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 .
Priority
The instant application is a 35 U.S.C. 371 national phase application filed 09/30/2022, and claims priority to International Application No. PCT/US2021/025107 (filing date 03/31/2021), which claims the benefit of the prior-filed United States
Provisional Patent Application Nos. 63/127,893 (filing date 12/18/2020), 63/118,579 (filing date 11/25/2020), and 63/003,842 (filing date 04/01/2020).
Status of Application/Claims
The amendment, filed 11/05/2025, is acknowledged. Claims 1-127 are canceled. Claims 128-143 are new. Claims 128-143 are currently pending and are examined on the merits herein.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 11/05/2025 has been fully considered by the examiner.
Withdrawn Objections/Rejections
Regarding the Specification objection for sequence compliance issues, applicant amendment has addressed the issue. Thus, the objection is withdrawn.
Regarding the Specification objection for trade names and/or trademark compliance issues, applicant amendment has addressed the issue. Thus, the objection is withdrawn.
Regarding the rejection for claims 121-127 under 35 U.S.C. 102 for anticipation: Claims 121-127 have been canceled. Thus, the rejection for claims 121-127 is withdrawn.
Regarding the non-statutory double patenting rejections for claims 121-127: Claims 121-127 have been canceled. Thus, the rejections for claims 121-127 are withdrawn.
Nucleotide and/or Amino Acid Sequence Disclosures
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiency - This application fails to comply with the requirements of 37 CFR 1.821 - 1.825. This application contains a “Sequence Listing” as a PDF file (37 CFR 1.821(c)(2)) or as physical sheets of paper (37 CFR 1.821(c)(3)). A copy of the "Sequence Listing" in computer readable form (CRF) has been submitted; however, the content of the CRF does not comply with one or more of the requirements of 37 CFR 1.822 through 1.824, as indicated in the "Error Report" that indicates the "Sequence Listing" could not be accepted. Refer to attachment or document "Computer Readable Form (CRF) for Sequence Listing – Defective" dated 11/18/2025. It is also noted that an attempt to correct sequence issues was submitted 11/14/2025 in response to a prior defective CRF report submitted 11/06/2025.
Required response – Applicant must provide:
A replacement "Sequence Listing" part of the disclosure, as described above in item 1); together with
An amendment specifically directing its entry into the application in accordance with 37 CFR 1.825(b)(2);
A statement that the "Sequence Listing" includes no new matter as required by 37 CFR 1.825(b)(5); and
A statement that indicates support for the amendment in the application, as filed, as required by 37 CFR 1.825(b)(4).
If the replacement "Sequence Listing" part of the disclosure is submitted according to item 1) a) or b) above, Applicant must also provide:
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3), and 1.125 inserting the required incorporation-by-reference paragraph, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter and
An amendment to the specification to remove the “Sequence Listing previously submitted as a PDF file (37 CFR 1.821(c)(2)) or as physical sheets of paper (37 CFR 1.821(c)(3))
If the replacement "Sequence Listing" part of the disclosure is submitted according to item 1) c) or d) above, Applicant must also provide:
A CRF in accordance with 1.821(e)(1) or 1.821(e)(2) as required by 37 CFR 1.825(b)(6)(ii); and
Statement according to item 2) a) or b) above.
Claim Objections
Claims 133, 136, and 141-142 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 137-140 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.
Claims 137 and 139 recite the limitation "the first Fc domain.” There is insufficient antecedent basis for this limitation in the claims. Additionally, it is unclear if the claims are intended to further limit the half-life extension domain of claim 128 to consisting only of an Fc domain or if the half-life extension domain can comprise additional domains/amino acids in addition to the Fc domain. Thus, the claims are rendered indefinite. For further examination, the claims are interpreted to comprise an Fc domain.
Claims 138 and 140 recite the limitations "(the) second Fc domain” and “the second Fc domain, respectively. There is insufficient antecedent basis for these limitations in the claims. Additionally, it is unclear if the claims are intended to further limit the half-life extension domain of claim 128 to consisting only of an Fc domain or if the half-life extension domain can comprise additional domains/amino acids in addition to the Fc domain. Thus, the claims are rendered indefinite. For further examination, the claims are interpreted to comprise an Fc domain.
[AltContent: textbox (Instant SEQ: 22 vs instant SEQ: 25 (HL1)
[img-media_image1.png])] Claim 137 is also rendered indefinite for the following: Claim 137 recites “wherein the first Fc domain comprises Y349C, T366S, L368A and Y407V mutations relative to SEQ ID NO: 22. Claim 139 is dependent upon claim 137 which recites that the first Fc domain comprises SEQ ID NO: 25. An alignment of instant SEQ ID NOs: 22 and 25 shows that, in addition to the Y349C, T366S, L368A and Y407V mutations listed in claim 137, SEQ ID NO: 25 of claim 139 also contains an N>A mutation (see arrow in alignment below). Thus, it is unclear if the first Fc (i.e., the first half life extension domain, HL1) of claim 137 is intended to encompass only the Y349C, T366S, L368A and Y407V mutations relative to SEQ ID NO: 22 or if the first Fc can have additional mutations, including the N>A mutation. Thus, the claim is rendered indefinite.
[AltContent: textbox (Instant SEQ: 22 vs instant SEQ: 26 (HL2)
[img-media_image2.png])]Claim 138 is also rendered indefinite for the following: Claim 138 recites “wherein the second Fc domain comprises S354C and T355W mutations relative to SEQ ID NO: 22. Claim 140 is dependent upon claim 138 which recites that the second Fc domain comprises SEQ ID NO: 26. An alignment of instant SEQ ID NOs: 22 and 26 shows that, in addition to the S354C and T366W mutations listed in claim 138, SEQ ID NO: 26 of claim 140 also contains an N>A mutation (see arrow in alignment below). Thus, it is unclear if the second Fc (i.e., the second half life extension domain, HL2) of claim 138 is intended to encompass only the S354C and T366W mutations relative to SEQ ID NO: 22 or if the second Fc can have additional mutations, including the N>A mutation. Thus, the claim is rendered indefinite.
