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 .
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 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.
Priority
The instant application, filed 10/21/2022, claims domestic benefit to US provisional application 63/368,573, filed 07/15/2022.
Status of Application, Amendments, and/or Claims
Applicant’s response and the declaration of Jiang Xia filed 12/31/2025 are acknowledged. Claims 1-3, 9-10, 14, and 16 are amended and claims 6 and 13 are cancelled. Claims 1-5, 7-12, and 14-22 are currently pending and are examined on the merits herein.
Withdrawn Objections and Rejections
The drawings were objected to. Applicant’s amendment of “Fig. 6” to “Fig. 6C” and the submission of a higher resolution Fig. 21 has overcome the objections and the objections are withdrawn.
Claim 14 was objected to. Applicant’s correction of “norborene” to “norbornene” has overcome the rejection and the rejection is withdrawn.
Claims 1, 3, and 10 were rejected under 35 USC 112(b). Applicant’s amendment to remove reference to “Lys6” in the claims has overcome the rejections and the rejections are withdrawn.
Claim 10 was rejected under 35 USC 112(b). Applicant’s amendment to part (b) to change “the modified Fc-III peptide” to “a modified Fc-III peptide” has overcome the rejection and the rejection is withdrawn.
Claims 6 and 13 were rejected under 35 USC 112(d). The cancellation of the claims has rendered the rejections moot and the rejections are withdrawn.
Claim 1 was rejected under 35 USC 103 over Yamada, US’395, Chen and Martos-Malonado and claim 2 was rejected under 35 USC 103 over Yamada, US’395, Chen, Martos-Malonado, Knerr, and Tong. Applicant’s amendment to independent claim 1, on which claim 2 depends, to require that the formula comprise Formula (I) has overcome the rejections and the rejections are withdrawn.
Claims 3-7 were rejected under 35 USC 103 over Encapsula Nano Sciences in view of Yamada, US’395, Chen, and Martos-Malonado. Applicant’s amendment to independent claim 3 to remove limitations concerning His5 and Lys6, requiring that the substitution be in Glu8 has overcome the rejections and the rejections are withdrawn.
Claim 8 was rejected under 35 USC 103 over Encapsula Nano Sciences in view of Yamada, US’395, Chen, Martos-Malonado, and Sun. Applicant’s amendment to independent claim 3 to remove limitations concerning His5 and Lys6, requiring that the substitution be in Glu8 has overcome the rejections and the rejections are withdrawn.
Claim 9 was rejected under 35 USC 103 over Encapsula Nano Sciences in view of Yamada, US’395, Chen, Martos-Malonado, Knerr, and Tong. Applicant’s arguments in the response and the declaration of Jiang Xia filed 12/31/2025 concerning unexpected results are persuasive and the rejection is withdrawn.
Claims 17-19 were rejected under 35 USC 103 over Park, Encapsula Nano Sciences, Yamada, US’395, Chen, and Martos-Malonado. Applicant’s amendment to independent claim 3, on which claims 17-19 depend, to remove limitations concerning His5 and Lys6, requiring that the substitution be in Glu8 has overcome the rejections and the rejections are withdrawn.
Claims 10-15 and 20-22 were rejected under 35 USC 103 over Kim, Bartels, Yamada, US’395, Chen, Martos-Malonado, and Smeenk. Applicant’s amendment to the claim to remove limitations concerning His5 and Lys6, requiring that the substitution be in Glu8 has overcome the rejections and the rejections are withdrawn.
Claim 16 was rejected under 35 USC 103 over Kim, Bartels, Yamada, US’395, Chen, Martos-Malonado, Smeenk, Knerr, and Tong. Applicant’s arguments in the response and the declaration of Jiang Xia filed 12/31/2025 concerning unexpected results are persuasive and the rejection is withdrawn.
The following grounds of objections are new or maintained and rejections new as necessitated by applicant’s amendment to the claims.
Nucleotide and/or Amino Acid Sequence Disclosures
The following drawings comprise amino acid sequences that have more than 4 amino acids and are not identified with an appropriate SEQ ID NO in either the drawing or the brief description of the drawings: 1B (F0), 4C, 21 (on the continued page which comprises a table, right column), 16B (the first Figure with a 6 His tail (HHHHHH), and 37.
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiencies and the required response to this Office Action are as follows:
Specific deficiency – Nucleotide and/or amino acid sequences appearing in the drawings are not identified by sequence identifiers in accordance with 37 CFR 1.821(d). Sequence identifiers for nucleotide and/or amino acid sequences must appear either in the drawings or in the Brief Description of the Drawings.
Required response – Applicant must provide:
Replacement and annotated drawings in accordance with 37 CFR 1.121(d) inserting the required sequence identifiers;
AND/OR
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required sequence identifiers into the Brief Description of the Drawings, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
Drawings
The drawings are objected to because of the following informalities:
The drawings contain colored the following colored figures without an approved petition. See the following figures:
In the replacement drawings of 12/31/2025, Figs: 5B, 6C, 7C, and 21.
In the drawings filed 10/21/2022 the following drawings are in color and were not replaced by black and white/grayscale figures in the 12/31/2025 filing, Figs: 8B, 16B, 23-24, 25A, 25B, 26, 42C, 43C, 44C, 45C, 46C, 47C, 48C, 49C, 50C, 51C, 52C, and 53C.
It is noted that Applicant filed a petition for colored figures on 12/31/2025; however, the petition was dismissed on 01/27/2026. An accepted petition or black and white/grayscale figures is required.
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Claim Objections
Claims 2, 9, and 16 are objected to for the following informalities: The claims contain structures that are low resolution making them difficult to read. Specifically see Formula XII in claim 2 (last figure). In claims 9 and 16, the figures are low resolution making them difficult to interpret. This is particularly the case as the structures recited indicate stereochemistry using wedge and dash notation and the poor resolution makes the notation difficult to identify.
Claim Rejections - 35 USC § 112(b)
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.
Claim 2 is 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 the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
As amended, claim 2 recites “or a modified azidoacetate motif” The term “modified azidoacetate motif” is a relative term which renders the claim indefinite. The term is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. The term “modified” indicates that the azidoacetate motif has been altered, but it is unclear to what extent the motif can be changed while still meeting the claim limitation.
For instance, in claim 2, Formula (VIII) is recited as follows and, according to the amended claim, comprises a modified azidoacetate motif in the Glu8 position (identified with a box):
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While the formula does have a modification at position Glu8, as claimed, the moiety at the position does not comprise an azide group at all. It is unclear whether this structure meets the limitation of a “modified azidoacetate motif” and to what extent the azidoacetate motif could be modified and still meet the limitation. Similarly, formulas (V), (VI), (VIII), (IX), (X), (XI), and (XII) all comprise motifs that do not comprise azide groups.
