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 .
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on November 25, 2025 has been entered.
Claims 1, 4, 14-16, 21, 24, 26, 28-31, 36-38 and 41-43 are pending.
Claims 41-43 are withdrawn from further consideration by the examiner, 37 C.F.R. 1.142(b) as being drawn to non-elected inventions.
Claims 1, 4, 14-16, 21, 24, 26, 28-31 and 36-38, drawn to an antibody that read on an azido and an acetyl as the particular reactive pair, a gamma heavy chain as the species of heavy chain, a kappa light chain as the species of light chain and an IgG as the species of antibody class, are being acted upon in this Office Action.
Priority
Applicant’ claim priority to provisional application 61/844, 771, filed July 10, 2013 and 61/890,121 filed October 11, 2013, is acknowledged.
Rejection and Objection Withdrawn
The rejection of claim 39 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph is withdrawn in view of the claim amendment.
The rejection of claims 13, 15, 16 and 28 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph is withdrawn in view of the claim amendment.
The provisional rejection of claims 1, 4, 13-16, 21, 24-31, 36-40 and 46 on the ground of nonstatutory obviousness-type double patenting as being unpatentable over claims 1, 3, 5-9, 14-15, 20-28
of copending Application No. 18/345,892 (now US Patent No. 12,144,869) is withdrawn in view of the amendment to claim 1.
The objection to claim 40 is withdrawn in view of the claim amendment filed on November 25, 2025.
Claim objection
Claim 1 is objected to because of the following informality: “…selected from the group consisting of (a)… Kabat numbering scheme; or…(b) …” should have been “…selected from the group consisting of (a)… Kabat numbering scheme; and…(b) …”.
Claim rejections under - 35 U.S.C. 112
The following is a quotation of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), first paragraph:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1, 4, 14-16, 21, 24, 26, 28-31 and 36-38 are 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 pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
The Written Description Guidelines for examination of patent applications indicates, “the written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical characteristics and/or other chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show applicant was in possession of the claimed genus.” (see MPEP 2163).
Claim 1 is drawn to any antibody conjugate comprising: any IgG antibody of the IgG class linked to any one or more therapeutic moieties via any one or more non-natural amino acids, wherein at least one of the one or more therapeutic moieties is any immunomodulatory gent, and wherein the antibody comprises a first site-specific non-natural amino acid residue and a second site-specific non-natural amino acid residue, wherein the first site-specific non-natural amino acid residue comprises an azide moiety and the second site-specific non-natural amino acid residue comprises an acetyl moiety, and wherein at least one non-natural amino acid is at a residue position selected from the group consisting of:
(a) is at heavy chain residues one of heavy chain site 19, 25, 40, 70, 119, 121, 136, 180, 190, 222, 241, or 404 according to the EU index of Kabat or Kabat numbering scheme; or
(b) is at light chain residues 7, 22, or 152 according to the Kabat numbering scheme.
Claim 4 encompasses antibody conjugate of claim 1, wherein the first site-specific non- natural amino acid residue is in a light chain polypeptide of the antibody and the second site- specific non-natural amino acid residue is in a heavy chain polypeptide of the antibody.
Claim 14 encompasses the antibody conjugate of claim 1, wherein the antibody comprises comprising a light chain selected from λ and κ.
Claim 15 encompasses the antibody conjugate of claim 1, wherein the IgG is selected from the group consisting of IgG1,IgG2, IgG3, and IgG4.
Claim 16 encompasses the antibody conjugate of claim 1, wherein the antibody is selected from the group consisting of Fv, Fab, (Fab')2, single chain Fv (scFv), single chain Fv-Fc (scFv-Fc), and full-length antibody.
Claim 21 encompasses the antibody conjugate of claim 1, wherein each non-natural amino acid residue is according to the formula:
PNG
media_image1.png
220
464
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Greyscale
Claim 24 encompasses the antibody conjugate of claim 21, wherein each L is a divalent linker selected from the group consisting of a bond, alkylene, substituted alkylene, heteroalkylene, substituted heteroalkylene, arylene, substituted arylene, heteroarlyene, and substituted heteroarylene.
Claim 26 encompasses the antibody conjugate of claim 1, wherein the antibody is linked to one or more drug or polymers.
Claim 28 encompasses the antibody conjugate of claim 1, wherein the antibody is linked to one or more single-chain binding domain (scFv).
Claim 29 encompasses the antibody conjugate of claim 1, wherein at least one of the one or more therapeutic moieties is linked to the antibody via the non-natural amino acid residue comprising an azide moiety.
Claim 30 encompasses the antibody conjugate of claim 1, wherein at least one of the one or more therapeutic moieties or labeling moieties is linked to the antibody via the non-natural amino acid residue comprising an acetyl moiety.
Claim 31 encompasses the antibody conjugate of claim 1, wherein at least one of the one of the one or more therapeutic moieties or labeling moieties is linked to the antibody via the non-natural amino acid residue comprising an azide moiety and at least one of the one of the one or more therapeutic moieties or labeling moieties is linked to the antibody via the non-natural amino acid residue comprising an acetyl moiety.
Claim 36 encompasses the antibody conjugate of claim 1, wherein the antibody is linked to the one or more therapeutic moieties via one or more linkers.
Claim 37 encompasses the antibody conjugate of claim 1, wherein the antibody conjugate has a melting temperature within about five degrees Celsius of a parent antibody.
Claim 38 encompasses the antibody conjugate of claim 1, wherein the antibody conjugate has a melting temperature that is at least about three degrees Celsius greater than that of a parent antibody.
Regarding “immunomodulatory agent”, the specification discloses:
[0301] Useful drug payloads include any cytotoxic, cytostatic or immunomodulatory drug. Useful classes of cytotoxic or immunomodulatory agents include, for example, antitubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cis-platin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, calmodulin inhibitors, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, maytansinoids, nitrosoureas, platinols, pore-forming compounds, purine antimetabolites, puromycins, radiation sensitizers, rapamycins, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like.
[0302] Individual cytotoxic or immunomodulatory agents include, for example, an androgen, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan, buthionine sulfoximine, calicheamicin, calicheamicin derivatives, camptothecin, carboplatin, carmustine (BSNU), CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin (formerly actinomycin), daunorubicin, decarbazine, DM1, DM4, docetaxel, doxorubicin, etoposide, an estrogen, 5-fluordeoxyuridine, 5-fluorouracil, gemcitabine, gramicidin D, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU), maytansine, mechlorethamine, melphalan, 6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone, nitroimidazole, paclitaxel, palytoxin, plicamycin, procarbizine, rhizoxin, streptozotocin, tenoposide, 6-thioguanine, thioTEPA, topotecan, vinblastine, vincristine, vinorelbine, VP-16 and VM-26.
[0310] In some embodiments, the payload is an immunomodulatory agent. The immunomodulatory agent can be, for example, ganciclovir, etanercept, tacrolimus, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil or methotrexate. Alternatively, the immunomodulatory agent can be, for example, a glucocorticoid (e.g., cortisol or aldosterone) or a glucocorticoid analogue (e.g., prednisone or dexamethasone).
[0311] In some embodiments, the immunomodulatory agent is an anti-inflammatory agent, such as arylcarboxylic derivatives, pyrazole-containing derivatives, oxicam derivatives and nicotinic acid derivatives. Classes of anti-inflammatory agents include, for example, cyclooxygenase inhibitors, 5-lipoxygenase inhibitors, and leukotriene receptor antagonists.
However, this list is exemplary and not limiting. The phrases “for example”, and “the like” are open ended. Further, “immunomodulatory” encompasses inhibitory as well as stimulatory, which are mutually exclusive. The disclosure fails to describe the common attributes or characteristics that identify the broad array of immunomodulatory agent that are contained within the genus.
The state of the art is such that immunomodulatory agent is complex.
For example, Wu et al (Acta Pharm Sin 12(12): 4287-4308, 2022; PTO 892) teaches that despite recent progress made in small molecule-based immunomodulators for cancer therapy, numerous obstacles remain to unlocking the full potential of immunotherapy. One of the challenges is to design compounds with high affinity to those targets without a stable or pocket-like active site. PD-L1 interacts with PD-1 through a hydrophobic, flat and extended (∼1.700 Å) interface, meaning that each of them does not have a deep binding pocket. One of the effective strategies to solve this problem is to design allosteric modulators. In addition, small molecule immunomodulators may exhibit less specificity than therapeutic antibodies, thus causing undesired adverse effects (e.g., ibrutinib and idelalisib). Nonetheless, a number of potent small molecule immunomodulators have been advanced to clinical stages, providing confidence to this field. Moreover, with the availability of crystal structures for many target proteins and the assistance of computer aided drug design technology, the prospects for discovering potent small molecule immunomodulators for cancer therapy seem to be more promising, e.g., compounds with improved specificity, and pharmacodynamic/pharmacokinetic/toxicological profiles. Another challenge for small molecule immuno-oncology agents is that many immunotherapeutic targets and pathways are interlinked, which means that modulating one target may affect additional immune signaling pathways. Taking IDO1 as an example, inhibition of IDO1 can affect Trp metabolism in the tumor microenvironment to enhance anti-tumor immunity. However, Trp may be compensated by other enzymes such as IDO2, TDO, which is one of the reasons for the poor efficacy of ECHO-301 (IDO1 inhibitor) in phase III clinical trials, see p. 4603, in particular.