For further examination, and in order to provide applicant with the broadest reasonable interpretation, claims 137 and 138 are interpreted to encompass the above listed Y349C, T366S, L368A, Y407V, S354C, and T366W mutations but not to require the N297A mutation, and are not limited to the above listed mutations. Accordingly, claims 139 and 140 are interpreted to provide the additional further limitation of comprising the N297A/”hole” mutation for SEQ ID NO: 25 of the claim 139 HL1/“first Fc domain” and an N297A/“knob” mutation for SEQ ID NO: 26 of the claim 140 HL2/“second Fc domain.”
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The 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.
Claims 128, 131, 137-138, and 143 are rejected under 35 U.S.C. 103 as being unpatentable over Askgene Pharma, Inc. – WO2019173832A2 (publication date: 09/12/2019; effective filing date: 03/09/2018; herein referred to as Askgene); further in view of Wojno, et al. The immunobiology of the interleukin-12 family: Room for discovery. Immunity Review (2019), 50, p.851-870 (herein referred to as Wojno); and, further in view of Chen, et al. Fusion protein linkers: Property, design and functionality. Adv. Drug Deliv. Rev. (2013), 65:10, p.1357-1369 (herein referred to as Chen).
Askgene teaches novel cytokine muteins and prodrugs and methods of making and using thereof, including using the cytokine muteins and prodrugs in a method of treating cancer or an infectious disease, or stimulating the immune system in a patient (title; abstract). The method of treating cancer includes treatment of solid cancer/tumors, including breast cancer, lung cancer, pancreatic cancer, esophageal cancer, medullary thyroid cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, colorectal cancer, and stomach cancer solid tumors (p.6, [0021]; p.27, [0106]). Specifically, Askgene teaches cytokine prodrugs wherein the cytokines are “masked” and metabolized in vivo, via cleavage, to become activated therapeutics (p.4, [0008]). Askgene teaches that such cytokine prodrugs provide benefits of fewer side effects, better in vivo pharmacokinetic profiles/longer half-life, and better target specificity; and, are thus, more efficacious as compared to prior cytokine therapeutics (p.11, [0046]). Askgene teaches that the prodrugs comprise a cytokine agonist moiety, a carrier moiety that can be an antigen-binding moiety/antibody for binding a target site, and a masking moiety (i.e., “cytokine antagonist”) that can be an extracellular domain (ECD) of a receptor for the cytokine wherein the receptor ECD inhibits the cytokine moiety’s biological functions while the receptor mask is binding to it (p.11, [0046]; p.13, [0057]). Askgene teaches that the prodrugs can be activated at a target site such as a tumor site in a patient by cleavage of the linker and consequent release of the cytokine mask from the prodrug, which exposes the previously masked cytokine moiety and allowing the cytokine to bind to its receptor on a target cell and exert its biological functions on the target cell (p.11, [0046]).
Askgene teaches prodrugs that are pro-inflammatory cytokine prodrugs (p.11, [0047]). Askgene teaches specific embodiments for IL-2 and IL-15 prodrugs, but also teaches that prodrugs for other cytokines, and in particular cytokines that are potent immune regulators and have strong side effects, are also contemplated and can be made according to the same principles as for IL-2 and IL-15 (p.11, [0048]). Askgene teaches IL-2 and IL-15 prodrugs wherein the masking moiety comprises an IL-2 or IL-15 receptor extracellular domain of their IL-2Rβ or IL-2Rγ receptors (p.13 – 14, [0058]). Askgene additionally teaches several linker sequences, including SEQ ID NO: 49 (i.e., GGGGSGGGGSGGGGS).
Askgene teaches that the carrier moiety can an antibody or antigen-binding fragment thereof that is a full-length antibody with two heavy chains and two light chains, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a disulfide linked Fv fragment, a single domain antibody, a nanobody, or a single chain variable fragment (scFv); and that the antigen-binding moiety can provide additional and potentially synergetic therapeutic efficacy to the cytokine (p.14, [0062]). Askgene teaches that the antibody may be of any heavy chain isotype or subtype including IgG1 Fc; and, that the antibody may be human, non-human, chimeric, or humanized (p.9, [0036]). Askgene teaches that the carrier moiety can improve the serum half-life of the cytokine agonist polypeptide and may also target the cytokine agonist polypeptide to a target site in the body, such as a tumor site (p.14, [0061]). Askgene teaches that strategies of forming heterodimers between the two heavy chains/Fc regions are well known: “For example, the two heavy chain polypeptides in the prodrug may form stable heterodimers through "knobs-into-holes" mutations. "Knobs-into-holes" mutations are made to promote the formation of the heterodimers of the antibody heavy chains and are commonly used to make bispecific antibodies. For example, the Fc domain of the antibody may comprise a T366W mutation in the CH3 domain of the "knob chain" and T366S, L368A, and/or Y407V mutations in the CH3 domain of the "hole chain." An additional interchain disulfide bridge between the CH3 domains can also be used, e.g., by introducing a Y349C mutation into the CH3 domain of the "knobs chain" and an E356C or S354C mutation into the CH3 domain of the "hole chain"… In other embodiments, the antibody moiety may comprise Y349C and/or T366W mutations in one of the two CH3 domains, and E356C, T366S, L368A, and/or Y407V mutations in the other CH3 domain. In certain embodiments, the antibody moiety may comprise Y349C and/or T366W mutations in one of the two CH3 domains, and S354C (or E356C), T366S, L368A, and/or Y407V mutations in the other CH3 domain, with the additional Y349C mutation in one CH3 domain and the additional E356C or S354C mutation in the other CH3 domain, forming an interchain disulfide bridge…” (p.15, [0064]).