As the scope of the claim is unclear, the metes and bounds are indefinite.
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 2 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 2 recites a modified Fc-III peptide of claim 1 according to SEQ ID NO: 4, wherein residue Glu8 is substituted with a glutamine derivative containing a phenyl azidoacetate motif at a side chain, wherein the Fc-III peptide is F1, F6, or a modified azidoacetate motif at a side chain, wherein the Fc-III peptide is f-F0, f-F4, f-F5, F7, F8, F9, F10, or F11. Claim 1; however, requires that the compound contain a phenyl azidoacetate motif at a side chain and be formula (I). As claim 2 includes additional motifs and formulas, the claim is broader than claim 1 and fails to further limit the claim upon which it depends.
Additionally, because claim 2 encompasses compounds that differ from formula (I), as recited in claim 1, the embodiments of claim 2 in which the peptide is F0, f-F4, f-F5, F7, F8, F9, F10, or F11 all do not include the limitations of the claim upon which it depends.
It is noted that claim 2 is included in the prior art rejections of the instant office action in an effort towards compact prosecution.
Claim Rejections - 35 USC § 112a – New Matter
The following is a quotation of the first paragraph 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 the first paragraph of pre-AIA 35 U.S.C. 112:
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 2 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), 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 or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. This is a new matter rejection.
Claim 2 has been amended to encompass a modified F-III peptide according to SEQ ID NO: 4, wherein residue Glu8 is substituted with a “modified azidoacetate motif” at a side chain. There is not support for a “modified azidoacetate motif” in the originally filed disclosure. The originally filed application considers only a phenyl azidoacetate motif at the recited position. It is noted that, while the originally filed disclosure does support the specific peptide structures that are recited in the claim, the disclosure does not identify these structures as having a “modified azidoacetate motif” nor does the disclosure suggest or identify that these structure fall under such category.
As the originally filed disclosure does not contemplate a “modified azidoacetate motif”, the claim to such a motif is considered to be new matter.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 2-3 and 7 are rejected under 35 U.S.C. 103 as being unpatentable over Encapsula NanoSciences (2018) Immunosome®-DBCO (PEGylated) Technical Information Version 1.1 in view of WO 2021/102052 A1 (Rastelli, L, et al) 27 May 2021, and Martos-Malonado, M.C., et al (2018) Selective N-terminal acylation of peptides and proteins with a Gly-His tag sequence Nature Communications 9(3307); 1-13.
It is noted that claim 2 is rejected under 35 USC 112(d) for being broader than the claim upon which it depends (claim 1) as discussed in detail above. While claim 1 is not rejected here, claim 2 is included in this rejection in an effort towards compact prosecution.
Encapsula NanoSciences teaches that during the past five decades, various types of chemistries have been used for conjugation of molecules, such as antibodies, peptides, proteins or other reactive ligands, to the surface of liposomes. In general, the conjugation can be achieved through the N-terminus, the C-terminus, or other available sulfur. Not all chemistries have the same yield and efficiency of conjugation and often reproducing biocompatible batches can be a challenge (page 1, paragraph 1). Encapsula NanoSciences further teaches that click chemistry is one of the most efficient and easiest conjugation chemistry available for coupling antibodies and other reactive ligands to the surface of liposomes. The conjugation chemistry is based on the reaction of dibenzocyclooctyne (DBCO) reagent with an azide linker to form a stable triazole. DBCO moiety can be on the antibody and the azide moiety can be on the liposomes or vice versa. This conjugation protocol is based on the reaction of the DBCO group on the liposomes with an azide linker on the antibody, peptide, or proteins (page 1, paragraph 2). A diagram of the click chemistry conjugation is shown on page 2 in which an antibody is labeled with an azide group forming an azide-activated antibody. The azide activated antibody is then mixed with liposomes containing DBCO on the surface forming an antibody conjugated liposome.
Encapsula NanoSciences teaches an immunosome-DBCO comprising hydrogenated soy PC, Cholesterol, DSPE-PEG(2000) and DSPE-PEG(2000)-DBCO at ratios of 57:38:4:1 and teaches the structure of DSPE-PEG(2000)-DBCO as follows (page 3):
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Encapsula NanoSciences teaches that the liposomes are in PBS buffer at a pH of 7.4 (page 3, Buffer and liposome size).
Encapsula NanoSciences teaches that the DBCO group is known to be hydrophobic and it buries itself in the lipid bilayer of the liposomes. Direct conjugation of a ligand to the liposomes containing DBCO has produced immunoliposomes with yield of above 60% which is quite acceptable and much higher than many other conjugation chemistries. Post-insertion of DBCO lipid conjugated ligands into the liposomes increases the yield to above 80% (page 5, technical notes, bullet 2).
Encapsula NanoSciences further teaches that reactions of DBCO and azides are more efficient at high concentrations and temperatures, for instance up to 37°C; however, In order to avoid denature of proteins, peptides, and antibodies, it is recommended to incubate molecules with liposomes at room temperature followed by refrigeration. Typical reaction times are less than 12 h; however, incubating for longer can improve efficiency (page 5, technical notes, bullets 3-4).
Encapsula NanoSciences teaches a preparation method in which 2.5 mol equivalents of DBCO-lipids in liposomes to 1 mol equivalent of azide containing protein are incubated at room temperature for 4 hours followed by overnight incubation at 4°C in a refrigerator (page 4, preparation method, conjugation protocol).
Encapsula NanoSciences, however, does not disclose that the azide labeling of the antibody to form the azide activated antibody comprises the step of incubating a modified Fc-III peptide, as claimed, with the antibody, yielding an acetylated antibody.
WO’052 teaches technologies for site-directed conjugation of various moieties of interest to target agents utilizing target binding moieties to provide high conjugation efficiency and selectivity. WO’052 teaches that the technologies provided are useful for preparing antibody conjugates (abstract). WO’052 further teaches that the technologies presented provide various advantages including improved efficiency and/or selectivity, reduced levels of heterogeneity, and/or reduced undesired transformations, and an avoidance of certain reaction conditions (page 2, [0005]). WO’052 further teaches that the technologies disclosed are useful for conjugating moieties to antibodies (page 26, [0089]).
WO’052 teaches compounds that comprise a first group comprising a target binding moiety that binds to a target agent, a reactive group, a moiety of interest, and optionally one or more linker moieties linking such groups/moieties. In some embodiments, the compound is useful as a reaction partner for conjugating moieties of interest to targets, e.g., various proteins. In come embodiments, the target binding moiety is part of a leaving group that is released upon contacting such a compound with a target and reacting a reactive group of the compound with a reactive group on a target, e.g., the -NH2 of a Lys residue of a target protein. As demonstrated, such compounds can provide improved conjugation efficiency, high selectivity, and fewer steps to conjugation products. WO’052 teaches that the compound has the structure of formula R-I: LG-RG-LRM-MOI; where LG is a targeting binding moiety that binds to a target agent; RG is a reactive group; LRM is a linker; and MOI is a moiety of interest.