The specification exemplifies:
Example 1: Multiple Non-Natural Amino Acid Incorporation into Heavy Chains
[0480] This example demonstrates that release-factor 1 (RF1)-attenuated cell-free protein synthesis (CFPS) reactions facilitate incorporation of up to 5 non-natural amino acids (nnAAs) per immunoglobulin G (IgG) heavy chain (HC) polypeptide.
[0481] Herceptin IgG heavy chain (HC) and light chain (LC) were expressed in CFPS reactions for 12 hr at 30° C. IgG HC DNA templates contained no TAG codon (WT), a TAG codon at Ser136, Asn297, or multiple TAG codons (1TAG=Ala118, 2TAG=Ala118/Val5, 3TAG=Ala118/Val5/Ser136, 4TAG=Ala118/Val5/Ser136/Asn297, STAG=Ala118/Val5/Ser136/Asn297/Asn384). Expression reactions were performed in the presence of .sup.14C-Leu for metabolic labeling of synthesized proteins. Samples of the expression reaction were analyzed by SDS-PAGE and autoradiography. The samples analyzed were non-boiled, non-reduced samples of the soluble fraction of the CFPS reaction (FIG. 1A.) and boiled, reduced samples of the total CFPS reaction (FIG. 1B).
[0482] In the presence of RF1, nnAA incorporation is up to 2 nnAAs per IgG HC polypeptide. FIG. 1B illustrates high yield of total nnAA-containing protein obtained in the reactions. These data exemplify a system that enables facile incorporation of multiple nnAAs per polypeptide.
Example 2: Expression of Herceptin IgG-HC with Two Non-natural Amino Acids
[0483] This example provides the design and expression of 45 HERCEPTIN heavy chains, each with two different non-natural amino acids at site-specific locations.
[0484] PCR template generation. Ten single TAG mutation heavy chain genes in pYD plasmid, R19, S25, A40, Y52, T117, S119, Y180, D221, K222 and F404, were used as templates for double TAG mutant template generation. Forty-five HC double TAG mutant expression templates as listed in Table 1 were generated by overlapping PCR, which is described in FIG. 2.
[0496] HC Double TAG Suppression with pN.sub.3F and pCH.sub.2N.sub.3F
[0497] 45 double TAG variants were expressed with PCR templates at 60 μl scale in 24 well plates in the presence of pN.sub.3F or pCH.sub.2N.sub.3F as described herein. The amber stop codons in the HC DNA sequences were suppressed by non-native amino acid charged tRNAPAZ/CUA. SBHS016 was used for TAG suppression since RF1 was degraded during cell-extract preparation, which resulted in higher TAG suppression efficiency by pN.sub.3F or pCH.sub.2N.sub.3F. SBEZ023 contains over-expressed tRNAPAZ/CUA, which was charged with pN.sub.3F or pCH.sub.2N.sub.3F by pN.sub.3FRS or pCNFRS.
[0498] The 45 double TAG variants were expressed with WT IgG as a control. The autoradiograms of IgG variant expression were shown in FIG. 3A and FIG. 4A. The full-length IgG protein yields were calculated and shown in FIG. 3B and FIG. 4B. The results demonstrated that some double TAG mutants could yield as much IgG as the WT sequence, especially Y52F404.
[0499] Of these, 8 double TAG mutants, R19Y52, R19F404, S25F404, A40F404, Y52F404, S119F404, Y180F404 and K222F404 were selected and subcloned into pYD317 plasmid for further examples below.
Example 3: Expression, Purification and Conjugation of HC:HC Combo Variants
[0500] This example provides expression, purification and drug conjugation of several HERCEPTIN IgG heavy chains with two site-specific non-natural amino acids each.
[0501] The cell free reaction mix in which the HC:HC combo variants were synthesized comprised an 80%:20% blend of cell free extracts made from an OmpT sensitive RF-1 attenuated E. coli strain, and an OmpT sensitive RF-1 attenuated E. coli strain which was engineered to produce an orthogonal CUA-encoding tRNA for insertion of a non-natural amino acid at an Amber Stop Codon.
[0503] The purified 4 nnAA HC:HC combo variants were conjugated as follows. DBCO-MMAF AB3627 or AB4285 (ACME Bioscience; Palo Alto, Calif.) was dissolved in DMSO to a final concentration of 5 mM. The compound was diluted with PBS to a concentration of 1 mM and then added to purified trastuzumab variants in IMAC elution buffer to achieve a final drug concentration of 100 μM. Mixture was incubated at RT (20° C.) for 17 hours. Reaction was stopped by adding Sodium Azide to final concentration of 100 mM and buffer exchanged using Zeba plates (Thermo Scientific; Waltham, Mass.) equilibrated in 1×PBS. Filtrate was then passed through a MUSTANG® Q plate (Pall Corp.; Port Washington, N.Y.) to remove endotoxin.
Example 4: Thermal Stability of Exemplary Antibody-Drug Conjugates
[0504] This example provides the thermal stability (Tm) of aglycosylated trastuzumab and trastuzumab conjugates.
[0508] Trastuzumab variants that exhibit a Tm1 and/or Tm2 within about 5° C. of unsubstituted trastuzumab are preferred.
Example 5: SKBR3 Cell Killing of Exemplary Antibody-Drug Conjugates
[0509] This example demonstrates that the exemplary antibody-drug conjugates from the examples above are effective in a cell-killing assay. The effects of the conjugated four site-specific non-natural heavy chain-heavy chain (HC-HC) antibodies, above, on cell killing were measured by a cell proliferation assay.
[0512] As shown in Table 3, the antibodies conjugated to two drugs were similarly or more effective than corresponding antibodies conjugated to single drugs at corresponding positions.
Example 6: Incorporation of Two Different Non-Natural Amino Acids in a Single IgG
[0513] This example provides single IgG molecules that incorporate two different non-natural amino acids at site-specific positions.
[0514] To make heteromeric antibodies in vivo or in vitro, heavy chain and light chain can be expressed in the same fermentation/reaction mixture. Here we describe a method to prefabricate light chain and add it back to the expression of heavy chain in the cell-free protein synthesis system to form heteromeric antibodies.
[0515] To incorporate two different nnAAs to IgG, both heavy chain and light chain plasmid were constructed with amber codons at desired sites, for example, trastuzumab LC T22, LC S63 and HC F404. The first non-natural amino acid, nnAA1, pAzidoF was incorporated during the cell-free protein synthesis of light chain as described below.
[0516] The cell free reaction mix in which trastuzumab variants were synthesized comprised a 85%:15% blend of cell free extracts made from an OmpT sensitive RF-1 attenuated E. coli strain which was engineered to over express DsbC (DsbC extract), and an OmpT sensitive RF-1 attenuated E. coli strain which was engineered to produce an orthogonal CUA-encoding tRNA (tRNA extract) for insertion of a non-natural amino acid at an Amber stop codon. The variants were expressed in a cell-free protein synthesis reaction as follows. Cell-free extracts were treated with 50 μM iodoacetamide for 30 min at RT (20° C.) and added to a premix containing all other components except for DNA encoding the variants of interest. The final concentration in the protein synthesis reaction was 30% cell extract, 2 mM para-azido phenylalanine (pAzF) (RSP Amino Acids), 0.37 mg/mL M jannaschii pAzF-specific amino-acyl tRNA synthetase (FRS), 2 mM GSSG, 8 mM magnesium glutamate, 10 mM ammonium glutamate, 130 mM potassium glutamate, 35 mM sodium pyruvate, 1.2 mM AMP, 0.86 mM each of GMP, UMP, and CMP, 2 mM amino acids (except 0.5 mM for Tyrosine and Phenylalanine), 4 mM sodium oxalate, 1 mM putrescine, 1.5 mM spermidine, 15 mM potassium phosphate, 100 nM T7 RNAP, and 10 μg/mL trastuzumab light chain DNA. After addition of DNA template, cell free reactions were incubated at 30° C. for 12 h.
[0517] The light chains were purified with protein L columns. The second non-natural amino acid, nnAA2, pAzMeF was then incorporated to heavy chain in the presence of prefabricated light chain containing pAzF using the same condition described above except that the pair of synthetase and nnAA switched to M jannaschii pAzMeF-specific amino-acyl tRNA synthetase and pAzMeF. 10 μg/mL trastuzumab heavy chain DNA F404 and 400 μg/mL prefabricated LC were added.
[0518] This process results in assembled IgG having pAzF on light chain and pAzMeF on heavy chain as shown in FIG. 6. Alternatively, we can also incorporate pAzMeF to light chain and incorporate pAzF to heavy chain.
[0519] Using this approach, any two different nnAAs can be incorporated into a single IgG. Different chemistries can be used to site specifically conjugate different drugs to IgG. Two orthogonal conjugations can be done in the same reaction.