Askgene teaches that the cytokine moiety and the masking moiety can be fused to the N-terminus or C-terminus of the light chains and/or heavy chains of the antigen-binding moiety (i.e., carrier; p.14, [0063]). Askgene teaches that the cytokine agonist polypeptide can be fused to the C-terminus of one of the heavy chains of an antibody via a (first) linker, and the cytokine’s mask is fused to the C-terminus of the other heavy chain of the antibody through a (second) cleavable peptide linker, wherein the two heavy chains contain mutations that allow the specific pairing of the two different heavy chains (i.e., association of a first half-life extension domain with a second half-life extension domain; p.15, [0065]).
Askgene additionally teaches that the cytokine moiety polypeptide can be fused to the carrier directly or through a cleavable or noncleavable linker; and, that the masking moiety can be fused to the cytokine or to the carrier through a cleavable peptide linker (p.12, [0050]).
Collectively, Askgene teaches a method of treating cancer in a subject by administering a masked cytokine comprising a protein heterodimer of a first polypeptide with a first half-life extension domain (instant “HL1”) linked to a masking moiety (instant “MM”) via a first linker (“L1”) and a second polypeptide with a second half-life extension domain (instant “HL2”) linked, via a second linker (instant “L2”) to a heterodimeric cytokine (instant “C”) comprised of two subunits covalently joined via a linker; and, wherein the first and second half-life extension domains associate with one another (instant claim 128); wherein the linker joining the two subunits of the cytokine is GGGGSGGGGSGGGGS (i.e., instant SEQ ID NO: 3; instant claim 131).
Askgene does not teach a method of cancer treatment wherein the masked cytokine is a masked IL-12 cytokine that comprises an IL-12p40 polypeptide covalently linked to an IL-12p35 polypeptide via a linker (instant claim 128); or, that the linker joining the IL-12 subunits is instant SEQ ID NO: 3 of GGGSGGGSGGGS (instant claim 131).
Wojno teaches that IL-12 has an important role for cancer and that expression of IL-12 has shown results of tumor regression and anti-tumor responses; but that there remain concerns about using cytokines as drugs due to side effects (p.861, col.2, paras.1-2). Wojno further teaches that these issues are exemplified in multiple trials that utilized IL-12 as a treatment for different cancers, which revealed significant toxicity and limited efficacy (p.862, col.1, para.1). Wojno also teaches that IL-12 is a heterodimer comprised of two subunits: the α subunit IL-12p35 and the β subunit IL-12p40 (p.851, col.2, para.2). Wojno further teaches that the IL-12 heterodimer utilizes a heterodimeric receptor complex for signaling made up of IL-12Rβ1 and IL-12Rβ2 (p.852, Fig.1 and legend).
Chen teaches fusion protein linkers, including flexible linkers, rigid linkers, and in vivo cleavable linkers (title; sections 3.1 – 3.3). Chen teaches that direct fusion of functional domains without a linker may lead to many undesirable outcomes, including misfolding of the fusion proteins, low yield in protein production, or impaired bioactivity (p.2, para.2). Chen teaches that linkers can be used to join protein drugs to carrier proteins such as antibodies in order to extend plasma half-lives and to achieve enhances therapeutic effects by drug targeting to specific types of cells (p.1, para.1). Chen teaches the use of flexible linkers for recombinant fusion proteins; that flexible linkers are useful when the domains require a certain degree of movement or interaction; that flexible linkers are generally composed of small, non-polar or polar amino acids which provides for flexibility and mobility of connecting functional domains; and that the most commonly used flexible linkers consist of stretches of glycine and serine residues (i.e., “GS linkers”) that have the sequence of (Gly-Gly-Gly-Gly-Ser)n (p.4, para.4).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to combine the teachings of Askgene with the teachings of Wojno by modifying the method of treating a cancer using a masked IL-2 or IL-15 cytokine as taught by Askgene by using an IL-12 cytokine taught by Wojno in order to arrive at the instantly claimed invention, because the combination of prior art elements according to known methods results in a predictable result and a beneficial outcome of reducing toxicity with administration of an IL-12 cytokine for cancer treatment.
It would have further been obvious to one of ordinary skill in the art to use Askgene’s SEQ ID NO: 49 (noncleavable) covalent cytokine domains linker GGGGSGGGGSGGGGS to covalently join the subunits of the IL-12 heterodimer (taught by Wojno) in order to tether the p40 and p35 subunits (taught by Wojno) to form a fusion protein (taught by Chen), because the combination of prior art elements according to known methods results in a predictable result of forming a biologically active IL-12 heterodimer for receptor stimulation (taught by Wojno) for IL-12 mediated cancer treatment (taught by Wojno). One of ordinary skill in the art would be motivated to do so because Chen teaches that flexible GS linkers of the formula GGGGSn provide a benefit of joining protein domains to facilitate interaction between the domains and one would have a reasonable expectation of success because Askgene’s SEQ ID NO: 49 linker is a GGGGSn linker.
Regarding the first and second half-life extension domains, it would have been prima facie obvious for one of ordinary skill in the art to further combine the teachings of Askgene by using a “knobs-in-holes” carrier comprised of two Fc polypeptides comprising the “knob” mutations and “holes” mutations, in order to arrive at the instantly claimed invention, because the combination of prior art elements taught by Askgene teaches that these mutations facilitate association of the two Fc chains to facilitate dimerization. One would be motivated to do so because this would produce a “carrier” domain that would provide the benefits of extended half-life for a masked cytokine “prodrug” for solid cancer treatment. One of ordinary skill in the art would have a reasonable expectation of success because Askgene teaches the specific amino residue mutations at amino acid positions of the instant claims.