WO’052 further teaches that the target binding moiety is or comprises the sequence DCAWHLGELVWCT (page 72, [0165]), which is the same as instant SEQ ID NO: 4:
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WO’052 further teaches that the target binding moiety can be connected to the rest of the molecule through the N- or C-terminus or through a side chain of an amino acid, which is shown as “X” in the disclosure. WO’052 also teaches that the two cysteine residues of the peptide form a disulfide bond. WO’052 teaches the following target binding moieties:
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Wherein X is an amino acid residue bonded to the rest of the compound or agent.
Based on these structures, WO’052 teaches that the reactive group, linker, and moiety of interest are at the position HI5, Val10, Leu6, or Glu8 position.
WO’052 further teaches that X can be a residue of:
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or
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Both of which could be considered to be glutamine derivatives which comprise a phenyl moiety.
WO’052 teaches the following structure which comprises the target binding moiety with X in the Glu8 position and the 2nd X residue above, demonstrating the structure of the cyclic peptide with the glutamine derivative:
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WO’052 further teaches that the target binding moiety is an antibody binding moiety. US’052 also teaches that the N-terminus and/or C-terminus are optionally capped, for instance with a -COOH or -NH2 group (page 69, [0161]).
WO’052 teaches that the reactive group can comprise an ester (page 105, [0224]) and that the moiety of interest can be an azide, -N3 (page 227, claim 180; page 232, claim 248).
WO’052 also teaches that the moiety of interest can be a detectable moiety, including an FITC moiety (page 232, claim 244; pages 118-119, [0278]) or an alkyne group where the moiety is -
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≡- (page 232, claim 249).
Martos-Maldonado teaches that methods for site-selective modification of peptides and proteins are required for different fields such as in the development of biopharmaceutical conjugates, e.g. PEGylation, lipidation, and antibody drug conjugates, bioimaging, medical diagnostics, and material sciences. Most proteins display multiple copies of the same side-chain at different locations. Reactions that target a particular function group, for example the primary amine in Lys or thiol in Cys, potentially modify all occurrences of this residue leading to the formation of a heterogenous mixture of modified proteins (page 2, left column, paragraph 1). Martos-Maldonado studied an N-terminal sequences GHHHn for the reaction with gluconolactone and 4-methyoxyphenyl esters as acetylating agents, facilitating the introduction of functionalities in a highly selective and efficient manner. Azides, biotin or a fluorophore are introduced at the N-termini of four unrelated proteins by effective and selective acetylation with 4-methyoxy phenyl esters (abstract). The phenyl esters studied for acetylation by Martos-Maldonado are as follows (page 5, Figure 2).
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Martos-Maldonado further demonstrates that the groups form an azidoacetyl group on amino acid chain after reaction with HEPES buffer (Figure 1 b):
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Martos-Malonado demonstrates that the disclosed phenyl esters result in azidoacetyl groups on peptides.
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to modify the method disclosed by Encapsula NanoSciences by adding the azide label to the antibody prior to conjugation with DSPE-PEG(2000)-DBCO using the modified Fc-III peptide taught WO’052, particularly where the side chain comprises a phenyl azidoacetate motif as taught by WO’052 and further supported by Martos-Maldonado. An ordinarily skilled artisan would have been motivated to use the conjugation methods disclosed by WO’052 in order to achieve the benefits of improved conjugation efficiency, high selectivity, and fewer steps to conjugation products. It would have further been obvious to use the phenyl azidoacetate side chain, as suggested by WO’052, based on the teachings of Martos-Maldonado, which demonstrates the use of such esters in the formation of azidoacetyl groups which provide an exposed azide that could react with the DBCO of the DSPE-PEG(2000)-DBCO lipid. An ordinarily skilled artisan would have had a reasonable expectation of success because Encapsula NanoSciences teaches the use of azide labeled antibodies and WO’052 teaches methods of conjugating functional groups, including azide, to antibodies. Additionally, both WO’052 and Martos-Maldonado the use of azide phenyl esters as reactive moieties.
Regarding claim 2, it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to modify the Fc-III peptide taught by the combination of Encapsula NanoScience, WO’052, and Martos-Maldonado to have the azidoacetyl group to the position 3- or 4- on the phenyl based on the teachings of WO’052 and Martos-Maldonado, rendering obvious the structures of instant Formulas (I) and (IV). Specifically, WO’052 teaches a modified glutamine with a phenyl having an attachment point in position 3- and Martos Maldonado demonstrates attachment in position 4-, opposite of other moieties. Additionally, WO’052 teaches that the moiety of interest can be -
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≡-, suggesting the structure of Formula (VIII).
Claims 4 and 5 are rejected under 35 U.S.C. 103 as being unpatentable over Encapsula NanoSciences (2018) Immunosome®-DBCO (PEGylated) Technical Information Version 1.1 in view of WO 2021/102052 A1 (Rastelli, L, et al) 27 May 2021, and Martos-Malonado, M.C., et al (2018) Selective N-terminal acylation of peptides and proteins with a Gly-His tag sequence Nature Communications 9(3307); 1-13 as applied to claim 3 above, and in further view of US 2021/0130395 A1 (Xiao, H., et al) 06 May 2021.
The combination of Encapsula NanoSciences, WO’052, and Martos-Malonado teach the method of claim 3 as discussed in detail above.
As discussed above, Encapsula NanoSciences teaches that reactions of DBCO and azides are more efficient at high concentrations and temperatures, for instance up to 37°C; however, In order to avoid denature of proteins, peptides, and antibodies, it is recommended to incubate molecules with liposomes at room temperature followed by refrigeration. Typical reaction times are less than 12 h; however, incubating for longer can improve efficiency (page 5, technical notes, bullets 3-4). Encapsula NanoSciences teaches a preparation method in which 2.5 mol equivalents of DBCO-lipids in liposomes to 1 mol equivalent of azide containing protein are incubated at room temperature for 4 hours followed by overnight incubation at 4°C in a refrigerator (page 4, preparation method, conjugation protocol).
US’395 teaches methods for proximity-induced site specific conjugation of a target agent to an antibody comprising: providing an affinity compound having a proximity-reactive motif, wherein the affinity compound is conjugated to the target agent, and bringing the affinity compound into proximity of the antibody for a sufficient period of time to covalently link the affinity compound to said antibody (page 1, [0006]). US’395 further teaches the proximity-reactive motif comprises a non-canonical amino acid (ncAA) has the ability to crosslink with an amino acid residue of the antibody, including histidine, serine, threonine, tryptophan, tyrosine, lysine, or cysteine. In some aspects, the ncAA has a reactive side chain (page 1, [0008]).
