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 .
Election/Restrictions
Applicant’s election without traverse of Group I (claims 1-6, 9-13, 16-18, 21-25, 30-32, and 34-37) and the species of (i) an N-linked glycosylation site and (ii) glycan trimming by endoglycosidase in the reply filed on 05/20/2026 is acknowledged.
Claim Status
Claims 14-15, 19-20, 26-29, 33, 38-42, 44-52, and 54-56 have been cancelled as requested in the amendment filed on 05/20/2026. Following the amendment, claims 1-6, 9-13, 16-18, 21-25, 30-32, 34-37, 43, and 53 are pending in the instant application.
Claims 43, and 53 stand as withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention and claims 11-12 and 17-18 stand as withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected species (i.e., chemical trimming or O-linked glycosylation) of invention in the Response filed 05/20/2026, there being no allowable generic or linking claim.
Claims 1-6, 9-10, 13, 16, 21-25, 30-32, and 34-37 are under examination in the instant office action.
Priority
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged.
Claims 1-6, 9-10, 13, 16-17, 21-25, 30-32, and 34-37 have an effective filing date of May 11, 2021 corresponding to PRO 63/187,376.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 01/07/2026 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Furthermore, it is noted that the listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Claim Interpretation
It is specifically noted that the sequence language of claim 9, reciting “an N-linked glycosylation site having an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-17” is being interpreted under broadest reasonable interpretation (BRI) as any sequence comprising at least two consecutive amino acid residues of SEQ ID NOs: 1-17 and an N-linked glycosylation site (i.e., an asparagine residue). The recitation of “an amino acid sequence” allows for truncations/mutations of the recited SEQ ID NOs such that a reference sequence meets the limitation when the reference sequence comprises (i) an asparagine residue and (ii) two consecutive amino acids comprised within SEQ ID NOs: 1-17.
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 22-24 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 the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 22 recites the limitation "the glycan" in line 1. There is insufficient antecedent basis for this limitation in the claim because it is unclear as to what “the glycan” is intended to refer to. Specifically, it is unclear if the “the glycan” is intended to refer to: (i) “the trimmed core glycan” of claim 21 from which claim 22 depends; (ii) “an unnatural glycan” of claim 21 from which claim 22 depends; or (iii) “the glycan” of claim 1 from which claim 22 ultimately depends. Additionally, claim 22 recites the limitation "the trimmed core of the N-glycan" in lines 1-2. There is insufficient antecedent basis for this limitation in the claim because “the trimmed core of the N-glycan” compounds on the recitation of “the glycan” which lacks antecedent basis. Thus, claim 22 is indefinite. For the purpose of applying art, claim 22 is being interpreted as follows: “[t]he method of claim 21, wherein the trimmed core glycan is a trimmed core N-glycan”.
Claim 23 recites the limitation "the trimmed core N-glycan" in line 2. There is insufficient antecedent basis for this limitation in the claim because neither of claims 1 or 21 from which claim 23 ultimately depends, recite a trimmed core N-glycan and the recitation of “the trimmed core of the N-glycan” in claim 22 lacks antecedent basis. Thus, claim 23 is indefinite.
Claim 24 recites the limitation "the at least one of said one or more modification site amino acid residues" in lines 3-4. There is insufficient antecedent basis for this limitation in the claim because claim 1, from which claim 24 depends, does not recite “one or more modification site amino acid residues”. Thus, claim 24 is indefinite. For the purpose of applying art, claim 24 is being interpreted as follows: “[t]he method of claim 1, wherein in step (b) the reagent comprises an amine-containing reagent that becomes covalently linked via an isopeptidic bond between an amine group of the amine-containing reagent and acyl group on a side chain of the modification site amino acid residue”.
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 23 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 23 recites "wherein a galactose moiety comprising a chemical handle is transferred onto the trimmed core N-glycan using a galactosyltransferase"; however claim 21 from which claim 23 depends recites "transferring a galactose moiety comprising a chemical handle onto the trimmed core glycan using a β-1,4-galactosyltransferase". Thus, claim 23 fails to further limit claim 21, as claim 23 recites "a galactosyltransferase", which is broader than "a β-1,4-galactosyltransferase". Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-6, 10, 13, 16, 21-25, 30-32, 34, and 36 are rejected under 35 U.S.C. 103 as being unpatentable over non-patent literature by Walker et. al. (Bioconjugate Chem., 2019, 30, 2452-2457; herein after referred to as "Walker") in view of non-patent literature by Agarwal and Bertozzi (Bioconjugate Chem., 2015, 26, 176-192; herein after referred to as "Agarwal") and non-patent literature by Van Geel et. al. (Bioconjugate Chem., 2015, 26, 2233-2242; herein after referred to as "Geel").