Regarding bioactivation of the masked IL-12 cytokine, Askgene specifically teaches that the cytokine moiety polypeptide can be fused to the carrier directly or through a cleavable or noncleavable linker; and, that the masking moiety can be fused to the cytokine or to the carrier through a cleavable peptide linker (p.12, [0050]); and, that the cytokine is activated by cleavage of the linker joined to the masking moiety. Askgene does not specifically teach an embodiment wherein it is explicitly stated that the cytokine’s linker is noncleavable and that the masking moiety’s linker is cleavable. It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Askgene to make the cytokine’s linker cleavable and the masking moiety’s linker noncleavable, in order to arrive at the instantly claimed invention, because the combination of prior art elements results in a predictable result of cleavage of either the cytokine or the masking moiety in order to facilitate dissociation of the cytokine from the masking moiety for bioactivation. The decision to cleave the cytokine from the “prodrug” would be obvious to try given that the ultimate goal is to produce a cleavage that would release the cytokine from the masking moiety. One of ordinary skill in the art would have a reasonable expectation of success because making the cytokine’s linker cleavable or the masking moiety’s linker cleavable would predictably result in dissociation of the cytokine and masking moiety and would make the cytokine available for receptor binding and activation in order to treat the solid tumor.
Claims 129 is rejected under 35 U.S.C. 103 as being unpatentable over Askgene, Wojno, and Chen, as applied to claim 128 above; further in view of Merck Patent GmbH – US20070154453A1 (publication date: 07/05/2007; effective filing date: 12/30/2005; herein referred to as Merck); and, further in view of Xencor, Inc. – WO2020072821A2 (effective filing date: 10/03/2018; herein referred to as Xencor).
The combination of Askgene/Wojno/Chen teaches a method of treating cancer in a subject by administering a masked IL-12 cytokine comprising an IL-12p40/IL-12p35 heterodimer with a first polypeptide with a first half-life extension domain (instant “HL1”) linked to a masking moiety (instant “MM”) via a first linker (“L1”) and a second polypeptide with a second half-life extension domain (instant “HL2”) linked, via a second linker (instant “L2”) to the IL-12 heterodimeric cytokine (instant “C”) subunits covalently joined via a linker; and, wherein the first and second half-life extension domains associate with one another, as described in detail for instant claim 128 above.
The combination of Askgene/Wojno/Chen does not teach that the IL-12p40 polypeptide comprises instant amino acid SEQ ID NO: 60 (instant claim 129).
[AltContent: textbox (Instant SEQ: 60 (IL-12p40) vs Merck (IL-12p40)
[img-media_image3.png])]Merck teaches IL-12p40 amino acid SEQ ID NO: 7 which aligns with instant SEQ ID NO: 60 with 99.7% identity wherein the only variation from applicant’s sequence is that Merck’s SEQ ID NO: 7 retains a cysteine at position 252 (see alignment below):
Thus, Askgene/Wojno/Chen and Merck do not teach that the IL-12p40 subunit comprises a C252S mutation.
Xencor teaches that IL-12 is a promising cytokine for cancer treatment, but that it has faced hurdles in clinical trials due to toxicity (p.1, [0003]). IL-12/Fc fusion proteins comprising both IL-12p40 and IL-12p35 subunits linked to half-life extension domains for cancer treatment, including several variants wherein the IL-12p40 subunit comprises mutations that modulate receptor affinity and potency and that removes free cysteines that may bond with other free cysteines leading to heterogeneity and undesirable immunogenicity, including an IL-12p40 variant comprising a C252S mutation for the purpose of removing the C252 free cysteine (p.6, [0011]; p.14, [0041]; p.15, [0042 – 0043]; p.151, [00477 – 00479]; p.277 –278, [00881]). Xencor teaches that the C252S mutation can be used alone or in combination with any other IL-12p40 variants (p.278, [00881]). Xencor further teaches that IL-12p40 variants comprising the C252S mutation exhibited improved potency compared to variants without the mutation (p.151, [00479]; p.193, Fig.67A-D).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Askgene/Wojno/Chen with the teachings of Merck and Xencor by using Merck’s SEQ ID NO: 7 that encodes the IL-12p40 subunit and to modify the IL-12p40 subunit to have a C252S mutation (taught by Xencor), in order to arrive at the instantly claimed invention because the combination of prior art elements results in a predictable result of encoding a IL-12p40 polypeptide that has a C252S mutation. Further, one would be motivated to mutate C252S in order to receive the expected benefit (as taught by Xencor) that the C252S mutation would remove a free cysteine from the IL-12p40 subunit to reduce heterogeneity and immunogenicity, and to improve potency of the IL-12 cytokine (as taught by Xencor) for the IL-12p40 subunit for the masked IL-12 cytokine in a method of treating cancer (as taught by Askgene/Wojno/Chen). One of ordinary skill in the art would have a reasonable expectation of success because the combination of Merck and Xencor teach the amino acid sequence for an improved IL-12p40 cytokine; Askgene, Wojno, and Xencor all teach IL-12 as a promising cytokine for cancer treatment; and, Askgene/Wojno/Chen and Xencor teach fusion proteins comprising IL-12p40 and IL-12p35 subunits as well as their linkage to half-life extension domains.
Claims 130 is rejected under 35 U.S.C. 103 as being unpatentable over Askgene, Wojno, and Chen, as applied to claim 128 above; and, further in view of UniProt. P29459 – IL12A_HUMAN. 01/23/2002, p.1-7 (herein referred to as UP-IL12A).
The combination of Askgene/Wojno/Chen teaches a method of treating cancer in a subject by administering a masked IL-12 cytokine comprising an IL-12p40/IL-12p35 heterodimer with a first polypeptide with a first half-life extension domain (instant “HL1”) linked to a masking moiety (instant “MM”) via a first linker (“L1”) and a second polypeptide with a second half-life extension domain (instant “HL2”) linked, via a second linker (instant “L2”) to the IL-12 heterodimeric cytokine (instant “C”) subunits covalently joined via a linker; and, wherein the first and second half-life extension domains associate with one another, as described in detail for instant claim 128 above.
The combination of Askgene/Wojno/Chen does not teach that the IL-12p35 polypeptide comprises instant amino acid SEQ ID NO: 2 (instant claim 130).