Example 7: A Single IgG Conjugated to Two Different Warheads
[0520] This example provides a single IgG conjugated to two different warhead moieties. A light chain with a first nnAA is expressed, purified and then conjugated to a first drug. A second nnAA (same or different) is then incorporated to into a heavy chain in the presence of conjugated LC. The generated IgG has a drug on the light chain and a site on heavy chain available for a second conjugation. The process is outlined in FIG. 7
[0521] In this example, a light chain is conjugated to one drug, monomethyl auristatin F (MMAF) and a heavy chain is conjugated to another drug, SN38.
[0522] pAzF was incorporated to LC at T22 or S63 according to the previous example. LC was then purified and incubated with MMAF at 1 to 5 molar ratio at room temperature for 16 hours. pAzMeF was incorporated to HC at F404 position in the presence of 400 μg/mL conjugated LC. The reaction condition was same as described in previous example. The IgG was then purified by protein A column.
[0523] The purified IgG was conjugated to second drug, SN38, after purification. 5 μM IgG and 25 μM SN38 were incubated at room temperature for 16 hours to generate ADC. This ADC was confirmed two MMAF on LC and two SN38 on HC by mass spectrometry analysis as provided in FIG. 8A (T22 pAzF) and FIG. 8B (S63 pAzF).
Example 8: Incorporating Non-Natural Amino Acids in Heavy Chains and Light Chains
[0524] This example demonstrates heavy chains incorporating at least one non-natural amino acid, light chains incorporating at least one non-natural amino acid, and antibodies with the heavy chains and light chains. The resulting antibodies have at least four non-natural amino acids at site specific locations. Table 4 provides sites for non-natural amino acids in heavy chains and light chains.
[0525] To demonstrate the feasibility of making antibodies and antibody-drug conjugates with at least four site-specific non-natural amino acids, DNA encoding trastuzumab heavy chain and light chain with amber were cloned into expression vector pYD317 separately. TAG codon was inserted by overlapping PCR mutagenesis at the nucleotides corresponding to the amino acid serine at positions S136, Y180, S190, and F404 on heavy chain, and S7 and T22 on light chain. To incorporate four pAzMeF in one IgG, the concentration of pCNFRS was increased by about two-fold.
[0526] The cell free reaction mix in which the HC/LC combo variants were synthesized was a 80%:20% blend of cell free extracts made from an OmpT sensitive RF-1 attenuated E. coli strain, and an OmpT sensitive RF-1 attenuated E. coli strain that was engineered to produce an orthogonal CUA-encoding tRNA for insertion of a non-natural amino acid at an Amber Stop Codon. The variants were expressed in a cell-free protein synthesis reaction as follows (based on the method described in Zawada et al., 2011, Biotechnol. Bioeng. 108(7)1570-1578) with the modifications described below.
[0527] Cell-free extracts were treated with 50 μM iodoacetamide for 30 min at RT (20° C.) and added to a premix containing all other components except for DNA encoding the variants of interest. The final concentration in the protein synthesis reaction was 30% cell extract, 1 mM para-azido methyl phenylalanine (pAzMeF) (RSP Amino Acids), 0.37 mg/mL M jannaschii pAzMeF-specific amino-acyl tRNA synthetase (FRS), 2 mM GSSG, 0.29 mg/mL PDI (Mclab), 30 μg/mL E. coli DsbC, 8 mM magnesium glutamate, 10 mM ammonium glutamate, 130 mM potassium glutamate, 35 mM sodium pyruvate, 1.2 mM AMP, 0.86 mM each of GMP, UMP, and CMP, 2 mM amino acids (except 0.5 mM for tyrosine and phenylalanine), 4 mM sodium oxalate, 1 mM putrescine, 1.5 mM spermidine, 15 mM potassium phosphate, 100 nM T7 RNAP, 2.5 μg/mL trastuzumab variant light chain DNA, 7.5 μg/mL trastuzumab-(His).sub.6 variant heavy chain DNA. After addition of DNA template, cell free reactions were incubated at 30° C. for 12h on a shaker at 650 rpm in Flower plates (m2p-labs #MTP-48-B). All variants were scaled up to 9 ml in flower plates (1.5 mL X 6 replicates) and purified using Protein Maker.
Example 9: Purification of 4nnAA Aglycosylated Trastuzumab Variants
[0528] Antibodies with four site-specific non-natural amino acids in heavy chains and light chains (4nnAA HC/LC combos) were purified with the following three step procedure:
[0529] IgG Capture: To capture the IgG, 8 ml of crude cell-free for each variant was first diluted 1:0.5 with equilibration buffer (50 mM sodium phosphate, pH 7) and spun at 11,000×g for 30 minutes. The supernatant was then passed through a 0.45 micron syringe filter prior to being loaded with a 2 minute residence time onto a pre-equilibrated 1 mL MabSelect Sure HiTrap (GE Lifesciences). The column was then washed with 7.5 CV (column volume) of wash buffer (100 mM sodium phosphate and 800 mM Arginine, pH 7) followed by 7.5CV of equilibration buffer. Each variant was then eluted with 4CV elution buffer (100 mM sodium citrate and 300 mM Arginine, pH 3). The elution pool was adjusted to pH 4.6 by addition of 20% (v/v) of 1M Tris, pH 9.
[0530] Aggregate Removal: To remove product related impurities, the 4.8 ml of MabSelect purified IgG was passed through a 1 mL Capto Adhere HiTrap (GE Lifesciences) that had previously been equilibrated with 83.3 mM sodium citrate, 167 mM Tris, 250m M Arginine, pH 4.6. The column was then washed with an additional 7.5 CV of the same buffer. The flowthrough and wash were collected and neutralized to pH 7 by addition of 10% (v/v) of 1 M Tris, pH 9.
[0531] Buffer Exchange: The collected Capto Adhere purified pool was concentrated and buffer exchanged into PBS using the Amicon Ultra-15 (Millipore) centrifugal filter unit by repeated dilution with PBS and subsequent concentration.
Example 10: Conjugation of Aglycosylated Trastuzumab Variants to MMAF
[0532] This example provides conjugation of the antibodies with at least four site-specific non-natural amino acids to the warhead moiety MMAF.
[0533] The purified 4 nnAA HC/LC combo variants were conjugated as follows. DBCO-MMAF AB3627 or AB4285 (ACME Bioscience; Palo Alto, Calif.), shown above, were dissolved in DMSO to a final concentration of 5 mM. The compounds were diluted with PBS to a concentration of 1 mM and then added to purified trastuzumab variants in an immobilized metal ion affinity chromatography (IMAC) elution buffer to achieve a final drug concentration of 100 μM. Mixture was incubated at RT (20° C.) for 17 hours. Reaction was stopped by adding sodium azide to a final concentration of 100 mM and buffer exchanged using Zeba plates (Thermo Scientific; Waltham, Mass.) equilibrated in 1×PBS. Filtrate was then passed through a MUSTANG® Q plate (Pall Corp.; Port Washington, N.Y.) to remove endotoxin.
Example 11: Thermal Stability of Exemplary Antibody-Drug Conjugates
[0534] This example provides the thermal stability (Tm) of aglycosylated trastuzumab and trastuzumab variants.
However, the specification does not disclose a correlation between the structure of the various immunomodulatory agent conjugated to which IgG class of antibody at a residue in the heavy chain 19, 25, 40, 70, 119, 121, 136, 180, 190, 222, 241 or 404 or a light chain residue 7, 22 or 152, numbering according to the Kabat numbering scheme via a first and second non-natural amino acid each comprising an azide moiety or an acetyl moiety for treating any and all subject.
Other than a single aglycosylated IgG trastuzumab with four site-specific non-natural amino acids in heavy chains (HC) and light chains at specific heavy and light chain residues conjugated to just DBCO-MMAF and SN38 by incorporating two different non-natural amino acids para-azido phenylalanine (pAzF) and para-azido methyl phenylalanine (pAzMeF), the specification does not describe the binding specificity of all antibody conjugate comprising any IgG class, e.g., IgG1, IgG2, IgG3 or IgG4 linked to one or more undisclosed immunomodulatory (stimulatory or inhibitory) agent via any one or more non-natural amino acids at heavy chain and light chain residues conjugated to the undisclosed immunomodulatory agent (claims 1 and dependent claims thereof) for treating any and all subject, any subject afflicted with any cancer or breast cancer.
The specification does not describe sufficient relevant identifying characteristics (such as: i. Complete structure, i.e., amino acid sequence of the heavy and light chain variable domains, ii. Partial structure, i.e., the six CDRs, iii. Physical and/or chemical properties, iv. Functional characteristics, binding specificity of all IgG class antibodies conjugated to any immunomodulatory agent (claim 1) or drug or polymer (claim 26). The specification does not describe a representative number of species falling within the scope of the genus or structural common to the members of the genus so the one of skill in the art can visualize or recognize member of the genus of the actual claimed antibody conjugate themselves for treating all subject afflicted with any cancer or breast cancer. Based upon the limited disclosure, it does not appear that Applicants were in possession of the genus of antibody conjugate as set forth in claims 1, 4, 14-16, 21, 24, 26, 28-31, 36-38 at the time of filing.