Walker teaches that site-specific modification of native antibodies has proven advantageous, as it enhances the properties of antibody-based bioconjugates without the need to manipulate the genetic code; however, native antibody modification is typically limited to strategies that introduce a single functional handle (Abstract). The authors addressed this limitation by designing heterobifunctional substrates for microbial transglutaminase (MTG) that contain both azide and methyltetrazine “click” handles and the structure-conjugation relationships for these substrates were evaluated using the Her2-targeted antibody trastuzumab (Id.; emphasis added). MTG recognizes glutamine 295 (Q295) within the heavy chain of aglycosylated, human IgGs, and co-treatment with peptide:N-glycosidase F (PNGase F) removes the N-linked glycan at asparagine 297 (N297) and facilitates efficient bioconjugation (i.e., Q295 is two amino acids away from the N297 glycosylation site); by supplying non-natural acyl acceptor substrates, this natural function has been co-opted for site-specific, homofunctional antibody modification (Page 2453, Column 1, First Full Paragraph; emphasis added). To validate the 2-to-1 conjugation, a multimilligram scale synthesis of conjugate T5 was performed, wherein conjugate T5 was isolated via HIC and recovered in high purity at an overall yield of 1.1 mg (48%) and successful conjugation of 2 substrates per antibody was confirmed via matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS); treatment of trastutuzmab with PNGase F to remove the N-linked glycan at N297 produced a decrease of approximately 1630 Da in the molecular weight of the heavy chain fragment, and simultaneous treatment with PNGase F, MTG, and linker 5 resulted in a 500 Da increase in the molecular weight of the aglycosylated control, consistent with incorporation of one linker per heavy chain (Page 2454, Column 1, Second Full Paragraph; emphasis added). Dual “click” modification of conjugate T5 was demonstrated via fluorescent SDS-PAGE analysis, wherein conjugate 5 was reacted with a Förster resonance energy transfer (FRET) pair of fluorophores, DBCO-modified carboxyrhodamine 101 and TCO-modified sulfo-Cy5; upon excitation with 488 nm light, carboxyrhodamine 101 displays a characteristic fluorescence emission at 523 nm and upon excitation with 633 nm light, sulfo-Cy5 displays a characteristic fluorescence emission at 655 nm (Page 2454, Column 1, Third Full Paragraph). Dual modification leads to intramolecular energy transfer and subsequent emission at 655 nm (sulfo-Cy5) upon excitation at 488 nm (carboxyrhodamine 101); this data validates that these chemistries (SPAAC and IEDDA) are mutually orthogonal and, therefore, enable the one-pot synthesis of bifunctional antibody conjugates (Id.). The authors synthesized, characterized, and tested the conjugation efficiency of five heterobifunctional substrates for MTG and identified spacer flexibility alpha to the primary amine as the critical structural component for efficient conjugation; a heterobifunctional, dual “click” conjugate was synthesized and characterized at multimilligram scale and this conjugate was used to demonstrate the mutually orthogonal nature of the SPAAC and IEDDA reactions (Page 2455, Column 1, Last Paragraph). This powerful feature was leveraged for the one-pot synthesis of a bifunctional ADC containing a maytansine-derived cytotoxic payload and hydrophobicity-masking PEG side chain, and this bifunctional antibody conjugate was shown to induce Her2-specific toxicity in an in vitro cell viability assay; taken together, these data demonstrate the power of substrate design in developing new approaches to site-specific antibody modification and the conjugates described may find utility in the field of pre-targeted, bioorthogonal labeling for the delivery of therapeutic cargo and imaging agents (Page 2455, Column 2, First Partial Paragraph). Furthermore, iterative methodology for linker synthesis, could be used to elaborate on the principles outlined in this work, which could enable the synthesis of antibody conjugates that contain multiple functionalities, including combinations of imaging agents, drug payloads, and stabilizing PEG chains; ultimately work in this area could yield multifunctional ADCs with high drug loading, diagnostic capabilities, and optimized pharmacokinetics (Id.).