[AltContent: textbox (Instant SEQ: 2 (IL-12p35) vs UniProt (IL-12A P29459)
[img-media_image4.png])]UP-IL12A teaches P29459: IL-12A, which is the human sequence for the IL-12 p35 subunit and instant SEQ ID NO: 2, at 100% identity (p.5; see alignment below):
UP-IL12A also teaches that IL12A (i.e., IL-12p35) heterodimerizes with IL12B(i.e., IL-12p40) to form the IL-12 cytokine to exert its biological effects through the IL-12 receptor subunits (p.1).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Askgene/Wojno/Chen with the teachings of UP-IL12A by using UP-IL12A’s mature “P29549 IL-12A” amino acid sequence that encodes the IL-12p35 subunit of the masked cytokine (as taught by Askgene/Wojno/Chen), in order to arrive at the instantly claimed invention because the combination of prior art elements results in a predictable result of encoding the a human IL-12p35 polypeptide for the masked IL-12 cytokine in a method of treating cancer. One of ordinary skill would have a reasonable expectation of success because Wojno and UP-IL12A both teach that the functional IL-12 cytokine is comprised of the IL-12B/IL-12p40 and IL-12A/IL-12p35 subunits.
Claim 132 is rejected under 35 U.S.C. 103 as being unpatentable over Askgene, Wojno, and Chen; further in view of Merck and Xencor; and, further in view of UP-IL12A.
The combination of Askgene/Wojno/Chen teaches a method of treating cancer in a subject by administering a masked IL-12 cytokine comprising an IL-12p40/IL-12p35 heterodimer with a first polypeptide with a first half-life extension domain (instant “HL1”) linked to a masking moiety (instant “MM”) via a first linker (“L1”) and a second polypeptide with a second half-life extension domain (instant “HL2”) linked, via a second linker (instant “L2”) to the IL-12 heterodimeric cytokine (instant “C”) subunits covalently joined via a linker; and, wherein the first and second half-life extension domains associate with one another, as described in detail for instant claim 128 above.
The combination of Askgene/Wojno/Chen does not teach that the IL-12 cytokine comprises instant SEQ ID NO: 64 (instant claim 132).
Instant SEQ ID NO: 64 (as recited in instant claim 132) is an amino acid sequence that encodes for a fusion protein comprising an IL-12p40 subunit joined to an IL-12p35 subunit via the flexible “GS linker” GGGGSGGGGSGGGGS (i.e., instant SEQ ID NOs: 60 (p40) –3 (linker) – 2 (p35); see sequence fusion below):
[AltContent: textbox (Instant SEQ: 64 vs fusion of instant SEQ: 60 (p40)/SEQ:3 (linker)/SEQ: 2 (p35)
[img-media_image5.png])]
The combination of Askgene, Wojno, and Chen teaches a masked cytokine wherein a polypeptide chain is comprised of an Fc half-life extension domain fused to a IL-12 cytokine via a cleavable linker, wherein the IL-12 cytokine is comprised of a p40 subunit linked to the p35 subunit using the flexible fusion protein linker GGGGSGGGGSGGGGS, as described in detail above for claim 128.
The combination of Askgene/Wojno/Chen, Merck, and Xencor teaches the IL-12p40 subunit for the IL-12 cytokine for the masked cytokine as described in detail above for claim 129.
The combination of Askgene/Wojno/Chen and UP-IL12A teaches the IL-12p35 subunit of the IL-12 cytokine for the masked cytokine as described in detail for claim 130 above.
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Askgene/Wojno/Chen with the teachings of Merck/Xencor and the teachings of UP-IL12A by using the flexible GS linker GGGGSGGGGSGGGGS (as taught by Askgene/Chen) to join the p40 and p35 subunits to produce a functional IL-12 heterodimeric cytokine (as taught by Wojno, Xencor, and UP-IL12A) for the IL-12 cytokine for a masked IL-12 cytokine (as taught by Askgene/Wojno/Chen) for IL-12-mediated cancer treatment (as taught by Askgene/Wojno/Chen and Xencor). Further, one would be motivated to link the p40 and p35 subunits using the flexible GS linker, as described in detail above, to facilitate their interaction (as taught by Chen). One of ordinary skill in the art would have a reasonable expectation of success because the combination of prior art elements results in the predictable result of producing a biologically functional IL-12 cytokine and because the p40, flexible linker, and p35 sequences are all taught by the prior art as described above.
Claim 134 is rejected under 35 U.S.C. 103 as being unpatentable over Askgene, Wojno, and Chen; and, further in view of Jounce Therapeutics, Inc. – WO2021163064A2 (effective filing date: 02/14/2020; herein referred to as Jounce).
The combination of Askgene/Wojno/Chen teaches a method of treating cancer in a subject by administering a masked IL-12 cytokine comprising an IL-12p40/IL-12p35 heterodimer with a first polypeptide with a first half-life extension domain (instant “HL1”) linked to a masking moiety (instant “MM”) via a first linker (“L1”) and a second polypeptide with a second half-life extension domain (instant “HL2”) linked, via a second linker (instant “L2”) to the IL-12 heterodimeric cytokine (instant “C”) subunits covalently joined via a linker; and, wherein the first and second half-life extension domains associate with one another, as described in detail for instant claim 128 above.
The combination of Askgene/Wojno/Chen does not teach that the first linker, which links the masking moiety to the first half-life extension domain, comprises a non-cleavable linker of instant amino acid SEQ ID NO: 14 (instant claim 134).