It is well known in the art that antibodies bind protein from one species and do notbind to the same protein from another species.
For example, Yu et al (Investigative Ophthalmology & Visual Science 49(2): 522-527, February 2008; PTO 892) teach bevacizumab, which is a humanized anti-human VEGF-A mAb A.4.6.1 binds specifically to human VEGF-A, the same antibody does not bind to mouse VEGF-A (see page 522, right col., page 523, Figure 1, in particular).
Poosarla et al (Biotechn. Bioeng., 114(6): 1331-1342, 2017; PTO 892) teach substantial diversity in designed mAbs (sharing less than 75% sequence similarity to all existing natural antibody sequences) that bind to the same 12-mer peptide, binding to different epitopes on the same peptide. Said reference further teaches “most B-cell epitopes... in nature consist of residues from different regions of the sequence and are discontinuous...de novo antibody designs against discontinuous epitopes present additional challenges...". (See entire reference.)
Nejadmoghaddam (Avicenna Journal of Medical Biotechnology 2(1): 3-23, 2019; PTO 892) discusses major obstacles of antibody-drug conjugates include off-target toxicity, tumor marker selection, antibody specificity, adequately affinity and receptor-mediated internalization are major aspects of choice, cytotoxic payload (e.g., up to 7 drugs per antibody), cytotoxic payload linkage strategy, aqueous solubility, non-immunogenic and stability in storage and bloodstream, see entire document, abstract, p. 15, in particular.
Tsuchikama (of record, Protein Cell 9(1): 33-46, 2018; PTO 1449) teaches that the choice of target antigens, payloads (drug) are important; antibody-payload conjugation methods and linker chemistry are also crucial elements for producing successful ADCs. In particular, instability of the linker and heterogeneity of the product (i.e., broad distribution of DARs) often negatively impacts ADC efficacy and therapeutic window, which often leads to difficulty or limitation in the optimization of clinical application and eventual failure in clinical trials, see p. 42-43. Thus, the art teaches there is a high level of unpredictability in the art of antibody-drug conjugate (ADCs).
Vas-Cath Inc. v. Mahurkar, 19 USPQ2d 1111, makes clear that “applicant must convey with reasonable clarity to those skilled in the art that, as of the filing date sought, he or she was in possession of the invention. The invention is, for purposes of the ‘written description’ inquiry, whatever is now claimed.” (see page 1117). The specification does not “clearly allow persons of ordinary skill in the art to recognize that [he or she] invented what is claimed.” (see Vas-Cath at page 1116).
Adequate written description requires more than a mere statement that it is part of the invention and reference to a potential method for isolating it. See Fiers v. Revel, 25 USPQ2d 1601, 1606 (CAFC 1993) and Amgen Inc. v. Chugai Pharmaceutical Co. Ltd., 18 USPQ2d 1016.
One cannot describe what one has not conceived. See Fiddles v. Baird, 30 USPQ2d 1481, 1483. In Fiddles v. Baird, claims directed to mammalian FGF’s were found unpatentable due to lack of written description for the broad class. The specification provided only the bovine sequence. Thus, the specification fails to describe these DNA sequences.
Therefore, only (1) an antibody conjugate comprising: an IgG antibody that binds to HER2 wherein the antibody is linked to one or more therapeutic moieties via any one or more non-natural amino acids, wherein at least one of the one or more therapeutic moieties is a cytotoxic agent, and wherein the antibody comprises a first site-specific non-natural amino acid residue and a second site-specific non-natural amino acid residue, wherein the first site-specific non-natural amino acid residue comprises an azide moiety and the second site-specific non-natural amino acid residue comprises an acetyl moiety, and wherein at least one non-natural amino acid is at a residue position selected from the group consisting of: (a) heavy chain residues one of heavy chain site 19, 25, 40, 70, 119, 121, 136, 180, 190, 222, 241, or 404 according to the EU index of Kabat or Kabat numbering scheme; and (b) light chain residues 7, 22, or 152 according to the Kabat numbering scheme, (2) the antibody conjugate above wherein the light chain comprises at least one site-specific non-natural amino acid p-azido-phenylalanine (pAzF) or para-azido methyl phenylalanine (pAzMeF) at position 7, 22 or 152, (3) The antibody conjugate above wherein the therapeutic moieties are MMAF and SN38, but not the full breadth of the claims meets the written description provision of 35 U.S.C. § 112, first paragraph.
Claims 1, 4, 14-16, 21, 24, 26, 28-31 and 36-38 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for (1) an antibody conjugate comprising: an IgG antibody that binds to HER2 wherein the antibody is linked to one or more therapeutic moieties via any one or more non-natural amino acids, wherein at least one of the one or more therapeutic moieties is a cytotoxic agent, and wherein the antibody comprises a first site-specific non-natural amino acid residue and a second site-specific non-natural amino acid residue, wherein the first site-specific non-natural amino acid residue comprises an azide moiety and the second site-specific non-natural amino acid residue comprises an acetyl moiety, and wherein at least one non-natural amino acid is at a residue position selected from the group consisting of: (a) heavy chain residues one of heavy chain site 19, 25, 40, 70, 119, 121, 136, 180, 190, 222, 241, or 404 according to the EU index of Kabat or Kabat numbering scheme; and (b) light chain residues 7, 22, or 152 according to the Kabat numbering scheme, (2) the antibody conjugate above wherein the light chain comprises at least one site-specific non-natural amino acid p-azido-phenylalanine (pAzF) or para-azido methyl phenylalanine (pAzMeF) at position 7, 22 or 152, (3) The antibody conjugate above wherein the therapeutic moieties are MMAF and SN38 for treating a subject having breast cancer, does not reasonably provide enablement for any antibody conjugate as set forth in claims 1, 4, 14-16, 21, 24, 26, 28-31 and 36-38 for treating any subject, any subject afflict with any cancer or breast cancer. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
Factors to be considered in determining whether undue experimentation is required to practice the claimed invention are summarized In re Wands (858 F2d 731, 737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988)). The factors most relevant to this rejection include the nature of the invention, the state of the prior art, the relative skill of those in the art, the amount of direction or guidance disclosed in the specification, the presence or absence of working examples, the predictability or unpredictability of the art, the breadth of the claims, and the quantity of experimentation which would be required in order to practice the invention as claimed.
Enablement is not commensurate in scope with claims as how to make use any antibody conjugate above for treating any subject, any subject afflict with any cancer or breast cancer without undue experimentation.
Claim 1 is drawn to any antibody conjugate comprising: any IgG antibody of the IgG class linked to any one or more therapeutic moieties via any one or more non-natural amino acids, wherein at least one of the one or more therapeutic moieties is any immunomodulatory gent, and wherein the antibody comprises a first site-specific non-natural amino acid residue and a second site-specific non-natural amino acid residue, wherein the first site-specific non-natural amino acid residue comprises an azide moiety and the second site-specific non-natural amino acid residue comprises an acetyl moiety, and wherein at least one non-natural amino acid is at a residue position selected from the group consisting of:
(a) is at heavy chain residues one of heavy chain site 19, 25, 40, 70, 119, 121, 136, 180, 190, 222, 241, or 404 according to the EU index of Kabat or Kabat numbering scheme; or
(b) is at light chain residues 7, 22, or 152 according to the Kabat numbering scheme.
Claim 4 encompasses antibody conjugate of claim 1, wherein the first site-specific non- natural amino acid residue is in a light chain polypeptide of the antibody and the second site- specific non-natural amino acid residue is in a heavy chain polypeptide of the antibody.
Claim 14 encompasses the antibody conjugate of claim 1, wherein the antibody comprises comprising a light chain selected from λ and κ.
Claim 15 encompasses the antibody conjugate of claim 1, wherein the IgG is selected from the group consisting of IgG1,IgG2, IgG3, and IgG4.
Claim 16 encompasses the antibody conjugate of claim 1, wherein the antibody is selected from the group consisting of Fv, Fab, (Fab')2, single chain Fv (scFv), single chain Fv-Fc (scFv-Fc), and full-length antibody.
Claim 21 encompasses the antibody conjugate of claim 1, wherein each non-natural amino acid residue is according to the formula:
PNG
media_image1.png
220
464
media_image1.png
Greyscale
Claim 24 encompasses the antibody conjugate of claim 21, wherein each L is a divalent linker selected from the group consisting of a bond, alkylene, substituted alkylene, heteroalkylene, substituted heteroalkylene, arylene, substituted arylene, heteroarlyene, and substituted heteroarylene.
Claim 26 encompasses the antibody conjugate of claim 1, wherein the antibody is linked to one or more drug or polymers.
Claim 28 encompasses the antibody conjugate of claim 1, wherein the antibody is linked to one or more single-chain binding domain (scFv).
Claim 29 encompasses the antibody conjugate of claim 1, wherein at least one of the one or more therapeutic moieties is linked to the antibody via the non-natural amino acid residue comprising an azide moiety.