However, Walker does not explicitly teach or suggest: (i) trimming a glycan linked to a glycosylation site amino acid residue of a glycoprotein whereby a core of the glycan remains linked to the glycosylation site amino acid residue; (ii) transferring a galactose moiety comprising a chemical handle onto the trimmed core glycan, transferring an unnatural glycan substrate onto the trimmed core glycan, or removing fucose from the trimmed core glycan; nor (iii) attaching a functional group to the chemical handle of a galactose moiety transferred to the trimmed core glycan. These deficiencies are remedied by Agarwal and Geel.
Agarwal teaches that with advances in the fields of bioorthogonal chemistry and protein engineering, there is growing interest in producing ADCs by site-specific conjugation to the
antibody, yielding more homogeneous products that have demonstrated benefits over their heterogeneous counterparts in vivo; the authors chronicle the development of a multitude of
site-specific conjugation strategies for assembly of ADCs and provide a comprehensive account of key advances and their roots in the fields of bioorthogonal chemistry and protein engineering (Abstract). Human IgG molecules have a conserved glycosylation site at each N297 residue in the CH2 domain, and the inherent site specificity of this post-translational modification and decades of research on glycoengineering make the pendant N-glycans a convenient target for site-specific conjugation; the glycosylation site is sufficiently far from the variable region that conjugation to attached glycans is unlikely to impact antigen binding, however glycosylation is a heterogeneous post translational modification, rendering the generation of homogeneous glycans for chemical modification a formidable challenge (Page 182, Column 2, Last Paragraph through Page 183, Column 1, First Paragraph). Transglutaminase catalyzes amide bond formation between glutamine side chains and small molecules containing a primary amine, and it is notable for its relaxed small molecule substrate specificity; work on using microbial transglutaminase to modify antibodies was first published by Schibli, whose group showed that Q295, the natural amino acid residue that is situated near the N297 glycosylation site (i.e, Q295 is two amino acids away from the glycosylation site), could be a substrate for transglutaminase, but only if the N-glycan was first removed wherein such treatment of an IgG makes Q295 more sterically accessible and increases the flexibility of the loop in which the residue is situated (Page 187, Column 1, Last Paragraph). Treatment of rituximab or an anti-L1 CAM antibody with the glycosidase PNGase F followed by transglutaminase in the presence of various amine-containing small molecules led to isopeptide bond formation at Q295; alternatively, transglutaminase treatment of the N297Q mutant yielded products modified at Q295 and Q297, resulting in up to four small molecule additions per antibody (Page 187, Column 1, Last Paragraph through Page 187, Column 2, First Paragraph). The Schibli group collaborated with Innate Pharma to use microbial transglutaminase for production of ADCs; in the initial study, amine-functionalized small molecules were directly coupled to antibodies in the presence of transglutaminase, and when this approach was applied to PNGase-treated IgG1 using amine-functionalized auristatin derivatives, only 50−80% of heavy chains were modified despite the presence of 40 equivalents of drug-linker; hypothesizing that transglutaminase would more readily accept a smaller substrate with a clickable functional group, the authors instead modified several antibodies with an azido-PEG-amine to yield fully modified conjugates wherein subsequent reaction with a cyclooctyne-functionalized MMAE yielded a uniform product with a DAR of 2 by MS analysis of the heavy chain, suggesting >95% coupling efficiency (Page 187, Column 2, First Full Paragraph). Thus, Agarwal suggests that reducing steric hinderance at N297 (e.g., by removing the N-glycan completely) makes Q295 available to transglutaminase for site-specific conjugation.