Jounce teaches antibodies and fusion proteins, wherein the antibody fragment can be an Fc region that is linked to another protein via a linker; wherein the conjugate a specific embodiment has the formula of a drug-linker-antibody conjugate where the drug is a cytotoxic agent that can be a cytokine (p.65, [0245]; p.65, [0242]); and, wherein the linker is a cleavable or noncleavable linker (p.65; [0245]); and, wherein the conjugate can be used in a method of treating cancer, including solid tumors (p.75, [0285]). Jounce also specifically teaches SEQ ID NO: 3 of SGGSGGGSG which is “GS linker” that comprises instant SEQ ID NO: 14 of GGSGGGSG (p.165; Table of Certain Sequences: SEQ ID NO: 3).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Askgene/Wojno/Chen with the teachings of Jounce by modifying the first polypeptide comprising a first half-life extension domain (HL1), a first linker (L1), and a masking moiety (MM) as for a masked IL-12 cytokine (as taught by Askgene/Wojno/Chen) by using noncleavable linker SGGSGGGSG, as taught by Jounce, to join the masking moiety to the Fc half-life extension domain, to arrive at the instantly claimed invention, because the combination of prior art elements would result in the predictable result of producing an polypeptide where a masking moiety is linked covalently to an Fc half-life extension domain. One of ordinary skill in the art would be motivated to do so because Chen teaches flexible “GS linkers” are used to join functional proteins to create fusion proteins. One of ordinary skill would also have a reasonable expectation of success because Askgene, Chen, and Jounce teach fusion of functional proteins using flexible GS linkers.
Claim 135 is rejected under 35 U.S.C. 103 as being unpatentable over Askgene, Wojno, and Chen; and, further in view of CytomX Therapeutics, Inc. – US10336824B2 (publication date: 10/27/2016; effective filing date: 03/13/2015; herein referred to as CytomX).
The combination of Askgene/Wojno/Chen teaches a method of treating cancer in a subject by administering a masked IL-12 cytokine comprising an IL-12p40/IL-12p35 heterodimer with a first polypeptide with a first half-life extension domain (instant “HL1”) linked to a masking moiety (instant “MM”) via a first linker (“L1”) and a second polypeptide with a second half-life extension domain (instant “HL2”) linked, via a second linker (instant “L2”) to the IL-12 heterodimeric cytokine (instant “C”) subunits covalently joined via a linker; and, wherein the first and second half-life extension domains associate with one another, as described in detail for instant claim 128 above.
The combination of Askgene/Wojno/Chen does not teach that the second linker comprises a proteolytically peptide of instant amino acid SEQ ID NO: 44 (instant claim 135).
CytomX teaches activatable antibodies and methods of manufacturing activatable antibodies that comprise masking moieties (MM), a cleavable moiety (CM), and an antibody or antigen binding fragment thereof (AB) that binds PDL1 for use in cancer treatment, including solid cancers (p.59, col.54, paras.1-3); wherein, the cleavable moiety (CM) is a polypeptide that is cleavable by a protease and activated in the tumor microenvironment (p.164, col.264, para.1); wherein the activatable antibody has the format MM – CM – AB or AB – CM – MM; and, wherein prior to cleavage the MM interferes with binding and when cleaved the MM no longer interferes with binding (p59, col.53, para.1). Further, CytomX specifically teaches multiple cleavable linker sequences comprising instant SEQ ID NO: 44 of ISSGLLSGRS; for example, see CytomX SEQ ID NO: 377, 394, 901-911 (p.50, cols.35 –36 through p.51, col.37; p.158 -- 159, Table 18).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Askgene/Wojno/Chen with the teachings of CytomX by modifying the IL-12 masked cytokine (as taught by Askgene/Wojno/Chen) to use a cleavable linker comprising ISSGLLSGRS (as taught by CytomX) in order to activate a fusion protein drug for the treatment of solid cancers (as taught by Askgene and CytomX). One of ordinary skill in the art would be motivated to do so because Askgene and CytomX teach activatable drugs conjugated to antigen-binding fragments wherein the drug is activated via cleavage of a protease-cleavable linker in the tumor microenvironment. One of ordinary skill in the art would have a reasonable expectation of success because CytomX teaches various cleavable amino acid sequences comprising the cleavable polypeptide sequence of ISSGLLSGRS.
As discussed in detail above for instant claim 128, regarding bioactivation of the masked IL-12 cytokine, Askgene specifically teaches that the cytokine moiety polypeptide can be fused to the carrier directly or through a cleavable or noncleavable linker; and, that the masking moiety can be fused to the cytokine or to the carrier through a cleavable peptide linker (p.12, [0050]); and, that the cytokine is activated by cleavage of the linker joined to the masking moiety. Askgene does not specifically teach an embodiment wherein it is explicitly stated that the cytokine’s linker is noncleavable and that the masking moiety’s linker is cleavable. It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Askgene (along with the teachings of CytomX) to make the cytokine’s linker cleavable and the masking moiety’s linker noncleavable, in order to arrive at the instantly claimed invention, because the combination of prior art elements results in a predictable result of cleavage of either the cytokine or the masking moiety in order to facilitate dissociation of the cytokine from the masking moiety for bioactivation. The decision to cleave the cytokine from the “prodrug” would be obvious to try given that the ultimate goal is to produce a cleavage that would release the cytokine from the masking moiety. One of ordinary skill in the art would have a reasonable expectation of success because making the cytokine’s linker cleavable or the masking moiety’s linker cleavable would predictably result in dissociation of the cytokine and masking moiety and would make the cytokine available for receptor binding and activation in order to treat the solid tumor.
Claims 139-140 are rejected under 35 U.S.C. 103 as being unpatentable over Askgene, Wojno, and Chen; further in view of UniProt. P0DOX5 – IGG1_HUMAN. 07/18/2018, p.1-5 (herein referred to as UP-IGG1); and, further in view of Wang, et al. Silencing Fc domains in T cell-engaging bispecific antibodies improves T-cell trafficking and antitumor potency. Cancer Immunol. Res (2019), p.2013-2024 (herein referred to as Wang).
The combination of Askgene/Wojno/Chen teaches a method of treating cancer in a subject by administering a masked IL-12 cytokine comprising an IL-12p40/IL-12p35 heterodimer with a first polypeptide with a first half-life extension domain (instant “HL1”) linked to a masking moiety (instant “MM”) via a first linker (“L1”) and a second polypeptide with a second half-life extension domain (instant “HL2”) linked, via a second linker (instant “L2”) to the IL-12 heterodimeric cytokine (instant “C”) subunits covalently joined via a linker; and, wherein the first and second half-life extension domains associate with one another, as described in detail for instant claim 128 above. The combination of Askgene/Wojno/Chen further teaches the first and second Fc domains that are comprised of IgG1 Fc polypeptides comprising “knobs-in-holes” mutations that facilitate association of the two Fc half-life extension polypeptides, as described in detail above for instant claims 137 and 138.
