anhydride, the anhydrides form covalent bonds with primary amines as shown
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see Fig 2. For maleic anhydrides (FIG. 1A), the resultant covalent bond can be cleaved under physiological conditions, see col. 5, lines 57-61. Citraconic and cis-aconitic-derived maleamic acids each have one substitution and are more pH-labile. Maleamic acids derived from disubstituted dimethyl maleic anhydride are the most pH-labile, see col. 5, line 64-67. Thus the results as argued are expected and obvious variation of the reference teachings.
For these reasons, the rejection is maintained.
Claims 28-29 and 88 are rejected under 35 U.S.C. 103 as being unpatentable over WO2013177421 publication (of record, published November 28, 2013; PTO 892) in view of US Patent 7,019,113 (newly cited, issued March 28, 2006; PTO 892) and Huang et al (of record, Materials Science & Engineering C 102: 558-568, 2019; PTO 892) as applied to claims 1, 3, 5-8, 15, 35, 72, 85, 86, 87 and 89 mentioned above and further in view of Lu et al (of record, US20180055944, published March 1, 2018; PTO 892).
The combine teachings of WO2013177421 publication, the ‘113 patent and Huang have been discussed supra. The WO2013177421 publication further teaches that the albumin-PEI conjugate via disulfide linkages, see p. 20, para. [00118].
The references above do not teach that the protein conjugate wherein human serum albumin comprises the amino acid sequence of SEQ ID NO: 1 as per claims 28, 29 and 88.
However, Lu teaches human serum albumin comprising the amino acid sequence of SEQ ID NO: 1, which is identical to the claimed SEQ ID NO: 1, see para [0005], sequence alignment below:
ALIGNMENT:
Query Match 100.0%; Score 3103; Length 585;
Best Local Similarity 100.0%;
Matches 585; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAE 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAE 60
Qy 61 NCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEV 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 NCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEV 120
Qy 121 DVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 DVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP 180
Qy 181 KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK 240
Qy 241 VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPA 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPA 300
Qy 301 DLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKC 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 DLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKC 360
Qy 361 CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVST 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVST 420
Qy 421 PTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 PTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES 480
Qy 481 LVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 LVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT 540
Qy 541 KEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL 585
|||||||||||||||||||||||||||||||||||||||||||||
Db 541 KEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL 585
Lu teaches HSA contains a free thiol at C34, which is ideal to conjugate active drug molecules and peptides to it through a sulfhydryl reactive group such as maleimide, see para. [0002]. Albumin has been used as carrier for hydrophobic drug molecules, see para. [0003]. The HSA can be linked to various proteins or peptides of interest directly or indirectly via a linker. Such linkers include cleavable ones and non-cleavable ones, see para. [0024], [0026] to [0027].
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filling date of the claimed invention to substitute the human serum albumin in the conjugate of WO2013177421 publication, the ‘113 patent and Huang for another, e.g., human serum albumin comprising SEQ ID NO 1 as taught by Lu in order to link the PEI to the cysteine residue at position 34 through a sulfhydryl reactive group such as maleimide.
One of ordinary skill in the art would have had a reasonable expectation of success in conjugating PEI to HSA via C34 because Lu teaches HSA contains a free thiol at C34, which is ideal to conjugate any protein or drug to it through a sulfhydryl reactive group such as maleimide, see para. [0002].
One of ordinary skill in the art would have had a reasonable expectation of success in making such substitution because of their structural similarity as Lu teaches that human serum albumin has been used as carrier for various proteins and drug molecules, see para. [0003]. In this case, simple substitution of one known element HSA for another HSA comprises SEQ ID NO: 1 would obtain predictable results; a person with ordinary skill has good reason to pursue known options within his or her technical grasp. Note: MPEP 2141 KSR International CO. v. Teleflex Inc. 82 USPQ 2d 1385 (Supreme Court 2007).