Geel teaches that the vast majority of ADCs in the clinic are based on conjugation of payload to naturally available amino acid side-chains (lysine, cysteine), leading to a stochastic distribution of drug-antibody ratio (DAR) between 0 and 8 (or even higher), and that it has been demonstrated that random conjugation has a negative impact on efficacy, and as a consequence the therapeutic index remains low (Page 2233, Column 1, First Paragraph). Two main strategies can enhance the therapeutic index of a given mAb-payload combination: (a) site-specific conjugation and (b) enhancing stability; site-specific conjugation is typically achieved by engineering of a specific amino acid (or sequence) into an antibody, serving as the anchor point for payload attachment (Page 2233). However, re-engineering of protein sequence and site optimization is a laborious exercise and expression yields may be compromised (typically ∼50% yield reduction for a cysteine-engineered mAb, while expression yields for genetic encoding of an unnatural amino acid typically reach 1 g/L maximally), which has a significant impact on the cost of goods of the ADC (Page 2233, Column 2, First Partial Paragraph). Additionally, it has been amply demonstrated that ADCs obtained by conjugation to cysteine side chains often display limited stability in circulation, leading to premature disconnection of the cytotoxic payload before the tumor site is reached (Id.). The authors report a unique and robust chemoenzymatic technology for efficient mAb-to-ADC conversion by anchoring a payload to the antibody’s glycan at precisely one azide, a unique anchor point is introduced for copper-free click conjugation with a payload wherein the versatility of this technology, termed GlycoConnect, is demonstrated across a range of different mAb isotypes and linker-payload combinations both in vitro and in vivo; seamless upscaling of GlycoConnect corroborated manufacturability, whereas the superiority of the native glycosylation site for conjugation was demonstrated through in vivo efficacy assessment of a series of ADCs based on glycosylation mutants (Pages 2233-2234). The authors disclose a chemoenzymatic ligation strategy to generate DAR2 ADCs, based on the strategy depicted in Figure 1 (Fig. 1A reproduced below): (1) trimming of all glycan isoforms (complex, hybrid, high-mannose) with an endoglycosidase, thereby liberating the core GlcNAc; (2) enzymatic transfer of a galactose residue harboring a reactivity-enhanced azide to reduce conjugation stoichiometry and incubation time; and (3) copper-free click conjugation with bicyclononyne (BCN), a cyclooctyne with minimal
PNG
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lipophilicity to reduce aggregation. Regarding the endoglycosidase, Geel teaches that a mAb obtained by recombinant expression in a mammalian system will display high glycan heterogeneity, and therefore effective trimming of all N-glycans present on any IgG requires access to an endoglycosidase with excellent substrate tolerability further wherein high activity of the endoglycosidase (or more precise: endo-β-N-acetylglucosaminidase) is also desirable; endoglycosidase endo S2 would displayed the desirable combination of substrate tolerability and activity wherein incubation of trastuzumab with only 1% (w/w) of enzyme was sufficient for full hydrolysis of the glycans (i.e., hydrolysis of N,N’ diacetyl chitobiose bonds), including oligomannose structures, as judged by SDS-PAGE and mass spectrometric analysis (Page 2235, Column 1, Endoglycosidase Treatment). Specifically with regard to the transfer of a galactose residue to the core GlcNAc, it is noted that incubation of trimmed trastuzumab (II) with GalNAz 2 (0.4 mM) in the presence of only 1% (w/w) of GalT(Y289L) (i.e., a β-1,4-galactosyltransferase) and MnCl2 (10 mM) led to complete labeling after overnight incubation (Page 2235, Column 1, Galactosyl Transferase). The conditions and conjugation efficiencies of azido-modified trastuzumab variants with various BCN-linker-payload constructs are provided in Table 1 (see Page 2236). Thus, Geel discloses glycan trimming via endoglycosidase to make the core GlcNAc available for modification (i.e., the GlcNAc directly linked to Asn297), after which a glycosyltransferase may be used to attach an azido-modified sugar (e.g., UDP-GalNAz) that can be reacted with BCN to conjugate various linker-payloads to said azido-modified galactose residue. The advantage of glycan-remodeled ADCs versus randomly labeled ADCs becomes apparent in vivo; PDX mice with an average tumor size of 100 mm3 were intravenously injected with one of three different GlycoConnect ADCs (cleavable/noncleavable with maytansine or cleavable with MMAF) and compared to Kadcyla at 9 mg/kg (single dose bolus injection), followed by twice weekly tumor sizes measurements (Figure 5) (Page 2237, Column 1, First Full Paragraph). The results were as follows: (i) during the first week, all groups showed significant tumor shrinkage or even complete regression; (ii) after 1 week, tumor sizes