US’395 teaches that the affinity compound is a peptide derived from protein A (e.g. Z domain), protein G, or antibody binding peptides evolved from phage display, such as FcIII (page 2, [0017]).
US’395 teaches studies in which FPheK, was selectively introduced into an affinity peptide chain (page 11, Example 3). In a study, on the basis of the crystal structure of the antibody:FB complex, a 33 AA affinity peptide was modified at residue 25. A Lysine with an orthogonal protected group, 4-Mmt, was introduced at residue 25 while the other lysine groups were protected with tert-butyloycarbonyl (Boc groups). The resulting full length peptide was then coupled to an azido-lysine at the N-terminus followed by orthogonal deprotection of residue 25, where FPheK was formed yielding an azide-labeled FB containing an E25PheK mutation (AzFB). The peptide was then incubated with trastuzumab with 16 equivalents of AzFB for 48 hours to yield Tras with an azide functional moiety (Tras-Azide-FB) (pages 11-15, [0105]; Figure 5).
In studies with FcIII, FcIII-FPheK peptide was formed and then dissolved in PBS (pH=8.5)/DMF solution and open to air for disulfide bond formation. After that, 32 equivalents of cyclic FcIII-FPheK peptides were co-incubated with trastuzumab antibody at 37C for 2 days and purified by 100000 Da concentrator. The successful conjugation reaction was confirmed by ESI-MS analysis (page 12, [0110]).
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to modify the method taught by the combination of Encapsula NanoSciences, WO’052, and Martos-Malonado by incubating the antibody with the modified Fc-III peptide (claimed step a) or the acetylated antibody with the functionalized lipid (step b) at a temperature of between room temperature and 37°C for between 4 hours to 2 days in PBS buffer, based on the teachings of Encapsula NanoSciences and US’395. An ordinarily skilled artisan would have had a reasonable expectation of success as US’395 demonstrates that incubation of the antibody with the FcIII peptide is possible at 37C without denaturing the antibody.
The suggested incubation temperature of RT to 37°C and time of 4 hours to 2 days overlaps with the temperature and incubation duration of the instantly claimed invention rendering the instant claims obvious per MPEP 2144.05, which states that “In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists.”
Additionally, it would have been obvious to use the teachings of the prior art as a starting point and optimization that was routine in the art to determine the optimal incubation temperature and time for the functionalization of the antibody. MPEP 2144.05 II states that “Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. ‘[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.’” In this case it would have been obvious to use the teachings of the prior art which include incubation temperatures of 20°C to 37°C and times of 1 hour to 2 days as a starting point to determine the optimal conditions for incubating the antibody with the modified Fc-III peptide in an effort to achieve optimal site specific functionalization. Additionally, Encapsula NanoSciences also teaches that reactions of DBCO and azides are more efficient at high concentrations and temperatures, for instance 37°C.
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Encapsula NanoSciences (2018) Immunosome®-DBCO (PEGylated) Technical Information Version 1.1 in view of WO 2021/102052 A1 (Rastelli, L, et al) 27 May 2021, and Martos-Malonado, M.C., et al (2018) Selective N-terminal acylation of peptides and proteins with a Gly-His tag sequence Nature Communications 9(3307); 1-13 as applied to claim 3 above and in further view of Sun, L., et al (2021) Membrane-selective nanoscale pores in liposomes by a synthetically evolved peptide: implications for triggered release Nanoscale 13(28); 12185-12197.
The combination of Encapsula NanoSciences, WO’052, and Martos-Malonado teach the method of claim 3 as discussed in detail above.
The combination of applied references, however, do not disclose that the liposome comprises POPC, cholesterol, and NBD-PE in a molar ratio of 67:30:3, respectively.
Sun teaches peptides that form nanoscale pores in lipid bilayers have potential applications in triggered release, but only if their selectivity for target synthetic membranes over bystander biomembranes can be optimized. Previously, a novel family of a-helical pore-forming peptides, called “macrolittins”, which release macromolecular cargoes from phosphatidycholine (PC) liposomes at concentrations as low as 1 peptide per 1000 lipids were identified. Sun demonstrates that macrolittins have no measurable cytolytic activity against multiple human cell types even at high peptide concentrations. This unprecedented selectivity for PC liposomes over cell plasma membranes is explained, in part, by the sensitivity of macrolittin activity to physical chemical properties of the bilayer hydrocarbon core. In the presence of cells, macrolittins release all vesicle-entrapped cargoes, including proteins and small molecule drugs, which are then readily uptaken up by cells. Triggered release occurs without any direct effect of the peptide on the cells and without vesicle-vesicle or vesicle-cell interactions (abstract).
Sun teaches the preparation of doxorubicin containing liposomes made using POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and cholesterol with a mol/mol of 7/3 (page 4, liposome preparation, paragraph 4), indicating a mol:mol ratio of 70:30. Sun further teaches liposomes that were labeled with 0.5% NBD-PE and 0.5% rhomadime-PE dyes (page 5, paragraph 3).
Sun studied the encapsulation and release of doxorubicin (DXR) from the liposomes. Doxorubicin is a small molecule chemotherapy drug widely used against breast cancer, uterine, ovarian, lung, and cervical cancer. Sun chose cell lines from cervical cancer to test DXR releasing efficiency given that the cells are highly sensitive to DXR. First, it was confirmed that DXR encapsulation in the liposome was stable for 24 hours. Sun then studied the peptide-induced leakage from the liposomes and demonstrated that the peptide M159 was able to induce release of the DXR from the PC/cholesterol vesicles. Sun teaches that the experiments presented collectively constitute a demonstration that the selectivity of the macrolittins is sufficient to trigger the release of small molecules and macromolecules from liposomes in the presence of cells, such that the cargo is immediately made available to interact with cells (page 12, paragraph 3). Sun further teaches that future technologies that may be possible include affinity targeting of vesicles to sites of interest (page 15, paragraph 2).
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to substitute the liposomes in the method disclosed by the combination of Encapsula NanoSciences, WO’052, and Martos-Malonado with the POPC/cholesterol/NBD-PE liposomes of Sun. One of ordinary skill in the art would have been motivated to substitute the liposomes with the POPC/cholesterol/NBD-PE liposomes of Sun as Sun demonstrates that the POPC containing liposomes allow for triggered release of cargo. An ordinarily skilled artisan would have had a reasonable expectation of success as Encapsula NanoSciences teaches that DBCO is hydrophobic and buries itself in the lipid bilayer of the liposomes which would be expected to be present in the liposomes taught by Sun. Additionally, Encapsula NanoSciences teaches that post-insertion of DBCO lipid conjugated ligands into liposomes increase the yield to above 80% indicating that modification of the liposomes of Sun could occur after liposome formation. Furthermore, Sun suggests the affinity targeting of vesicles to sites of interest, which could be achieved with an antibody.