“The test of obviousness is not express suggestion of the claimed invention in any or all of the references but rather what the references taken collectively would suggest to those of ordinary skill in the art presumed to be familiar with them.” See In re Rosselet 146 USPQ 183, 186 (CCPA 1965).
“There is no requirement (under 35 USC 103(a)) that the prior art contain an express suggestion to combine known elements to achieve the claimed invention. Rather, the suggestion to combine may come from the prior art, as filtered through the knowledge of one skilled in the art.,” Motorola, Inc, v. Interdigital Tech. Corn., 43 USPQ2d 1481, 1489 (Fed. Cir. 1997).
Accordingly, the claimed invention as a whole was prima facie obvious to one of ordinary skill in the art before the effective filling date of the claimed invention especially in the absence of evidence to the contrary.
Given the lack of any additional rebuttal with respect to this rejection, the rejection is maintained for reasons of record.
Claim 38 is rejected under 35 U.S.C. 103 as being unpatentable over WO2013177421 publication (of record, published November 28, 2013; PTO 892) in view of US Patent 7,019,113 (newly cited, issued March 28, 2006; PTO 892) and Huang et al (of record, Materials Science & Engineering C 102: 558-568, 2019; PTO 892) as applied to claims 1, 3, 5-8, 15, 35, 72, 85, 86, 87 and 89 mentioned above and further in view of Atassi et al (of record, Methods Enzymology 25:546-553, 1972; PTO 1449 filed 5/23/24).
The combine teachings of WO2013177421 publication, Meyer and Huang have been discussed supra.
The references above do not teach that the protein conjugate wherein protecting group is
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as per claim 38.
However, Atassi teaches reversible masking of amino groups is an extremely valuable procedure for protecting these groups from side reactions and reversible masking of amino groups is useful for rendering hydrolysis with trypsin specific for cleavage at arginine residues in proteins and peptides, see p. 546, in particular. Atassi teaches using myoglobin or lysozyme as the protein model and allowing it to react with maleic anhydride or citraconic anhydride. The results of both with myosin and lysozyme showed that of the reagents studies, citraconic anhydride was the most satisfactory, yielding upon deblocking for both myoglobin and lysozyme homogenous preparations identical with the respective native proteins in biological properties and in conformational and hydrodynamic parameters, see p. 547, in particular. The citraconic anhydride (aka 2-methylmaleic anhydride) reacts with amino groups to give two reaction products.
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It would have been prima facie obvious to a person of ordinary skill in the art before the effective filling date of the claimed invention to combine the teachings of WO2013177421 publication and Huang in view of Atassi by substituting the 2,3-dimethylmaleic anhydride (DMA) for Atassi’s citraconic anhydride (aka 2-methylmaleic anhydride) having similar chemical structure as the protecting group to mask the positively charge amines of PEI in the conjugate of WO2013177421 publication at neutral pH and unmask at acidic pH.
One of ordinary skill in the art would have been motivated to mask the positively charge amine of PEI in HSA-PEI conjugate in order to lower cytotoxicity using citraconic anhydride because Atassi teaches that citraconic anhydride was the most satisfactory precursor for protecting group, yielding upon deblocking agent for any protein homogenous preparations identical with the respective native proteins in biological properties and in conformational and hydrodynamic parameters, see p. 547, in particular.
One of ordinary skill in the art would have had a reasonable expectation of success in making such substitution because of their structural similarity as Atassi teaches that citraconic anhydride reacting to amine group by undergo ring-opening, resulting in the protecting bound to the protein, see reaction scheme above.
It would have been obvious to one of ordinary skill in the art to try any of the specifically taught maleic anhydride or citraconic anhydride as precursor for protecting group to mask the charge of PEI as a person with ordinary skill has good reason to pursue known options within his or her technical grasp. Note: MPEP 2141 KSR International CO. v. Teleflex Inc. 82 USPQ 2d 1385 (Supreme Court 2007). Indeed, the common sense of those skilled in the art demonstrates why some combinations would have been obvious where others would not.