The combination of Askgene/Wojno/Chen does not teach that the first Fc domain comprises instant amino acid SEQ ID NO: 25 (instant claim 139); or, that the second Fc domain comprises instant amino acid SEQ ID NO: 26 (instant claim 140).
[AltContent: textbox (Instant SEQ: 22 vs instant SEQ: 25 (HL1)
[img-media_image6.png])] In addition to the HL1 and HL2 “knobs-in-holes” mutations taught by Askgene above (i.e., Y349C, T366S, L368A, Y407V, S354C, and T366W), instant SEQ ID NO: 25 of instant claim 139 and SEQ ID NO: 26 of instant claim 140 harbor a N>A mutation compared to Fc SEQ ID NO: 22 (see arrows in alignments below):
[AltContent: textbox (Instant SEQ: 22 vs instant SEQ: 26 (HL2)
[img-media_image7.png])]Thus, the combination of Askgene/Wojno/Chen does not teach an IgG1 Fc sequence (i.e., a backbone sequence) comprising the N>A mutation that, with the “knobs-in-holes” mutation of Askgene, would arrive at a first Fc domain of instant SEQ ID NO: 25 and a second Fc domain of instant SEQ ID NO: 26.
UP-IGG1 teaches the human IgG1 human immunoglobulin gamma-1 heavy chain amino acid sequence P0DOX5 (p.4), which provides the backbone sequence for human IgG1 Fc, except for the “knobs-in-holes” mutations taught by Askgene, and the N>A mutation.
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Askgene/Wojno/Chen with the teachings of UP-IGG1 by using the human IgG1 Fc taught by UP-IGG1 to encode the human IgG1 Fc half-life extension domains and to modify the first and second half-life extension Fc domains to include the “knobs-in-holes” mutations taught by Askgene, to arrive at a first Fc half-life extension domain and a second half-life extension domain that arrives at the HL1 and HL2 domains of instant claims 128, 139, and 140 except for the N>A mutations, because the combination of prior art elements teaches the IgG1 sequence and in order to achieve the expected benefit of the “knobs-in-holes”-mediated association between the two half-life extension domains (taught by Askgene) for the IL-12 masked cytokine for cancer treatment (taught by Askgene/Wojno/Chen). One of ordinary skill in the art would have a reasonable expectation of success because Askgene teaches that the half-life extension domains can be derived from human IgG1 Fc regions, UP-IgG1 teaches the human IgG1 Fc amino acid sequence, and Askgene teaches the specific mutations and positions of the “knobs-in-holes” mutations.
Askgene/Wojno/Chen and UP-IgG1 do not teach a human IgG1 Fc sequence that comprises the N>A mutation (as it corresponds to instant SEQ ID NO: 22, and amino acid position 77 of the above alignment).
Wang teaches mutations introduced into Fc domains to eliminate FcγR binding in order to avoid or reduce antigen-independent cytokine release syndrome (CRS; p.2013 – 2014). Wang also teaches that human IgG contains a single conserved site in the CH2 domain of Fc, Asn297, for N-linked glycosylation and that glycans at this site determine the interaction between IgG Fc domain and Fcγ receptors. Wang also teaches that the N297A mutation removes the N-glycosylation, thereby preventing binding to Fcγ receptors (p.2013, col.2, para.2).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Askgene/Wojno/Chen and UP-IGG1 with the teachings of Wang by modifying the human IgG1 Fc half-life extension domains to comprise the N>A mutation (i.e., N297A of full IgG1 in instant SEQ ID NO: 22) as taught by Wang, in order to arrive at the instantly claimed invention, in order to receive the expected benefit, as taught by Wang, that the N297A mutation prevents binding of human IgG1 Fc to FcγRs to avoid or reduce CRS with administration of the IL-12 masked cytokine that comprises Fc half-life extension domains. One of ordinary skill in the art would have a reasonable expectation of success because UP-IGG1 teaches the human IgG1 sequence and Wang teaches the position of the N>A mutation.
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 conflicting claims 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); 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 nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) 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 www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
US11718655B2
Claims 128-132, 134-135, 137-140, and 143 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-9 of U.S. Patent No. US11718655B2 (herein referred to as Pat’655); and further in view of Askgene, Wojno, Chen, Merck, Xencor, UP-IL12A, Jounce, CytomX, UP-IGG1, and Wang.
[AltContent: textbox (Pat’655 SEQ: 154 vs Instant SEQ ID NO: 22
[img-media_image8.png])] Pat’655 teaches a masked IL-12 cytokine having a first H-linker-C polypeptide wherein H is an Fc domain and C is an IL-12 cytokine; and, a second H-linker-MM polypeptide wherein H is an Fc and MM is an extracellular domain of IL-12Rβ2; and, wherein one Fc domain comprises the “knobs-in-holes” mutations Y349C, T366S, L368A, and Y407V and the other Fc domain comprises the “knobs-in-holes” mutations S354C and T366W relative to Fc amino acid SEQ ID NO: 154, which is identical to instant SEQ ID NO: 22 except for the N>A mutation (Pat’655 claim 1; instant claims 128 and 137-138; see alignment below):
Pat’655 also teaches the linker amino acid sequence ISSGLLSGRS of SEQ ID NO: 281, which is identical to the cleavable linker amino acid sequence of SEQ ID NO: 44 (Pat’655 claim 3; instant claim 135).