Sun teaches a POPC/cholesterol ratio of 70:30 and the inclusion of 0.5% NBD-PE as a fluorescent label. It would have been obvious to use these POPC/cholesterol/NBD-PE amounts disclosed by Sun and optimization that is routine in the art to determine the optimal ratio of POPC/cholesterol/NBD-PE for use in the liposomes suggested by the combination of Encapsula NanoSciences, WO’052, and Martos-Malonado, and Sun. See MPEP 2144.05 II. In this case, Sun teaches an approximate ratio of 70:30:0.5 of POPC/cholesterol/NBD-PE. It would have been obvious to use these amounts as a starting point to determine the optimal amount of POPC/cholesterol/NBD-PE for the liposomes.
Claims 2, 10-12, 14-15 and 20-22 are rejected under 35 U.S.C. 103 as being unpatentable over Kim, C.H., et al (2012) Synthesis of bispecific antibodies with genetically encoded unnatural amino acids J Am Chem Soc. 134(24): 9918-9921 in view of Bartels, L., et al (2019) Preparation of bispecific antibody-protein adducts by site-specific chemo-enzymatic conjugation Methods 154; 93-101, WO 2021/102052 A1 (Rastelli, L, et al) 27 May 2021, Martos-Malonado, M.C., et al (2018) Selective N-terminal acylation of peptides and proteins with a Gly-His tag sequence Nature Communications 9(3307); 1-13, and Smeenk, M.L.W., et al (2021) Recent developments in biorthogonal chemistry and the orthogonality within Current Opinion in Chemical Biology 60; 79-88.
It is noted that claim 2 is rejected under 35 USC 112(d) for being broader than the claim upon which it depends (claim 1) as discussed in detail above. While claim 1 is not rejected here, claim 2 is included in this rejection in an effort towards compact prosecution.
Kim teaches that there had been considerable interest in the generation of bispecific antibodies that simultaneously bind two different antigens. A number of recombinant strategies have been developed to synthesize bispecific antibodies including a number of chemical approaches which largely exploit the reactivity of lysine or cysteine restudies within the antibody. However, lysine modification often yields heterogeneous products due to multiple reactive surface lysines in an antibody; and while cysteine-based approaches are more selective, the reaction is complicated due to multiple disulfide bonds in the antibody molecule. Recently, a novel chemical strategy has been reported in which heterodimeric peptides with a branched azetidinone linker were fused to the antibody in a site specific manner. Kim reports a general method to generate chemically defined homogenous bispecific antibodies (page 9918, left column, paragraph 1).
Kim uses genetically encoded unnatural amino acids with orthogonal chemical reactivity relative to the canonical twenty amino acids to site-specifically modify antibody fragments. Specifically, a tRNA/aminoacyl-tRNA synthetase pair was used to site-specifically incorporate pAcF at defined sites in each of two Fab fragments in response to an amber nonsense code. The mutant Fab fragments were then selectively coupled by a stable oxime bond to bifunctional linkers with an aloxy-amine on one terminus and an azide or cyclooctyne group on the other (Figure 1B). The two Fab-linker conjugates were then linked to obtain the heterodimer through copper-free [3+2] Huisgen-cycloaddition, Click reaction (Figure 1C). Kim teaches that this approach has a number of advantages over recombinant technologies and conventional coupling chemistries. For example, the use of biorthogonal chemistries produces homogenous, chemically-defined products; variable linker lengths and conjugation sites on the antibody can be easily optimized to ensure flexibility and good efficacy for each specific application; and the molecular approach easily and rapidly allows for combinational generation of diverse heterodimers including antibodies, enzymes, cytokines, etc. (page 9918, paragraph bridging columns).
Kim teaches the synthesis of a heterodimeric bispecific antibody consisting of an anti-HER2 Fab derived from trastuzumab linked to the Fab of monoclonal antibody UCHT1, which specifically binds to human CD3 on CD8/CD3 positive cytotoxic lymphocytes. Such a construct is expected to recruit T lymphocytes to tumor cells and form a pseudo-immunological synapse that results in the activation of T lymphocytes and the subsequent lysis of the target cells (page 9919, left column, paragraph 4; page 9918, right column, paragraph 2).
Kim genetically incorporated pAcF into anti-HER2 and anti-CD3 antibody fragments. Bifunctional linkers (50-fold molar equivalents) were then coupled to the modified antibody Fabs. The two Fab-linker conjugates were separately buffer exchanged into PBS, pH 7.4, then mixed at a 1:1 ratio at 10 mg/mL and incubated at 37°C for the copper-free click reaction. The reaction was monitored by SDS-PAGE and a band at ~100kDa was observed, corresponding to the molecular weight of the Fab dimer. After 48 hours, about 70% of the starting material was consumed (page 9919, left column, paragraph 2-paragraph 4). Figure 2A provides time-course analysis of the ligation reaction and demonstrates that dimer formation is observed as early as 6 hours (page 9919; Figure 2A).
Figures 1A-B provides a schematic of the pAcF functional group and the polyethylene glycol linkers used in the formation of the bispecific antibody and demonstrate the use of PEG4 and PEG5:
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Figure 1C provides a schematic of the reaction in which a first Fab and second Fab are each site-specifically connected to linkers. The Fab-linkers are then incubated together in PBS at 37°C to form the bispecific antibody.
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Kim further demonstrates that the heterodimer can recruit T cells to kill target cancer cells in an in vitro cytotoxicity assay (page 9920, left column, paragraph 2). Kim also discusses clinical successes in which bispecific antibody therapies have been reported and even approved as therapies (page 9918, left column, paragraph 1). Teachings which, when combined, suggest the administration of the bispecific antibodies to treat cancers.
The teachings of Kim differ from those of the instantly claimed invention in that the bispecific antibody of Kim is formed from Fab fragments of the antibodies, not full antibodies. Additionally, Kim does not disclose that the site specific conjugation of the antibodies includes incubating a modified Fc-III peptide according to the instant claims.
Bartels teaches that, historically, bispecific antibodies have been constructed through the genetic fusion of additional binding domains to the constant domains of the antibody heavy- or light chains. Bartels presents an alternative method for the introduction of additional functional domains to an antibody: site-specific chemo-enzymatic conjugation. A method which relies on a combination of site-specific transpeptidases and biorthogonal chemistries. Transpeptidases are used to site-specifically introduce chemical handles, which can then be used to couple new functional groups by means of a biorthogonal chemical reaction. After introducing the chemical handle in the antibody, any functional group of interest may then be attached. The modularity of this conjugation method allows for a ‘plug-and-play’ approach to prepare new antibody conjugates, thus bypassing the need for laborious genetic fusions (abstract). Figure 2 (page 95) provides a schematic of antibody fusion with click chemistry and demonstrates bispecific antibodies formed using intact antibodies.