gradually increased for mice treated with Kadcyla but not for any of the GlycoConnect ADCs; (iii) only after approximately 3 weeks, reappearance and regrowth of tumors became apparent for mice treated with trastuzumab-vc-PABA-MMAF, while both ADCs based on maytansine (cleavable or noncleavable) showed complete tumor regression for the whole length of the study (60 days) (Id.). This study thus clearly demonstrates the superiority of GlycoConnect conjugation technology versus a randomly conjugated ADC based on the same components, wherein the latter observation becomes particularly interesting in view of the higher drug loading of Kadcyla (average DAR3.5) versus GlycoConnect ADCs (DAR2; the modified N297 site of each heavy chain in the antibody is conjugated to a drug-linker) (Id.). The authors also note that no difference in blood clearance was observed (Page 2237, Column 1, Second Full Paragraph). Thus, the resulting ADCs were found to be homogeneous and highly hydrolytically stable, while displaying negligible aggregation, and benchmark in vitro and in vivo efficacy studies against the marketed product Kadcyla, based on the same antibody and payload components, generated strong biological data in favor of the GlycoConnect ADCs, despite the lower DAR (Page 2238, Column 2). GlycoConnect technology is readily applicable to any off-the-shelf monoclonal antibody (turnaround in <1 week), while delivering site-specific, stable, and highly efficacious ADCs, and therefore GlycoConnect shows high promise as the next generation ADC generation technology for targeted therapy with improved therapeutic index (Id.). Thus, Geel discloses trimming a glycan at Asn297 using an endoglycosidase such that the core of the glycan remains at Asn297, followed by antibody modification at Asn297 via the core GlcNAc, wherein each antibody heavy chain is modified to comprise, generally, GlcNAc(Fuc)GalNAc-Azide which may be reacted with a cyclooctyne group (e.g., BCN) via ligation reaction with various payload-linkers.
In the test of whether it is “obvious to try” there must be:
(1) a finding in the art at the time of filing of the invention that there had been a recognized problem or need in the art;
(2) a finding that there had been a finite number of identified, predictable potential solutions to the recognized need or problem;
(3) a finding that one of ordinary skill in the art could have pursued the known potential solutions with a reasonable expectation of success.
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to modify the method of Walker, wherein the N-glycan at Asn297 is completely removed to make the glutamine residue(s) in close proximity available for transglutaminase-mediated conjugation by supplying non-natural acyl acceptor substrates for site-specific, homofunctional antibody modification, such that instead of removing the N-glycan, the N-glycan is trimmed such that a core of the N-glycan (i.e., GlcNAc) remains linked to the Asn residue, after which (i) now available glutamine may be used for conjugation and/or the timed core of the N-glycan may be used for conjugation (e.g., transferring a galactose moiety comprising a chemical handle onto the trimmed core glycan and attaching a functional group to said chemical handle via, for example, click chemistry). One would have been motivated to make such a modification because: (i) Walker teaches that site-specific modification of native antibodies (e.g., adding payloads and/or imaging agents such as chromophores) has proven advantageous, as it enhances the properties of antibody-based bioconjugates without the need to manipulate the genetic code; (ii) Agarwal suggests that reducing steric hinderance at N297 (e.g., by removing the N-glycan) makes Q295 available to transglutaminase for site-specific conjugation; and (iii) Geel discloses trimming a glycan at Asn297 using an endoglycosidase such that the core of the glycan remains at Asn297, followed by antibody modification at Asn297 via the core GlcNAc, wherein each antibody heavy chain is modified to comprise, generally, GlcNAc(Fuc)GalNAc-Azide (GalNAc-Azide is a functionalized galactose moiety transferred via β-1,4-galactosyltransferase) which may be reacted with a cyclooctyne group (e.g., BCN) via ligation reaction with various payload-linkers. One of ordinary skill in the art would have a reasonable expectation of success of generating homogenous, conjugated antibodies useful for (i) therapeutic applications with the conjugation of payloads and/or (ii) diagnostics/imaging with the conjugation of detectable agents, because Agarwal suggests that removing the steric hinderance at Asn297 cause by the presence of the full N-glycan structure opens up glutamine for conjugation (e.g., via known methods disclosed by Walker) and one of ordinary skill in the art would recognize that trimming the N-glycan at Asn297 via endoglycosidase (via known methods disclosed by Geel) would remove the bulk of the steric hinderance opening glutamine for conjugation and allowing for additional site-specific modifications at Asn297.
Claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over Walker et. al. (Bioconjugate Chem., 2019, 30, 2452-2457; herein after referred to as "Walker"), Agarwal and Bertozzi (Bioconjugate Chem., 2015, 26, 176-192; herein after referred to as "Agarwal"), and Van Geel et. al. (Bioconjugate Chem., 2015, 26, 2233-2242; herein after referred to as "Geel"), as applied to claims 1-6, 10, 13, 16, 21-25, 30-32, 34, and 36 above, and in further view of WO 2015/184002 A1 (herein after referred to as “Wu”).
The method of claim 1 is rendered obvious by the combination of Walker, Agarwal, and Geel. However, it is noted that none of the cited references explicitly teach or suggest an N-linked glycosylation site having an amino acid residue selected from the group consisting of SEQ ID NOs: 1-17. This deficiency is remedied by Wu.
Wu teaches a composition of anti-HER2 glycoantibodies comprising a homogeneous population of anti-HER2 IgG molecules having the same N-glycan on each of Fc; the anti-HER2 glycoantibodies of the invention can be produced from anti-HER2 monoclonal antibodies by Fc glycoengineering wherein the anti-HER2 glycoantibodies have improved therapeutic values with increased ADCC activity or increased Fc receptor binding affinity compared to the corresponding monoclonal antibodies that have not been glycoengineered (Paragraph 0008). In preferred embodiments, the N-glycan is attached to the Asn-297 of the Fc region and in some embodiments, the anti-HER2 glycoantibody described herein comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 1, and a light chain having the amino acid sequence set forth in SEQ ID NO: 2 wherein, in a preferred embodiment, the glycoantibody comprises a light chain sequence and a heavy chain sequence of Trastuzumab (Paragraphs 0010-0011; emphasis added). It is specifically noted that the heavy chain sequence residues 294-302 of Wu SEQ ID NO: 1 corresponds to instant SEQ ID NO: 1; thus, Wu teaches that trastuzumab (the antibody modified by transglutaminase in the Walker and Geel references) comprises an exact match to instant SEQ ID NO: 1.
Thus, it would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to use the method rendered obvious by Walker, Agarwal, and Geel to modify a glycoprotein comprising an N-linked glycosylation site having an amino acid sequence of SEQ ID NO: 1. One would have been motivated to apply the method to such glycoproteins because both Walker and Geel teach the site-specific modification of (i) glutamine or (ii) asparagine comprising a trimmed core GlcNAc, respectively, and the heavy chain of trastuzumab, as taught by Wu, comprises an exact match to instant SEQ ID NO: 1 (which comprises the N-glycosylation site) and thus one of ordinary skill in the art would have a reasonable expectation of success of modifying trastuzumab at glutamine and/or asparagine comprising a trimmed core GlcNAc.
Claim 35 is rejected under 35 U.S.C. 103 as being unpatentable over Walker et. al. (Bioconjugate Chem., 2019, 30, 2452-2457; herein after referred to as "Walker"), Agarwal and Bertozzi (Bioconjugate Chem., 2015, 26, 176-192; herein after referred to as "Agarwal"), and Van Geel et. al. (Bioconjugate Chem., 2015, 26, 2233-2242; herein after referred to as "Geel"), as applied to claims 1-6, 10, 13, 16, 21-25, 30-32, 34, and 36 above, and in further view of non-patent literature by Wang et. al. (Annu. Rev. Biochem., 2019, 88, 433-459; herein after referred to as “Wang”).
The method of claim 1 is rendered obvious by the combination of Walker, Agarwal, and Geel. However, it is noted that none of the cited references explicitly teach or suggest modifying a naturally occurring antibody while retaining its primary sequence, core glycan, and binding specificity. This deficiency is remedied by Wang.