“The test of obviousness is not express suggestion of the claimed invention in any or all of the references but rather what the references taken collectively would suggest to those of ordinary skill in the art presumed to be familiar with them.” See In re Rosselet 146 USPQ 183, 186 (CCPA 1965).
“There is no requirement (under 35 USC 103(a)) that the prior art contain an express suggestion to combine known elements to achieve the claimed invention. Rather, the suggestion to combine may come from the prior art, as filtered through the knowledge of one skilled in the art.,” Motorola, Inc, v. Interdigital Tech. Corn., 43 USPQ2d 1481, 1489 (Fed. Cir. 1997).
Accordingly, the claimed invention as a whole was prima facie obvious to one of ordinary skill in the art before the effective filling date of the claimed invention especially in the absence of evidence to the contrary.
Applicants’ arguments filed December 2, 2025 have been fully considered but are not found persuasive.
Applicants’ position is that Claim 1 is currently amended. Claim 88 is cancelled without prejudice. Claim 89 is added as a new claim.
All of the rejections are based upon Lee. However, Applicant asserts that Lee is not relevant to instant claim 1 and is an inappropriate piece of art for producing an obviousness type rejection. Lee teaches HSA linked to PEI600, but Lee is silent with respect to any "protecting group". The Office Action acknowledges that Lee does not teach: 1) a negative zeta potential, 2) a covalent bond from the PEI to the protecting group and 3) the protecting group being represented by Formula 1. However, the Office Action seems to be stating that Lee does in fact teach a protecting group and that the protecting group of Lee can be swapped for the protecting group of Meyer and Huang.
Applicant, however, is of the opinion that a careful reading of Lee does not indicate that there is any protecting group. Lee merely encapsulates the HSA-PEI in a lipid nanoparticle (LNP). It is true that claim 1 recites "comprising" and so an LNP can be part of the composition, but this does not negate the fact the Lee is silent with respect to any type of "protecting group". The Office Action seems to be arguing that the LNP itself is a kind of pH-responsive protecting group, but this is not so. Lee states that conjugation of PEI600 to HSA results in a "hyper-cationized pH-responsive albumin- polymer conjugate (APC)". Thus, the HSA-PEI is "pH-responsive" not the LNP. PEI600 is well- known in the art to be pH-sensitive on its own, in that it becomes more positive at low pH. This is what Lee is referring to when he states that the APC is "pH-responsive". This has nothing to do whatsoever with the LNP.
The LNP is merely a carrier. The term "protecting group" is well-known in the art of Chemistry. A protecting group is a temporary chemical modification to a functional group in a molecule that renders it unreactive. The LNP does not protect the PEI. This is evident because the LNPs still have a positive zeta potential once the HSA-PEI is incorporated, while they were neutral before. This means that the PEI is not protected and can still react. Indeed, this is why Lee puts the HSA-PEI in the LNP in the first place, so that the PEI can improve the transfection of the LNP. Lee states this explicitly in paragraph [008], "The incorporation of hyper-cationized albumin- polymer conjugates (APC) increases the transfection efficiency of LN formulations." The HSA- PEI is meant to improve LNP transfection, so the PEI is clearly still active and not protected. If the LNP does not protect, it certainly does not deprotect at a pH value of less than 7. Essentially in calling an LNP a "protecting group" the Office Action has given no patentable weight to this well-known term. The protecting group is protecting the positive charge of the PEI so that it cannot react. The claim has previously stated that the protein conjugate has a negative zeta potential, indicated that the full positive charge of the PEI has been blocked (i.e., protected). Blocking/protecting the positive charge of the PEI is what the protecting group does. It is thus clear that a LNP is not actually a protecting group, but merely a carrier.