The teachings of WO’052 and Martos-Malonado are as discussed above.
As discussed in detail above in the rejection of claim 3, the combination of WO’052 and Martos-Malonado render obvious the claimed modified Fc-III peptide and its use in functionalizing antibodies for site-specific conjugation. Martos-Maldonado demonstrates that the disclosed phenyl esters result in azidoacetyl groups on peptides. As such, one of ordinary skill in the art would reasonably expect that incubating the Fc-III peptides with an antibody would result in an azidoacetylated antibody intended for site specific conjugation.
Additionally, WO’052 further teaches that he provided technologies can provide antibody-antibody conjugates as demonstrated in Figure 24, where a trastuzumab-cetuximab bispecific antibody is shown (page 5, [0030]; page 126, [0299]; page 369, [0872]) .
Smeenk teaches that the emergence of biorthogonal reactions has greatly advanced research in the fields of biology and medicine. They are not only valuable for labeling, tracking, and understanding biomolecules within living organisms, but also are important for constructing advanced bioengineering and drug delivery systems. Smeenk provides a review of different biorthogonal reactions that have recently been developed, the methods of cellular incorporation, and the strategies to create orthogonality within the biorthogonal landscape (abstract).
Smeenk further teaches that biorthogonal ‘click’ reactions must meet a set of defined criteria in that they must proceed with minimal interference in the biological system, be chemoselective, stable, fast, and high yield (page 79, right column, paragraph 3).
In Figure 2 (page 81), Smeenk provides a summary of experimental reaction rates of a set of biorthogonal reactions as found in the literature and supplemented the table with some computational predicted rates as reported in the prior art (page 82, left column, paragraph 1). The table shows that azide groups (represented by the number “1”) form stable conjugates with Alkyne, DBCO, BCN, and Phosphine methyl ester groups (represented by the letters “A”, “B”, “C”, and “J”). The table also provides reaction pairs that form stable conjugates that do not react with those specific for azide groups, including methyl tretazine and norbornene (represented by “11” and “D”, respectively).
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to modify the method of Kim to use full length antibodies as disclosed by Bartels in place of Fab fragments. It would have further been obvious to modify the antibodies using the modified Fc-III peptides disclosed by the combination of WO’052 and Martos-Malonado, which would be expected to result in azidoacetylated antibodies providing azide functional groups for conjugating. It would have also been obvious to substitute the functionalities on the PEG linkers disclosed by Kim by substituting the alkoxy-amine on the terminus that binds to the antibody with a group that reacts with an azide group, including DBCO as taught by Smeenk, and to substitute the azide and cyclooctyne groups on the other terminus of the linkers with an alternative click chemistry pair that would not react with the azide or DBCO groups, including methyl tetrazine and norbornene as taught by Smeenk.
It would have been obvious to use full length antibodies with a reasonable expectation of success as Bartels demonstrates that bispecific antibodies can be formed by tethering full length antibodies together using site specific conjugation methods including click chemistry.
An ordinarily skilled artisan would have been motivated to use the modified Fc-III peptide taught by the combination of WO’052 and Martos-Malonado to modify the antibodies for conjugation in order to achieve improved conjugation efficiency, high selectivity, and fewer steps to conjugation products as taught by WO’052 while avoiding the need for potentially laborious genetic fusions as taught by Bartels. An ordinarily skilled artisan would have had a reasonable expectation of success as Kim and Bartels both teaching site-specific introduction of functional groups on antibodies for conjugation to form bispecific antibodies and the methods taught by the combination of WO’052 and Martos-Malonado result in site specific conjugation of a functional group on antibodies, specifically an acetyl azide group.
One of ordinary skill in the art would have been motivated to modify the functionalities on the PEG linkers disclosed by Kim by substituting the aloxy-amine on the terminus that binds to the antibody with a group that reacts with an azide group, including DBCO, in order to attach the linker to the azide group on the azidoacetylated antibodies. An ordinarily skilled artisan would have further been motivated to substitute the azide and cyclooctyne groups on the other terminus of the linkers with an alternative click chemistry pair, including methyl tetrazine and norbornene as taught by Smeenk that so that the groups would not react with the azide or DBCO groups. An ordinarily skilled artisan would have had a reasonable expectation of success as Smeenk demonstrates that these click chemistry reactant pairs are able to form stable conjugates. Additionally, Martos-Malonado also demonstrates azido functionalized groups conjugated with DBCO (page 4, right column, paragraph 5) further supporting the use of DBCO to conjugate the linkers to the antibodies.
Regarding claim 2, it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to modify the Fc-III peptide taught by the combination of Kim, Bartels, WO’052, Martos-Maldonado, and Smeenk to have the azidoacetyl group to the position 3- or 4- on the phenyl based on the teachings of WO’052 and Martos-Maldonado, rendering obvious the structures of instant Formulas (I) and (IV). Specifically, WO’052 teaches a modified glutamine with a phenyl having an attachment point in position 3- and Martos Maldonado demonstrates attachment in position 4-, opposite of other moieties. Additionally, WO’052 teaches that the moiety of interest can be -
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≡-, suggesting the structure of Formula (VIII).
Claims 17-19 are rejected under 35 U.S.C. 103 as being unpatentable over Park, J.W., et al (2001) Tumor targeting using anti-HER2 immunoliposomes Journal of Controlled Release 74; 95-113 in view of Encapsula NanoSciences (2018) Immunosome®-DBCO (PEGylated) Technical Information Version 1.1, WO 2021/102052 A1 (Rastelli, L, et al) 27 May 2021, Martos-Malonado, M.C., et al (2018) Selective N-terminal acylation of peptides and proteins with a Gly-His tag sequence Nature Communications 9(3307); 1-13.
Park teaches the generation of anti-HER2 immunoliposomes consisting of long circulating liposomes linked to anti-HER2 monoclonal antibody fragments to provide targeted drug delivery to HER2-overexpressing cells (abstract).
Park teaches that, as a target antigen, HER2 is a readily accessible cell surface receptor and, when overexpressed, provides a basis for selective immunotargeting of tumor cells. As an oncogene product, HER2 plays an important role in the development and progression of many breast and other cancers. HER2 is overexpressed in 20-30% of breast and ovarian cancers, most commonly via gene amplification, and overexpression is associated with poor prognosis in these patients. In addition to breast and ovarian cancer, HER2-overexpression also occurs frequently in a number of other carcinomas. In normal adult tissues, HER2 occurs only at low levels in certain epithelial cell types. When present in cancer, HER2-amplification and overexpression typically display a relatively homogenous distribution within primary breast tumors, and also appears to be retained or increased at metastatic sites, suggesting a continuous requirement for HER2-overexpression throughout the malignant process (paragraph bridging page 96).