Wang teaches that a typical IgG antibody is composed of two light and two heavy chains that are associated to form three protein domains: two identical Fab regions specific for antigen binding and an Fc domain (constant or crystallizable IgG fragment) responsible for engaging various Fc receptors in antibody effector functions; the Fab domain and the Fc domain are connected by a flexible hinge region and the IgG–Fc domain is a homodimer, in which the two third constant domains (CH3 domains) are paired through noncovalent interactions, while each of the two second constant domains (CH2 domains) carries an N-linked oligosaccharide (N-glycan) at the conserved N-glycosylation site (Asn-297) (Page 434, Introduction, First Paragraph; emphasis added). Structural analysis of N-glycans released from the Fc domain of human polyclonal IgG (i.e., naturally occurring antibodies) and recombinant mAbs has indicated that the Fc glycans are of the typical biantennary complex type with a considerable level of structure heterogeneity wherein more than 30 different Fc oligosaccharides in which the core heptasaccharide bears 0, 1, or 2 terminal galactose moieties have been characterized, and most are fucosylated (Page 434 Last Paragraph through Page 435 First Paragraph; see Figure 1). Natural and recombinant antibodies are usually produced as heterogeneous mixtures of glycoforms that are extremely difficult to separate or enrich to isolate pure forms, and therefore methods that can lead to the production of structurally well-defined homogeneous glycoforms of antibodies are needed for both functional studies and the development of more efficient antibody-based therapeutics; several aspects of antibody glycosylation have been explored to control and modulate the glycosylation pattern of antibodies including (i) a genetic approach that focuses on controlling protein glycosylation by manipulating the N-glycan biosynthetic pathways in different host expression systems and (ii) in vitro glycan remodeling via chemoenzymatic synthesis that could lead to highly homogeneous antibody glycoforms wherein glycoengineering of Fc N-glycans is emerging as an attractive method for site-specific antibody–drug conjugation (Page 435, Last Paragraph). Thus, Wang teaches that glycosylation sites are conserved in natural antibodies as well as recombinantly produced antibodies.
Thus, it would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to use the method rendered obvious by Walker, Agarwal, and Geel to modify a naturally occurring antibody wherein the primary sequence, core glycan, and binding specificity are maintained. One would have been motivated to apply the method to naturally occurring antibodies because Wang teaches that glycosylation sites are conserved in natural antibodies as well as recombinantly produced antibodies (e.g., trastuzumab) and both Walker and Geel teach the site-specific modification of (i) glutamine or (ii) asparagine, respectively, wherein Geel specifically teaches modification of asparagine comprising a trimmed core GlcNAc without altering the primary sequence of the antibody and maintaining the binding specificity; thus one of ordinary skill in the art would have a reasonable expectation of success of modifying naturally occurring antibodies at glutamine and/or asparagine, wherein the asparagine comprises a trimmed core GlcNAc, without altering the primary sequence and while preserving binding specificity.
Claim 37 is rejected under 35 U.S.C. 103 as being unpatentable over Walker et. al. (Bioconjugate Chem., 2019, 30, 2452-2457; herein after referred to as "Walker"), Agarwal and Bertozzi (Bioconjugate Chem., 2015, 26, 176-192; herein after referred to as "Agarwal"), and Van Geel et. al. (Bioconjugate Chem., 2015, 26, 2233-2242; herein after referred to as "Geel"), as applied to claims 1-6, 10, 13, 16, 21-25, 30-32, 34, and 36 above, and in further view of WO 2014/066733 A2 (herein after referred to as “Agnew”).
The method of claim 1 is rendered obvious by the combination of Walker, Agarwal, and Geel. However, none of the cited references explicitly teach or suggest a kit for site-specific modification comprising an endoglycosidase, a transglutaminase, and instructions for performing the method of claim 1. This deficiency is remedied by Agnew.