A skilled artisan reading Lee would learn nothing about protection or pH sensitivity of a protecting group, but merely that LNP encapsulation is a mode of delivery and that the PEI improves the transfection of the LNP. The Office Action argues that a skilled artisan would have been motivated to reversibly protect the HSA-PEI of Lee with the DMMAn of Meyer or the DMA of Huang, but there is no motivation in Lee to protect the HSA-PEI.
Without protection being recited in Lee there is no reason a skilled artisan would even look to Meyer and Huang. In fact, since Lee's whole reason for incorporating the HSA-PEI into LNPs is so that the PEI can actively improve the transfection of the LNPs, protecting (i.e., blocking the PEI) would be counterproductive. The combination with Meyer and Huang is motivated only by hind-sight reasoning and not by anything in Lee that would suggest such a combination. When the term "protecting group" is given is proper meaning Lee becomes irrelevant and the case for prima facia obviousness falls apart.
For the sake of completeness it should be noted that the instant application provides data that blocking with the protecting group of claim 38 (citraconic anhydride) is superior to other blocking groups. A dimethyl maleic anhydride such as is taught by Meyer and Huang was tested in the instant application. In paragraph [0430] it states "dimethyl maleic anhydride gave a smear probably suggesting removal of this masking prior and during this analysis." This indicates that dimethyl maleic anhydride deprotects even at a pH around or above 7 and not only at acidic pH. This early deprotecting was not observed with citraconic anhydride protection. It should be noted that new claim 89 recites that the protecting group does not deprotect "at a pH value of 7 or greater". Thus, Meyer and Huang cannot render this claim obvious.
Atassi is brought as teaching citraconic anhydride, but a skilled artisan could not have predicted from Atassi the superiority of this protecting agent over other protecting agents known in the art (e.g., dimethyl maleic anhydride). Unexpected results are evidentiary proof of non-obviousness. The unexpected superiority of citraconic anhydride certainly renders claim 38 non-obvious.
Just as claim 1 is non-obvious so too all claims that depend from claim 1 are also non-obvious.
In response, the argument with respect to Lee and Huang have been addressed above and incorporated here by reference.
In response to applicant's argument that citraconic anhydride is superior, the fact that the inventor has recognized another advantage which would flow naturally from following the suggestion of the prior art cannot be the basis for patentability when the differences would otherwise be obvious. See Ex parte Obiaya, 227 USPQ 58, 60 (Bd. Pat. App. & Inter. 1985).
For these reasons, the rejection is maintained.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 6 and 85 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which applicant regards as the invention.
Claims 6b, 6c recite the limitation "said protein" in claim 1. There is insufficient antecedent basis for this limitation in the claim. Further, the phrase “said protein” renders the claim indefinite because it is unclear said protein is referring to the human serum albumin or the PEI. Replacing “protein” in claims 6b and 6c with “HSA” would obviate this rejection.
Claims 6f recite the limitation "the protein" in claim 1. There is insufficient antecedent basis for this limitation in the claim.
Claim 85 recites the limitation "said protein" in claim 1. There is insufficient antecedent basis for this limitation in the claim. Amending claim 85 to recite “said HSA” would obviate this rejection.
Claim objection
Claim 1 is objected to because of the following informalities:
A, multiple “optionally” should be deleted.
B. “any combination” should have been “a combination”.
C. “selected from…or…and…or R and R1 are bound together so as to form a cyclic ring” should have been “selected from the group consisting of…and R and R1 are bound together so as to form a cyclic ring”.
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 PHUONG HUYNH whose telephone number is (571)272-0846. The examiner can normally be reached on 9:00 a.m. to 6:30 p.m. The examiner can also be reached on alternate alternative Friday from 9:00 a.m. to 5:30 p.m.
If attempts to reach the examiner by telephone are unsuccessful, the examiner's supervisor, Misook Yu, can be reached at 571-270-3497. The fax phone number for the organization where this application or proceeding is assigned is 571-272-0839.
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/PHUONG HUYNH/ Primary Examiner, Art Unit 1641
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