Monoclonal antibodies directed against HER2 offer one strategy for targeted anticancer therapy. One such antibody, muMAb 4D5, specifically binds HER2 in its extracellular domain (ECD), and inhibits the growth of HER2-overexpressing breast cancer cells in vitro and in animal models. A humanized version of this antibody, trastuzumab, was developed to retain these properties while reducing the potential for immunogenicity. In clinical studies in advanced breast cancer, trastuzumab induced antitumor responses as a single agent and was particularly effective when combined with anthracycline or taxane based chemotherapy (paragraph bridging pages 96-97).
Park teaches that immunoliposomes represent a logical strategy to achieve drug delivery to tumor cells by linking liposomes to monoclonal antibody fragments against tumor-associated antigens. Thus, Park developed anti-HER2 ILs for targeted intracellular drug delivery (page 97, right column, paragraph 1).
Park concludes that anti-HER2 immunoliposomes represent a promising technology for tumor-targeted drug delivery.
Park, however, does not disclose that the anti-HER2 immunoliposomes are synthesized according to the method of instant claim 3.
The teachings of Encapsula NanoSciences, WO’052, and Martos-Malonado are as discussed above and the combination of the references teach the method of claim 3 as discussed in detail above in the rejection of claim 3.
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to substitute the immunoliposome in the method of Park with an antibody-conjugated liposome prepared by the method taught by the combination of Encapsula NanoSciences, WO’052, and Martos-Malonado. An ordinarily skilled artisan would have been motivated to use the liposome preparation method taught by the combination of Encapsula NanoSciences, WO’052, and Martos-Malonado in order to achieve rapid, efficient, site-specific labeling of the liposome to the antibody. An ordinarily skilled artisan would have had a reasonable expectation of success as the combination of Encapsula NanoSciences, WO’052, and Martos-Malonado teaches antibody functionalized liposomes and Park teaches the use of antibody-functionalized liposomes for the treatment of cancers.
Response to Arguments
Applicant’s arguments and the declaration of Jiang Xia filed 12/31/2025 have been fully considered in so far as they apply to the rejections of the instant office action, but were not persuasive.
It is first noted that applicant’s arguments concerning the references Yamada and Chen have been fully considered but are moot as the rejections of the instant office action do not rely on these references for any teaching.
With regards to Martos-Maldonado, the declaration and response argue that the method of acetylation in Martos-Maldonado is achieved through the introduction of a Gly-His tag at the N-terminus of the engineered recombinant proteins instead of naïve antibodies, which results in N-terminal selective acetylation through reaction with phenyl ester. In contrast, applicant used antibody affinity peptides to achieve acetylation through the reaction between Lys248 (K248) in the antibody and phenyl ester in the peptide.
While Martos-Maldonado does demonstrate the reactivity of the phenyl esters in acetylating a recombinant protein not an antibody, Martos-Maldonado is not required to teach each and every limitation of the claimed invention in order to establish a prima facie case of obviousness. Rather, the rejection depends on the combination of the applied references and what the combination would have suggested to one of ordinary skill in the art prior to the effective filing date of the claimed invention. In this case, WO’052 is applied in the rejection to demonstrate site specific conjugation of antibodies using peptide targeting agents including a peptide with the sequence of Fc-III. As discussed in the rejection, WO’052 also suggests the modification of the peptide in the Glu8 position with a glutamine derivative that comprises a phenyl group as well as the use of esters to conjugate an azide moiety to an antibody. Martos-Maldonado is used in the rejection to further support the structure of the phenyl azidoacetate motif and demonstrate successful acetylation of proteins using the moiety.
Applicant further argues that, in the instant disclosure, applicant has substituted amino acids at the L6 and H5 sites (F2 (formula (II) and F3 (formula (III)) when phenol ester reactive groups and discovered that both F2 and F3 peptides showed poorer site-specific modification on antibodies as shown in Fig. 1D. Applicant argues that; therefore, one of ordinary skill in the art would not have been able to combine the references to achieve obvious results. Specifically, applicant uses modification of E8 in the FcIII peptide to achieve highly efficient acetylation of K248.
Fig. 1D from the instant disclosure is shown below for convenience. :
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The disclosure identifies the figure as Coomassie blue stained gel and fluorescent image to show successful acetylation by peptide F1. Lane M is a molecular weight marker; lane 1 is IgG Fc (5 ug); lane 2 is IgG Fc (5ug) and peptide F1; lane 3 is IgG Fc (5ug) and peptide F2; lane 4 is IgG Fc (5ug) and peptide F3.
While the results do demonstrate that the peptide of F1 results in significantly more acetylation compared to F2 and F3, the results provided by applicant are not commensurate in scope with the instantly rejected claims.
MPEP 716.02(d) states “Whether the unexpected results are the result of unexpectedly improved results or a property not taught by the prior art, the "objective evidence of nonobviousness must be commensurate in scope with the claims which the evidence is offered to support." In other words, the showing of unexpected results must be reviewed to see if the results occur over the entire claimed range.”
Claim 3 is drawn to a method of synthesizing an antibody-lipid conjugate comprising the use of a modified Fc-III peptide according to SEQ ID NO: 4, wherein the residue Glu8 is substituted with a glutamine derivative containing a phenyl azidoacetate motif at a side chain. As such, the claim encompasses any modified Fc-III peptide in which the Glu8 residue is substituted with any glutamine derivative so long as it contains a phenyl azidoacetate motif at any side chain.
The results provided by applicant demonstrate only a single species of the claimed peptide resulting in significantly higher amounts of acetylation, which is the compound F1.
The results provided by applicant further demonstrate that the structure of the modified Fc-III compound can significantly change the degree of acetylation achieved. For instance, the instant specification also discloses the compound F6, which comprises the following structure compared to F1:
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As shown, F6 is also a species of the instantly claimed compound of a modified Fc-III peptide according to SEQ ID NO: 4, where the Glu8 residue is substituted with a glutamine derivative containing a phenyl azidoacetate motif at a side chain. The results from 3C; however, suggest that the variation in the attachment point of the azidoacetate motif to the phenol in the species impacted the level of acetylation achieved by the peptide. For instance, see Fig 3C below, which has been annotated with a box to clearly show results where F1 and F6 were tested alone:
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The figure shows that F6 resulted in a lower amount of acetylation compared to F1. F6 differs from F1 in the structure of the phenyl azidoacetate motif, specifically in the location of the azidoacetate connection to the phenyl group, suggesting that the structure of the motif can also impact the degree of acetylation. This is further discussed in the specification which states that changing the 4-aminophenol (peptide F1) to 3-aminophenol (peptide F6) markedly decreased the acetylation yield (Fig. 3C), showing the importance of special organization of the electrophile. (page 53, lines 2-4).
While compound F1 demonstrates a significantly higher degree of acetylation compared to the same modification in the His5 or Leu6 position, this significant difference is not demonstrated over the entire scope of the instantly rejected claims.