Agnew teaches methods, compositions and kits for use in the site-specific labeling of glycoproteins comprising a combination of enzyme-mediated incorporation of modified sugars comprising a chemical handle and cycloaddition chemistry with a labeling molecule comprising a reactive group, a metal ion chelator, and/or a fluorophore (Abstract). Methods for labeling a glycoprotein are provided, the methods comprising: a) providing a glycoprotein comprising a terminal GlcNAc residue; b) providing a modified sugar comprising a chemical handle; c) contacting the glycoprotein with the modified sugar, wherein the modified sugar attaches to the terminal GlcNAc residue to provide a modified glycoprotein; d) providing a labeling molecule comprising a metal ion chelator group, a reactive group, and a fluorophore; e) contacting the modified glycoprotein with the labeling molecule, wherein the reactive group attaches to the chemical handle to provide a labeled glycoprotein; f) providing a radioactive metal ion; and g) contacting the labeled glycoprotein with the radioactive metal ion, wherein the metal ion associates with the chelator group to provide a radiolabeled glycoprotein (Page 5, Lines 14-26). In certain embodiments, the glycoprotein comprises an antibody or an Fc-fusion protein, wherein in certain embodiments the antibody is an IgA, an IgE, an IgD, an IgG, an IgM, or an IgY and/or the antibody has an affinity for a cell-associated (Page 5, Lines 27-30). In certain embodiments, prior to step (c), the method further comprises the steps of providing a glycoprotein comprising an oligosaccharide having a GlcNAc-GlcNAc linkage; providing an enzyme to cleave the oligosaccharide at the GlcNAc-GlcNAc linkage; and contacting the glycoprotein with the enzyme to provide a glycoprotein comprising a terminal GlcNAc residue, wherein in certain embodiments, the enzyme is an endoglycosidase (Page 6, Lines 1-6). Kits are provided for use in the methods of the invention, wherein in certain embodiments, kits are provided for labeling a glycoprotein that include a modified sugar comprising a chemical handle, and a labeling molecule comprising a metal ion chelator group and a reactive group and, in further embodiments, the kits further comprise instructions for using the components in any of the methods of the invention (Page 24, Lines 4-8). In certain embodiments, the kits may further include one or more of the following: an endoglycosidase, a sialidase, a β-galactosidase, a galactosyl transferase, a mutant galactosyl transferase, a Y289L mutant galactosyl transferase, a glycoprotein, an antibody, an Fc-fusion protein, and a radioactive metal ion; in certain embodiments, the kits may further include one or more of the following: one or more buffers, detergents and/or solvents (Page 25, Lines 10-15). Thus, Agnew teaches methods for site-specific labeling of glycoproteins (e.g., antibodies) wherein such a method comprising glycan trimming with an endoglycosidase and subsequent labeling of the trimmed glycan using chemical handles (similar to the method of Geel) and kits for performing such methods comprising the necessary components (i.e., enzymes, labeling reagents, necessary buffers detergents, and/or solvents, and instructions for use).
It is specifically noted, with regard to the instructions for performing the method of claim 1 (i.e., printed matter), that to be given patentable weight the printed matter and associated product must be in a functional relationship. A functional relationship can be found where the printed matter performs some function with respect to the product to which it is associated. See Lowry, 32 F.3d at 1584, 32 USPQ2d at 1035 (citing Gulack, 703 F.2d at 1386, 217 USPQ at 404). Where a product merely serves as a support for printed matter, no functional relationship exists. These situations may arise where the claim as a whole is directed towards conveying a message or meaning to a human reader independent of the supporting product. Where the printed matter and product do not depend upon each other, no functional relationship exists. For example, in a kit containing a set of chemicals and a printed set of instructions for using the chemicals, the instructions are not related to that particular set of chemicals. In re Ngai, 367 F.3d at 1339, 70 USPQ2d at 1864. See MPEP 2111.05(I). Thus, in the instant case the recited instructions are not given patentable weight, as the printed matter and the product are not dependent upon each other and therefore no functional relationship exists.
Thus, it would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to make a kit for site-specific modification of antibodies, wherein the kit can be used for the method rendered obvious by Walker, Agarwal, and Geel. One would have been motivated to make such a kit because kits for the site-specific modification/labeling of antibodies are known in the art, as provided by Agnew, and said kits comprise the necessary components (i.e., enzymes, labeling reagents, necessary buffers detergents, and/or solvents, and instructions for use), and one of ordinary skill would recognize that an endoglycosidase and transglutaminase are enzymes required for (i) N-glycan trimming and (ii) glutamine conjugation in the method rendered obvious by Walker, Agarwal, and Geel.
Conclusion
Claims 1-6, 9-13, 16-18, 21-25, 30-32, 34-37, 43, and 53 are pending. Claims 11-12, 17-18, 43, and 53 are withdrawn. Claims 1-6, 9-10, 13, 16, 21-25, 30-32, and 34-37 are rejected. No claims are allowed.
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/ALYSSA RAE STONEBRAKER/Examiner, Art Unit 1642
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