Claim 30 encompasses the antibody conjugate of claim 1, wherein at least one of the one or more therapeutic moieties or labeling moieties is linked to the antibody via the non-natural amino acid residue comprising an acetyl moiety.
Claim 31 encompasses the antibody conjugate of claim 1, wherein at least one of the one of the one or more therapeutic moieties or labeling moieties is linked to the antibody via the non-natural amino acid residue comprising an azide moiety and at least one of the one of the one or more therapeutic moieties or labeling moieties is linked to the antibody via the non-natural amino acid residue comprising an acetyl moiety.
Claim 36 encompasses the antibody conjugate of claim 1, wherein the antibody is linked to the one or more therapeutic moieties via one or more linkers.
Claim 37 encompasses the antibody conjugate of claim 1, wherein the antibody conjugate has a melting temperature within about five degrees Celsius of a parent antibody.
Claim 38 encompasses the antibody conjugate of claim 1, wherein the antibody conjugate has a melting temperature that is at least about three degrees Celsius greater than that of a parent antibody.
Regarding “immunomodulatory agent”, the specification discloses:
[0301] Useful drug payloads include any cytotoxic, cytostatic or immunomodulatory drug. Useful classes of cytotoxic or immunomodulatory agents include, for example, antitubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cis-platin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, calmodulin inhibitors, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, maytansinoids, nitrosoureas, platinols, pore-forming compounds, purine antimetabolites, puromycins, radiation sensitizers, rapamycins, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like.
[0302] Individual cytotoxic or immunomodulatory agents include, for example, an androgen, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan, buthionine sulfoximine, calicheamicin, calicheamicin derivatives, camptothecin, carboplatin, carmustine (BSNU), CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin (formerly actinomycin), daunorubicin, decarbazine, DM1, DM4, docetaxel, doxorubicin, etoposide, an estrogen, 5-fluordeoxyuridine, 5-fluorouracil, gemcitabine, gramicidin D, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU), maytansine, mechlorethamine, melphalan, 6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone, nitroimidazole, paclitaxel, palytoxin, plicamycin, procarbizine, rhizoxin, streptozotocin, tenoposide, 6-thioguanine, thioTEPA, topotecan, vinblastine, vincristine, vinorelbine, VP-16 and VM-26.
[0310] In some embodiments, the payload is an immunomodulatory agent. The immunomodulatory agent can be, for example, ganciclovir, etanercept, tacrolimus, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil or methotrexate. Alternatively, the immunomodulatory agent can be, for example, a glucocorticoid (e.g., cortisol or aldosterone) or a glucocorticoid analogue (e.g., prednisone or dexamethasone).
[0311] In some embodiments, the immunomodulatory agent is an anti-inflammatory agent, such as arylcarboxylic derivatives, pyrazole-containing derivatives, oxicam derivatives and nicotinic acid derivatives. Classes of anti-inflammatory agents include, for example, cyclooxygenase inhibitors, 5-lipoxygenase inhibitors, and leukotriene receptor antagonists.
However, this list is exemplary and not limiting. The phrases “for example”, and “the like” are open ended. Further, “immunomodulatory” encompasses inhibitory as well as stimulatory, which are mutually exclusive. It is unpredictable which undisclosed immunomodulatory agent conjugated to IgG is effective for treating cancer.
Wu et al (Acta Pharm Sin 12(12): 4287-4308, 2022; PTO 892) teaches that despite recent progress made in small molecule-based immunomodulators for cancer therapy, numerous obstacles remain to unlocking the full potential of immunotherapy. One of the challenges is to design compounds with high affinity to those targets without a stable or pocket-like active site. PD-L1 interacts with PD-1 through a hydrophobic, flat and extended (∼1.700 Å) interface, meaning that each of them does not have a deep binding pocket. One of the effective strategies to solve this problem is to design allosteric modulators. In addition, small molecule immunomodulators may exhibit less specificity than therapeutic antibodies, thus causing undesired adverse effects (e.g., ibrutinib and idelalisib). Nonetheless, a number of potent small molecule immunomodulators have been advanced to clinical stages, providing confidence to this field. Moreover, with the availability of crystal structures for many target proteins and the assistance of computer aided drug design technology, the prospects for discovering potent small molecule immunomodulators for cancer therapy seem to be more promising, e.g., compounds with improved specificity, and pharmacodynamic/pharmacokinetic/toxicological profiles. Another challenge for small molecule immuno-oncology agents is that many immunotherapeutic targets and pathways are interlinked, which means that modulating one target may affect additional immune signaling pathways. Taking IDO1 as an example, inhibition of IDO1 can affect Trp metabolism in the tumor microenvironment to enhance anti-tumor immunity. However, Trp may be compensated by other enzymes such as IDO2, TDO, which is one of the reasons for the poor efficacy of ECHO-301 (IDO1 inhibitor) in phase III clinical trials, see p. 4603, in particular.
The specification exemplifies:
Example 1: Multiple Non-Natural Amino Acid Incorporation into Heavy Chains
[0480] This example demonstrates that release-factor 1 (RF1)-attenuated cell-free protein synthesis (CFPS) reactions facilitate incorporation of up to 5 non-natural amino acids (nnAAs) per immunoglobulin G (IgG) heavy chain (HC) polypeptide.
[0481] Herceptin IgG heavy chain (HC) and light chain (LC) were expressed in CFPS reactions for 12 hr at 30° C. IgG HC DNA templates contained no TAG codon (WT), a TAG codon at Ser136, Asn297, or multiple TAG codons (1TAG=Ala118, 2TAG=Ala118/Val5, 3TAG=Ala118/Val5/Ser136, 4TAG=Ala118/Val5/Ser136/Asn297, STAG=Ala118/Val5/Ser136/Asn297/Asn384). Expression reactions were performed in the presence of .sup.14C-Leu for metabolic labeling of synthesized proteins. Samples of the expression reaction were analyzed by SDS-PAGE and autoradiography. The samples analyzed were non-boiled, non-reduced samples of the soluble fraction of the CFPS reaction (FIG. 1A.) and boiled, reduced samples of the total CFPS reaction (FIG. 1B).
[0482] In the presence of RF1, nnAA incorporation is up to 2 nnAAs per IgG HC polypeptide. FIG. 1B illustrates high yield of total nnAA-containing protein obtained in the reactions. These data exemplify a system that enables facile incorporation of multiple nnAAs per polypeptide.
Example 2: Expression of Herceptin IgG-HC with Two Non-natural Amino Acids
[0483] This example provides the design and expression of 45 HERCEPTIN heavy chains, each with two different non-natural amino acids at site-specific locations.
[0484] PCR template generation. Ten single TAG mutation heavy chain genes in pYD plasmid, R19, S25, A40, Y52, T117, S119, Y180, D221, K222 and F404, were used as templates for double TAG mutant template generation. Forty-five HC double TAG mutant expression templates as listed in Table 1 were generated by overlapping PCR, which is described in FIG. 2.
[0496] HC Double TAG Suppression with pN.sub.3F and pCH.sub.2N.sub.3F
[0497] 45 double TAG variants were expressed with PCR templates at 60 μl scale in 24 well plates in the presence of pN.sub.3F or pCH.sub.2N.sub.3F as described herein. The amber stop codons in the HC DNA sequences were suppressed by non-native amino acid charged tRNAPAZ/CUA. SBHS016 was used for TAG suppression since RF1 was degraded during cell-extract preparation, which resulted in higher TAG suppression efficiency by pN.sub.3F or pCH.sub.2N.sub.3F. SBEZ023 contains over-expressed tRNAPAZ/CUA, which was charged with pN.sub.3F or pCH.sub.2N.sub.3F by pN.sub.3FRS or pCNFRS.
[0498] The 45 double TAG variants were expressed with WT IgG as a control. The autoradiograms of IgG variant expression were shown in FIG. 3A and FIG. 4A. The full-length IgG protein yields were calculated and shown in FIG. 3B and FIG. 4B. The results demonstrated that some double TAG mutants could yield as much IgG as the WT sequence, especially Y52F404.
[0499] Of these, 8 double TAG mutants, R19Y52, R19F404, S25F404, A40F404, Y52F404, S119F404, Y180F404 and K222F404 were selected and subcloned into pYD317 plasmid for further examples below.
Example 3: Expression, Purification and Conjugation of HC:HC Combo Variants
[0500] This example provides expression, purification and drug conjugation of several HERCEPTIN IgG heavy chains with two site-specific non-natural amino acids each.
[0501] The cell free reaction mix in which the HC:HC combo variants were synthesized comprised an 80%:20% blend of cell free extracts made from an OmpT sensitive RF-1 attenuated E. coli strain, and an OmpT sensitive RF-1 attenuated E. coli strain which was engineered to produce an orthogonal CUA-encoding tRNA for insertion of a non-natural amino acid at an Amber Stop Codon.