Additionally, it is noted that MPEP 716.02 states “A difference of degree is not as persuasive as a difference in kind – i.e., if the range produces ‘"a new property dissimilar to the known property,’" rather than producing a predictable result but to an unexpected extent.”
Regarding claim 2, applicant and the declaration further argues that applicant demonstrates that “only the F1 peptide (formula (I)), or F6, f-F4, f-F5, and F7-F11, obtained from modifying E8 can effectively achieve site-specific modification of antibodies.”
These results; however, are not apparent from the instant disclosure.
For instance, the disclosure identifies that the parental Fc-III peptide (f-F0), an F1 analog in which the ester group was changed to an unreactive amide group (f-F5), and the hydrolytic product (f-F4) were synthesized and fluorescently labeled at the N-termini. Compared with f-F0, both f-F4 and f-F5 showed decreased binding affinities (Kd of 11.7 nm vs 1.5uM and 2.0uM, respectively), suggesting that both would have decreased labeling capacity. Fig. 3B demonstrates that f-F5 alone does not provide any acetylation:
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While F7-F11, shown in Fig. 27A-27B, are shown to label antibodies to some degree (results from 27B shown below), the labeling of antibodies using a Fc-III peptide modified in position Glu8 would have been reasonably expected in view of the teachings of the applied prior art, particularly WO’052. Additionally, as demonstrated in instant Fig. 27B, shown below, the percent of label varied significantly based on the structure of the peptide as well as the pH of the solution/time incubated.
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With regards to claim 3, and its dependent claims, the declaration argues that liposome synthesis is a relatively known and open method, including the use of biorthogonal reactions to modify liposomes. Applicant’s invention utilizes Fc-III derived F1 to modify the Fc region of antibodies and then coupled lipids through azide-cyclooctyne biorthogonal reaction to form antibody-lipid conjugates. Regarding the acetylation method of F1 to antibodies, it is not a simple combination of the applied references as discussed above.
As discussed in detail above, while applicant does demonstrate that F1 results in significantly higher amounts of acetylation of antibodies, none of instant claims 3-5, 7-8, or 17-19 require that the Fc-III modified peptide have the structure of F1. Rather, the claims encompass any modified Fc-III peptide in which the residue of Glu8 is substituted with any glutamine derivative containing a phenyl azido acetate moiety at any side chain. As such, applicant’s arguments are not commensurate in scope with the instantly claimed invention.
With regards to claims 10-12, 14-15, and 20-22, applicant and the declaration argue that the molecular weight of a Fab is 50kDa, while the full length antibody has a molecular weight of 150kDa, so linking two Fabs together is different than linking two full length antibodies. Applicant argues that, generally, it is considered to be more difficult to conjugate proteins with larger molecular weights. Smeenk reports a method of conjugating two full length antibodies but does not use the proximity effect of antibody binding peptides to achieve site specific acetylation of antibodies. Applicant argues that, regarding the F1 method for acetylation, it is not a simple combination of the applied references as discussed above.
In response, while applicant argues that it is generally considered to be more difficult to conjugate proteins with larger molecular weights, applicant has provided no evidence to support the speculation that the art would view this as a more difficult strategy. Additionally, the rejections of record do not rely on the teachings of any of the applied references alone, including Kim, Bartels, or Smeenk. Rather, the rejections rely on the combination of applied references and what the references would have suggested to one of ordinary skill in the art prior to the effective filing date of the claimed invention. In this case, Kim is not required to teach the binding proteins of higher molecular weight (antibodies) as this limitation is taught and supported by Bartels which teaches the use of click chemistry for the formation of bispecific antibodies using intact antibodies. Furthermore, it is considered that the standard of obviousness is reasonable expectation of success and conclusive proof of efficacy is not required in order to establish a prima facie case of obviousness.
With regards to applicant’s arguments concerning the use of F1 as a means for acetylation, none of instant claims 10-12, 14-15, and 20-22 require that the Fc-III modified peptide have the structure of F1. Rather, the claims encompass any modified Fc-III peptide in which the residue of Glu8 is substituted with any glutamine derivative containing a phenyl azido acetate moiety at any side chain. As such, applicant’s arguments are not commensurate in scope with the instantly claimed invention.
Allowable Subject Matter
Claim 1 is allowed.
Claims 9 and 16 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
The following is a statement of reasons for the indication of allowable subject matter: The instant claims are drawn to a modified Fc-III peptide having the structure of formula (I), which is the peptide F1 of the instant disclosure. The claims also encompass the application of the F1 peptide in a method of synthesizing an antibody-lipid conjugate and a method of synthesizing a bispecific antibody complex. In searches of the prior art no peptide was found that is an identical match to the peptide F1 of formula I. The closest prior art identified is WO’052, which is discussed in detail in the rejections of the instant office action.
WO’052 teaches a modified Fc-III peptide and teaches that the peptide can be modified in position 5, 6, 8, or 10 and provides various moieties that can be attached to the peptide for the purpose of site-specific conjugation. In combination the reference suggest the use of glutamine derivative comprising a phenyl group and an ester with the active moiety being azide. The use of this moiety is further supported by the teachings of Martos-Malonado. While these references suggest that the peptide of the instantly claimed invention would be obvious to make and use, applicant has provided evidence that is sufficient to overcome the prima facie case of obviousness.
Specifically, applicant demonstrates in Fig. 1D, shown below, that the peptide F1 resulted in significantly higher antibody acetylation compared to F2 and F3, which comprise the same Fc-III structure and modified glutamine with the phenyl azidoacetate motif but in positions 5 and 6 as opposed to 8.
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These results demonstrate that the peptide F1 results in an unexpectedly high acetylation and demonstrates the criticality of the structure of the claimed peptide.
The criticality of the structure of the claimed peptide is further demonstrated in results that compare F1 to F6 which differs in the attachment point of the azidoacetate moiety on the phenyl ring. As discussed in the specification, changing the 4-aminophenol (peptide F1) to 3-aminophenol (peptide F6) markedly decreased the acetylation yield (Fig. 3C), showing the importance of special organization of the electrophile.
In searches of the prior art no art was identified that would have provided a reasonable expectation that a peptide with the exact structure claimed would result in a significant increase in Fc acetylation. While WO’052 does suggest that the modification can be made in position E8 of FcIII, WO’052 does not suggest that this would result in a significantly higher acetylation of an antibody or that the structure of the side chain motif would play a critical role in providing higher conjugation. As such, the structure claimed was found to be novel and non-obvious.
Conclusion
THIS ACTION IS MADE FINAL. 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 extension fee 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.
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/AUDREY L BUTTICE/Examiner, Art Unit 1647
/SCARLETT Y GOON/Supervisory Patent Examiner
Art Unit 1693