[0503] The purified 4 nnAA HC:HC combo variants were conjugated as follows. DBCO-MMAF AB3627 or AB4285 (ACME Bioscience; Palo Alto, Calif.) was dissolved in DMSO to a final concentration of 5 mM. The compound was diluted with PBS to a concentration of 1 mM and then added to purified trastuzumab variants in IMAC elution buffer to achieve a final drug concentration of 100 μM. Mixture was incubated at RT (20° C.) for 17 hours. Reaction was stopped by adding Sodium Azide to final concentration of 100 mM and buffer exchanged using Zeba plates (Thermo Scientific; Waltham, Mass.) equilibrated in 1×PBS. Filtrate was then passed through a MUSTANG® Q plate (Pall Corp.; Port Washington, N.Y.) to remove endotoxin.
Example 4: Thermal Stability of Exemplary Antibody-Drug Conjugates
[0504] This example provides the thermal stability (Tm) of aglycosylated trastuzumab and trastuzumab conjugates.
[0508] Trastuzumab variants that exhibit a Tm1 and/or Tm2 within about 5° C. of unsubstituted trastuzumab are preferred.
Example 5: SKBR3 Cell Killing of Exemplary Antibody-Drug Conjugates
[0509] This example demonstrates that the exemplary antibody-drug conjugates from the examples above are effective in a cell-killing assay. The effects of the conjugated four site-specific non-natural heavy chain-heavy chain (HC-HC) antibodies, above, on cell killing were measured by a cell proliferation assay.
[0512] As shown in Table 3, the antibodies conjugated to two drugs were similarly or more effective than corresponding antibodies conjugated to single drugs at corresponding positions.
Example 6: Incorporation of Two Different Non-Natural Amino Acids in a Single IgG
[0513] This example provides single IgG molecules that incorporate two different non-natural amino acids at site-specific positions.
[0514] To make heteromeric antibodies in vivo or in vitro, heavy chain and light chain can be expressed in the same fermentation/reaction mixture. Here we describe a method to prefabricate light chain and add it back to the expression of heavy chain in the cell-free protein synthesis system to form heteromeric antibodies.
[0515] To incorporate two different nnAAs to IgG, both heavy chain and light chain plasmid were constructed with amber codons at desired sites, for example, trastuzumab LC T22, LC S63 and HC F404. The first non-natural amino acid, nnAA1, pAzidoF was incorporated during the cell-free protein synthesis of light chain as described below.
[0516] The cell free reaction mix in which trastuzumab variants were synthesized comprised a 85%:15% blend of cell free extracts made from an OmpT sensitive RF-1 attenuated E. coli strain which was engineered to over express DsbC (DsbC extract), and an OmpT sensitive RF-1 attenuated E. coli strain which was engineered to produce an orthogonal CUA-encoding tRNA (tRNA extract) for insertion of a non-natural amino acid at an Amber stop codon. The variants were expressed in a cell-free protein synthesis reaction as follows. Cell-free extracts were treated with 50 μM iodoacetamide for 30 min at RT (20° C.) and added to a premix containing all other components except for DNA encoding the variants of interest. The final concentration in the protein synthesis reaction was 30% cell extract, 2 mM para-azido phenylalanine (pAzF) (RSP Amino Acids), 0.37 mg/mL M jannaschii pAzF-specific amino-acyl tRNA synthetase (FRS), 2 mM GSSG, 8 mM magnesium glutamate, 10 mM ammonium glutamate, 130 mM potassium glutamate, 35 mM sodium pyruvate, 1.2 mM AMP, 0.86 mM each of GMP, UMP, and CMP, 2 mM amino acids (except 0.5 mM for Tyrosine and Phenylalanine), 4 mM sodium oxalate, 1 mM putrescine, 1.5 mM spermidine, 15 mM potassium phosphate, 100 nM T7 RNAP, and 10 μg/mL trastuzumab light chain DNA. After addition of DNA template, cell free reactions were incubated at 30° C. for 12 h.
[0517] The light chains were purified with protein L columns. The second non-natural amino acid, nnAA2, pAzMeF was then incorporated to heavy chain in the presence of prefabricated light chain containing pAzF using the same condition described above except that the pair of synthetase and nnAA switched to M jannaschii pAzMeF-specific amino-acyl tRNA synthetase and pAzMeF. 10 μg/mL trastuzumab heavy chain DNA F404 and 400 μg/mL prefabricated LC were added.
[0518] This process results in assembled IgG having pAzF on light chain and pAzMeF on heavy chain as shown in FIG. 6. Alternatively, we can also incorporate pAzMeF to light chain and incorporate pAzF to heavy chain.
[0519] Using this approach, any two different nnAAs can be incorporated into a single IgG. Different chemistries can be used to site specifically conjugate different drugs to IgG. Two orthogonal conjugations can be done in the same reaction.
Example 7: A Single IgG Conjugated to Two Different Warheads
[0520] This example provides a single IgG conjugated to two different warhead moieties. A light chain with a first nnAA is expressed, purified and then conjugated to a first drug. A second nnAA (same or different) is then incorporated to into a heavy chain in the presence of conjugated LC. The generated IgG has a drug on the light chain and a site on heavy chain available for a second conjugation. The process is outlined in FIG. 7
[0521] In this example, a light chain is conjugated to one drug, monomethyl auristatin F (MMAF) and a heavy chain is conjugated to another drug, SN38.
[0522] pAzF was incorporated to LC at T22 or S63 according to the previous example. LC was then purified and incubated with MMAF at 1 to 5 molar ratio at room temperature for 16 hours. pAzMeF was incorporated to HC at F404 position in the presence of 400 μg/mL conjugated LC. The reaction condition was same as described in previous example. The IgG was then purified by protein A column.
[0523] The purified IgG was conjugated to second drug, SN38, after purification. 5 μM IgG and 25 μM SN38 were incubated at room temperature for 16 hours to generate ADC. This ADC was confirmed two MMAF on LC and two SN38 on HC by mass spectrometry analysis as provided in FIG. 8A (T22 pAzF) and FIG. 8B (S63 pAzF).
Example 8: Incorporating Non-Natural Amino Acids in Heavy Chains and Light Chains
[0524] This example demonstrates heavy chains incorporating at least one non-natural amino acid, light chains incorporating at least one non-natural amino acid, and antibodies with the heavy chains and light chains. The resulting antibodies have at least four non-natural amino acids at site specific locations. Table 4 provides sites for non-natural amino acids in heavy chains and light chains.
[0525] To demonstrate the feasibility of making antibodies and antibody-drug conjugates with at least four site-specific non-natural amino acids, DNA encoding trastuzumab heavy chain and light chain with amber were cloned into expression vector pYD317 separately. TAG codon was inserted by overlapping PCR mutagenesis at the nucleotides corresponding to the amino acid serine at positions S136, Y180, S190, and F404 on heavy chain, and S7 and T22 on light chain. To incorporate four pAzMeF in one IgG, the concentration of pCNFRS was increased by about two-fold.
[0526] The cell free reaction mix in which the HC/LC combo variants were synthesized was a 80%:20% blend of cell free extracts made from an OmpT sensitive RF-1 attenuated E. coli strain, and an OmpT sensitive RF-1 attenuated E. coli strain that was engineered to produce an orthogonal CUA-encoding tRNA for insertion of a non-natural amino acid at an Amber Stop Codon. The variants were expressed in a cell-free protein synthesis reaction as follows (based on the method described in Zawada et al., 2011, Biotechnol. Bioeng. 108(7)1570-1578) with the modifications described below.
[0527] Cell-free extracts were treated with 50 μM iodoacetamide for 30 min at RT (20° C.) and added to a premix containing all other components except for DNA encoding the variants of interest. The final concentration in the protein synthesis reaction was 30% cell extract, 1 mM para-azido methyl phenylalanine (pAzMeF) (RSP Amino Acids), 0.37 mg/mL M jannaschii pAzMeF-specific amino-acyl tRNA synthetase (FRS), 2 mM GSSG, 0.29 mg/mL PDI (Mclab), 30 μg/mL E. coli DsbC, 8 mM magnesium glutamate, 10 mM ammonium glutamate, 130 mM potassium glutamate, 35 mM sodium pyruvate, 1.2 mM AMP, 0.86 mM each of GMP, UMP, and CMP, 2 mM amino acids (except 0.5 mM for tyrosine and phenylalanine), 4 mM sodium oxalate, 1 mM putrescine, 1.5 mM spermidine, 15 mM potassium phosphate, 100 nM T7 RNAP, 2.5 μg/mL trastuzumab variant light chain DNA, 7.5 μg/mL trastuzumab-(His).sub.6 variant heavy chain DNA. After addition of DNA template, cell free reactions were incubated at 30° C. for 12h on a shaker at 650 rpm in Flower plates (m2p-labs #MTP-48-B). All variants were scaled up to 9 ml in flower plates (1.5 mL X 6 replicates) and purified using Protein Maker.
Example 9: Purification of 4nnAA Aglycosylated Trastuzumab Variants
[0528] Antibodies with four site-specific non-natural amino acids in heavy chains and light chains (4nnAA HC/LC combos) were purified with the following three step procedure:
[0529] IgG Capture: To capture the IgG, 8 ml of crude cell-free for each variant was first diluted 1:0.5 with equilibration buffer (50 mM sodium phosphate, pH 7) and spun at 11,000×g for 30 minutes. The supernatant was then passed through a 0.45 micron syringe filter prior to being loaded with a 2 minute residence time onto a pre-equilibrated 1 mL MabSelect Sure HiTrap (GE Lifesciences). The column was then washed with 7.5 CV (column volume) of wash buffer (100 mM sodium phosphate and 800 mM Arginine, pH 7) followed by 7.5CV of equilibration buffer. Each variant was then eluted with 4CV elution buffer (100 mM sodium citrate and 300 mM Arginine, pH 3). The elution pool was adjusted to pH 4.6 by addition of 20% (v/v) of 1M Tris, pH 9.
[0530] Aggregate Removal: To remove product related impurities, the 4.8 ml of MabSelect purified IgG was passed through a 1 mL Capto Adhere HiTrap (GE Lifesciences) that had previously been equilibrated with 83.3 mM sodium citrate, 167 mM Tris, 250m M Arginine, pH 4.6. The column was then washed with an additional 7.5 CV of the same buffer. The flowthrough and wash were collected and neutralized to pH 7 by addition of 10% (v/v) of 1 M Tris, pH 9.
[0531] Buffer Exchange: The collected Capto Adhere purified pool was concentrated and buffer exchanged into PBS using the Amicon Ultra-15 (Millipore) centrifugal filter unit by repeated dilution with PBS and subsequent concentration.
Example 10: Conjugation of Aglycosylated Trastuzumab Variants to MMAF
[0532] This example provides conjugation of the antibodies with at least four site-specific non-natural amino acids to the warhead moiety MMAF.
[0533] The purified 4 nnAA HC/LC combo variants were conjugated as follows. DBCO-MMAF AB3627 or AB4285 (ACME Bioscience; Palo Alto, Calif.), shown above, were dissolved in DMSO to a final concentration of 5 mM. The compounds were diluted with PBS to a concentration of 1 mM and then added to purified trastuzumab variants in an immobilized metal ion affinity chromatography (IMAC) elution buffer to achieve a final drug concentration of 100 μM. Mixture was incubated at RT (20° C.) for 17 hours. Reaction was stopped by adding sodium azide to a final concentration of 100 mM and buffer exchanged using Zeba plates (Thermo Scientific; Waltham, Mass.) equilibrated in 1×PBS. Filtrate was then passed through a MUSTANG® Q plate (Pall Corp.; Port Washington, N.Y.) to remove endotoxin.
Example 11: Thermal Stability of Exemplary Antibody-Drug Conjugates
[0534] This example provides the thermal stability (Tm) of aglycosylated trastuzumab and trastuzumab variants.
However, one species of antibody trastuzumab incorporated just two different non-natural amino acids p-azidophenylalanine (pAzidoF) and p-azidomethylphenylalanine (pAzMeF) at the specific location on the heavy and light chain conjugated to MMAE and SN38, is not representative of antibody conjugates for treating any and all subject afflicted with any cancer.
The specification does not teach sufficient relevant identifying characteristics (such as: i. Complete structure, i.e., amino acid sequence of the heavy and light chain variable domains, ii. Partial structure, i.e., the six CDRs, iii. Physical and/or chemical properties, iv. Functional characteristics, binding specificity and affinity of a genus of IgG class antibody conjugated to any immunomodulatory agent, or drug or polymer to enable one of skill in the art to make and use without undue experimentation.
The specification does not teach the binding specificity of all IgG class antibody that can treat any subject when conjugated to any immunomodulatory agent.
It is well known in the art that antibodies bind protein from one species and do notbind to the same protein from another species.
For example, Yu et al (Investigative Ophthalmology & Visual Science 49(2): 522-527, February 2008; PTO 892) teach bevacizumab, which is a humanized anti-human VEGF-A mAb A.4.6.1 binds specifically to human VEGF-A, the same antibody does not bind to mouse VEGF-A (see page 522, right col., page 523, Figure 1, in particular).
Poosarla et al (Biotechn. Bioeng., 114(6): 1331-1342, 2017; PTO 892) teach substantial diversity in designed mAbs (sharing less than 75% sequence similarity to all existing natural antibody sequences) that bind to the same 12-mer peptide, binding to different epitopes on the same peptide. Said reference further teaches “most B-cell epitopes... in nature consist of residues from different regions of the sequence and are discontinuous...de novo antibody designs against discontinuous epitopes present additional challenges...". (See entire reference.)
Nejadmoghaddam (Avicenna Journal of Medical Biotechnology 2(1): 3-23, 2019; PTO 892) discusses major obstacles of antibody-drug conjugates include off-target toxicity, tumor marker selection, antibody specificity, adequately affinity and receptor-mediated internalization are major aspects of choice, cytotoxic payload (e.g., up to 7 drugs per antibody), cytotoxic payload linkage strategy, aqueous solubility, non-immunogenic and stability in storage and bloodstream, see entire document, abstract, p. 15, in particular.
Tsuchikama (of record, Protein Cell 9(1): 33-46, 2018; PTO 1449) teaches that the choice of target antigens, payloads (drug) are important; antibody-payload conjugation methods and linker chemistry are also crucial elements for producing successful ADCs. In particular, instability of the linker and heterogeneity of the product (i.e., broad distribution of DARs) often negatively impacts ADC efficacy and therapeutic window, which often leads to difficulty or limitation in the optimization of clinical application and eventual failure in clinical trials, see p. 42-43. Thus, the art teaches there is a high level of unpredictability in the art of antibody-drug conjugate (ADCs).
There are insufficient in vivo working examples. One of skill in the art cannot predict which undisclosed immunomodulatory agent or drug or polymer conjugated to which IgG class via one or more non-natural amino acids, wherein the first non-natural amino acid residue comprises an azide moiety and the second non-natural amino acid comprises an acetyl moiety and wherein at least one non-natural amino acid is (a) a heavy chain residue 19, 25, 40, 70, 119, 121, 136, 180, 190, 222, 241, or 404 according to the EU index of Kabat or Kabat numbering scheme; or (b) is at light chain residues 7, 22, or 152 according to the Kabat numbering scheme is effective for treating any and all subject afflict with any cancer or breast cancer without undue experimentation.
Applicant is reminded that reasonable correlation must exist between the scope of the claims and scope of enablement set forth. The scope of the claims must bear a reasonable correlation with the scope of enablement. See In re Fisher, 166 USPQ 19 24 (CCPA 1970).
“Tossing out the mere germ of an idea does not constitute enabling disclosure. While every aspect of a generic claim certainly need not have been carried out by an inventor, or exemplified in the specification, reasonable detail must be provided in order to enable members of the public to understand and carry out the invention.” Genentech Inc. v. Novo Nordisk A/S, 42 USPQ2d 1001, 1005 (CA FC 1997).
In view of the lack of the predictability of the art to which the invention pertains as evidenced by Yu, Poosarla, Nejadmoghaddam, Nejadmoghaddam and Tsuchikama, the lack of guidance and direction provided by applicant, and the absence of in vivo working examples, it would require undue experimentation of one skilled in the art to make and use the claimed invention, commensurate in scope with the claims.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the claims at issue are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); and In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on a nonstatutory double patenting ground provided the reference application or patent either is shown to be commonly owned with this application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The USPTO internet Web site contains terminal disclaimer forms which may be used. Please visit http://www.uspto.gov/forms/. The filing date of the application will determine what form should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to http://www.uspto.gov/patents/process/file/efs/guidance/eTD-info-I.jsp.
Claims 1, 4, 14-16, 21, 24, 26, 28-31 and 36-38 are rejected on the ground of nonstatutory obviousness-type double patenting as being unpatentable over claims 1-6, 10-17, 19-20 of U.S. Patent No. 9,732,161. Although the conflicting claims are not identical, they are not patentably distinct from each other because the claims differ only in scope.
Instant specification defines “immunomodulatory agent” to include any cytotoxic, cytostatic or immunomodulatory drug. Useful classes of cytotoxic or immunomodulatory agents include, for example, antitubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cis-platin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, calmodulin inhibitors, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, maytansinoids, nitrosoureas, platinols, pore-forming compounds, purine antimetabolites, puromycins, radiation sensitizers, rapamycins, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like, see para. [0301].
Issued claim 1 recites an antibody fragment of the IgG class comprising an Fc protein, wherein the Fc protein comprises a polypeptide chain having a non-natural amino acid residue at heavy chain residue 404 according to the EU index of Kabat, or a post-translationally modified variant or an aglycosylated variant thereof, wherein the non-natural amino acid residue is selected from the group consisting of: ortho-substituted tyrosine, meta-substituted tyrosine, para-substituted phenylalanine, ortho-substituted phenylalanine, and meta-substituted phenylalanine, which correspond to instant claim 1.
The term “or” in instant claim 1 at lines 12, 13 and 14 does not require light chain residue substitution or more than one substitutions within the heavy or light chain.
The issued patent also teaches the conjugate Trastuzumab IgG HC-F404 DBCO-MMAF2, see Example 15.
Issued claim 2 recites an antibody fragment of the IgG class comprising an Fc protein, wherein the Fc protein comprises a polypeptide chain having a non-natural amino acid at heavy chain residue 404 according to the EU index of Kabat, or a post-translationally modified variant or an aglycosylated variant thereof, wherein each non-natural amino acid residue is according to the formula
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wherein each L is independently a divalent linker; each R is independently a reactive group, a therapeutic moiety or a labeling moiety, and wherein the non-natural amino acid residue is selected from the group consisting of: ortho-substituted tyrosine, meta-substituted tyrosine, para-substituted phenylalanine, ortho-substituted phenylalanine, and meta-substituted phenylalanine.
Issued claim 3. The antibody fragment of claim 2 wherein each R is a reactive group selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido and alkynyl.
Issued claim 4. The antibody fragment of claim 2 wherein each L is a divalent linker selected from the group consisting of a bond, alkylene, substituted alkylene, heteroalkylene, substituted heteroalkylene, arylene, substituted arylene, heteroarlyene and substituted heteroarylene.
Issued claim 5. The antibody fragment of claim 1, wherein the Fc protein is aglycosylated.
Issued claim 6. The antibody fragment of claim 5, wherein the Fc protein has a higher thermal stability (T.sub.m1) compared to the corresponding wild-type Fc protein, which corresponds to instant claims 37-38.
Issued claim 10. The antibody fragment of claim 5, wherein the Fc protein corresponds to a subclass selected from the group consisting of Ig1, IgG2, IgG3, and IgG4, which corresponds to instant claim 15.
Issued claim 11. The antibody fragment of claim 5, wherein said non-natural amino acid residue comprises a moiety selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido (instant claim 1) and alkynyl.
Issued claim 12. The antibody fragment of claim 5, wherein each non-natural amino acid residue is according to the formula ##STR00073## wherein each L is independently a phenylene; and each R is independently a functional group selected from the group consisting of a therapeutic moiety, a labeling moiety, amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido and alkynyl.
Issued claim 13. The antibody fragment of claim 5, wherein the non-natural amino acid residue is selected from the group consisting of: p-acetyl-L-phenylalanine (instant claim 1), O-methyl-L-tyrosine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine (instant claim 1), p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, p-propargyloxy-phenylalanine, and p-azidomethyl-L-phenylalanine.
Issued claim 14. The antibody fragment of claim 5, wherein the non-natural amino acid residue is p-azido-L-phenylalanine, which corresponds to instant claim 1.
Issued claim 15. The antibody fragment of claim 5, wherein the non-natural amino acid residue is p-azidomethyl-L-phenylalanine, which corresponds to instant claim 1.
Issued claim 16. An Fc protein conjugate comprising the antibody fragment of claim 5, wherein the Fc protein is linked to one or more therapeutic moieties or labeling moieties.
Issued claim 17. The Fc protein conjugate of claim 16, wherein the Fc protein is linked to one or more drugs or polymers, which corresponds to instant claim 26.
Issued claim 18. The Fc protein conjugate of claim 16, wherein the Fc protein is linked to one or more labeling moieties.
Issued claim 19. The Fc protein conjugate of claim 16, wherein said Fc protein is linked to one or more single chain binding domains (scFv), which corresponds to instant claim 16.
Issued claim 20. The Fc protein conjugate of claim 16, wherein said one or more therapeutic moieties or labeling moieties is linked to the non-natural amino acid, or a residue thereof, via one or more linkers, which corresponds to instant claim 24.
Claims 1, 4, 14-16, 21, 24, 26, 28-31 and 36-38 are rejected on the ground of nonstatutory obviousness-type double patenting as being unpatentable over claims 5, 10-17, 19-20 of U.S. Patent No. 10,501,558. Although the conflicting claims are not identical, they are not patentably distinct from each other because the claims differ only in scope.
Instant specification defines “immunomodulatory agent” to include any cytotoxic, cytostatic or immunomodulatory drug. Useful classes of cytotoxic or immunomodulatory agents include, for example, antitubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cis-platin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, calmodulin inhibitors, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, maytansinoids, nitrosoureas, platinols, pore-forming compounds, purine antimetabolites, puromycins, radiation sensitizers, rapamycins, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like, see para. [0301].
Issued claim 16 recites an Fc protein conjugate comprising the antibody fragment of claim 5, wherein the Fc protein is linked to one or more therapeutic moieties or labeling moieties, which corresponds to instant claim 1.
The issued patent also teaches IgG antibody such as brentuximab or trastuzumab conjugated to DBCO-MMAF at heavy chain residues K121, Y180 and LC N152, see col. 128, Example 13, Table 12A and Table 12B, Table 13. The term “or” in instant claim 1 at lines 12, 13 and 14 does not require light chain residue substitution or more than one substitutions within the heavy or light chain.
Issued claim 1 recites an antibody fragment of the IgG class comprising an Fc protein, wherein the Fc protein comprises a polypeptide chain having one or more non-natural amino acid residues at specific sites selected from the group consisting of heavy chain residues H241 and H222 according to the EU index of Kabat in the polypeptide chain, or a post-translationally modified variant or an aglycosylated variant thereof, wherein the non-natural amino acid residue is selected from the group consisting of: ortho-substituted tyrosine, meta-substituted tyrosine, para-substituted phenylalanine, ortho-substituted phenylalanine, and meta-substituted phenylalanine.
Issued claim 2 recites an antibody fragment of the IgG class comprising an Fc protein, wherein the Fc protein comprises a polypeptide chain having one or more non-natural amino acid residues at specific sites selected from the group consisting of heavy chain residues H241 and H222 according to the EU index of Kabat, wherein each non-natural amino acid residue is according to the formula
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95
172
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wherein each L is independently a divalent linker; each R is a reactive group; and wherein the non-natural amino acid residue is selected from the group consisting of: ortho-substituted tyrosine, meta-substituted tyrosine, para-substituted phenylalanine, ortho-substituted phenylalanine, and meta-substituted phenylalanine.
Issued claim 3. The antibody fragment of claim 2, wherein each R is a reactive group selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido and alkynyl, which corresponds to instant claim 1.
Issued claim 4. The antibody fragment of claim 2, wherein each L is a divalent linker selected from the group consisting of a bond, alkylene, substituted alkylene, heteroalkylene, substituted heteroalkylene, arylene, substituted arylene, heteroarylene and substituted heteroarylene, which corresponds to instant claim 24.
Issued claim 5. The antibody fragment of claim 1, wherein the Fc protein is aglycosylated.
Issued claim 6. The antibody fragment of claim 5, wherein the Fc protein has a higher thermal stability (Tm1) compared to the corresponding wild-type Fc protein, which corresponds to 37-38.
Issued claim 10. The antibody fragment of claim 5, wherein the Fc protein corresponds to a subclass selected from the group consisting of Ig1, IgG2, IgG3, and IgG4, which corresponds to instant claim 15.
Issued claim 11. The antibody fragment of claim 5, wherein said non-natural amino acid residue comprises a moiety selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido, and alkynyl.
Issued claim 12. The antibody fragment of claim 5, wherein each non-natural amino acid residue is according to the formula
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95
172
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wherein each L is independently a phenylene; and each R is independently a functional group selected from the group consisting of a therapeutic moiety, a labeling moiety, amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido, and alkynyl.
Issued claim 13. The antibody fragment of claim 5, wherein the non-natural amino acid residue is selected from the group consisting of: p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, p-propargyloxy-phenylalanine, and p-azidomethyl-L-phenylalanine.
Issued claim 14. The antibody fragment of claim 5, wherein the non-natural amino acid residue is p-azido-L-phenylalanine, which corresponds to instant claim 1.
Issued claim 15. The antibody fragment of claim 5, wherein the non-natural amino acid residue is p-azidomethyl-L-phenylalanine, , which corresponds to instant claim 1.
Issued claim 17. The Fc protein conjugate of claim 16, wherein the Fc protein is linked to one or more drugs or polymers, which corresponds to instant claim 26.
Issued claim 19. The Fc protein conjugate of claim 16, wherein said Fc protein is linked to one or more single chain binding domains (scFv), which corresponds to instant claim 28.
Issued claim 20. The Fc protein conjugate of claim 16, wherein said one or more therapeutic moieties or labeling moieties is linked to the non-natural amino acid, or a residue thereof, via one or more linkers, which corresponds to instant claim 36.
Conclusion
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to PHUONG HUYNH whose telephone number is (571)272-0846. The examiner can normally be reached on 9:00 a.m. to 6:30 p.m. The examiner can also be reached on alternate alternative Friday from 9:00 a.m. to 5:30 p.m.
If attempts to reach the examiner by telephone are unsuccessful, the examiner's supervisor, Misook Yu, can be reached at 571-270-3497. The fax phone number for the organization where this application or proceeding is assigned is 571-272-0839.
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/PHUONG HUYNH/ Primary Examiner, Art Unit 1641