Pat’655 does not teach a method of treating cancer comprising administering the IL-12 cytokine (instant claims 128, 137-138); a method wherein the IL-12 cytokine comprises an amino acid sequence that encodes an IL-12p40 polypeptide of instant SEQ ID NO: 60 (instant claim 129) that is linked to an IL-12035 polypeptide of instant SEQ ID NO: 2 (instant claim 130) via a linker of amino acid instant SEQ ID NO: 3 (instant claim 131); that the IL-12 cytokine comprises an amino acid sequence of SEQ ID NO: 64 (instant claim 132); that the first linker comprises a noncleavable linker of SEQ ID NO: 14 (instant claim 134); that the first and second Fc domains comprise an additional N>A mutation (instant claims 139 and 140, respectively); or, a method wherein the cancer is a solid tumor (instant claim 142).
Askgene teaches novel cytokine muteins and prodrugs and methods of making and using thereof, including using the cytokine muteins and prodrugs in a method of treating cancer or an infectious disease, or stimulating the immune system in a patient (title; abstract). The method of treating cancer includes treatment of solid cancer/tumors, including breast cancer, lung cancer, pancreatic cancer, esophageal cancer, medullary thyroid cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, colorectal cancer, and stomach cancer solid tumors (p.6, [0021]; p.27, [0106]). Specifically, Askgene teaches cytokine prodrugs wherein the cytokines are “masked” and metabolized in vivo, via cleavage, to become activated therapeutics (p.4, [0008]). Askgene teaches that such cytokine prodrugs provide benefits of fewer side effects, better in vivo pharmacokinetic profiles/longer half-life, and better target specificity; and, are thus, more efficacious as compared to prior cytokine therapeutics (p.11, [0046]). Askgene teaches that the prodrugs comprise a cytokine agonist moiety, a carrier moiety that can be an antigen-binding moiety/antibody for binding a target site, and a masking moiety (i.e., “cytokine antagonist”) that can be an extracellular domain (ECD) of a receptor for the cytokine wherein the receptor ECD inhibits the cytokine moiety’s biological functions while the receptor mask is binding to it (p.11, [0046]; p.13, [0057]). Askgene teaches that the prodrugs can be activated at a target site such as a tumor site in a patient by cleavage of the linker and consequent release of the cytokine mask from the prodrug, which exposes the previously masked cytokine moiety and allowing the cytokine to bind to its receptor on a target cell and exert its biological functions on the target cell (p.11, [0046]).
Askgene teaches prodrugs that are pro-inflammatory cytokine prodrugs (p.11, [0047]). Askgene teaches specific embodiments for IL-2 and IL-15 prodrugs, but also teaches that prodrugs for other cytokines, and in particular cytokines that are potent immune regulators and have strong side effects, are also contemplated and can be made according to the same principles as for IL-2 and IL-15 (p.11, [0048]). Askgene teaches IL-2 and IL-15 prodrugs wherein the masking moiety comprises an IL-2 or IL-15 receptor extracellular domain of their IL-2Rβ or IL-2Rγ receptors (p.13 – 14, [0058]). Askgene additionally teaches several linker sequences, including SEQ ID NO: 49 (i.e., GGGGSGGGGSGGGGS).
Askgene teaches that the carrier moiety can an antibody or antigen-binding fragment thereof that is a full-length antibody with two heavy chains and two light chains, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a disulfide linked Fv fragment, a single domain antibody, a nanobody, or a single chain variable fragment (scFv); and that the antigen-binding moiety can provide additional and potentially synergetic therapeutic efficacy to the cytokine (p.14, [0062]). Askgene teaches that the antibody may be of any heavy chain isotype or subtype including IgG1 Fc; and, that the antibody may be human, non-human, chimeric, or humanized (p.9, [0036]). Askgene teaches that the carrier moiety can improve the serum half-life of the cytokine agonist polypeptide and may also target the cytokine agonist polypeptide to a target site in the body, such as a tumor site (p.14, [0061]). Askgene teaches that strategies of forming heterodimers between the two heavy chains/Fc regions are well known: “For example, the two heavy chain polypeptides in the prodrug may form stable heterodimers through "knobs-into-holes" mutations. "Knobs-into-holes" mutations are made to promote the formation of the heterodimers of the antibody heavy chains and are commonly used to make bispecific antibodies. For example, the Fc domain of the antibody may comprise a T366W mutation in the CH3 domain of the "knob chain" and T366S, L368A, and/or Y407V mutations in the CH3 domain of the "hole chain." An additional interchain disulfide bridge between the CH3 domains can also be used, e.g., by introducing a Y349C mutation into the CH3 domain of the "knobs chain" and an E356C or S354C mutation into the CH3 domain of the "hole chain"… In other embodiments, the antibody moiety may comprise Y349C and/or T366W mutations in one of the two CH3 domains, and E356C, T366S, L368A, and/or Y407V mutations in the other CH3 domain. In certain embodiments, the antibody moiety may comprise Y349C and/or T366W mutations in one of the two CH3 domains, and S354C (or E356C), T366S, L368A, and/or Y407V mutations in the other CH3 domain, with the additional Y349C mutation in one CH3 domain and the additional E356C or S354C mutation in the other CH3 domain, forming an interchain disulfide bridge…” (p.15, [0064]).
Askgene teaches that the cytokine moiety and the masking moiety can be fused to the N-terminus or C-terminus of the light chains and/or heavy chains of the antigen-binding moiety (i.e., carrier; p.14, [0063]). Askgene teaches that the cytokine agonist polypeptide can be fused to the C-terminus of one of the heavy chains of an antibody via a (first) linker, and the cytokine’s mask is fused to the C-terminus of the other heavy chain of the antibody through a (second) cleavable peptide linker, wherein the two heavy chains contain mutations that allow the specific pairing of the two different heavy chains (i.e., association of a first half-life extension domain with a second half-life extension domain; p.15, [0065]).
Askgene additionally teaches that the cytokine moiety polypeptide can be fused to the carrier directly or through a cleavable or noncleavable linker; and, that the masking moiety can be fused to the cytokine or to the carrier through a cleavable peptide linker (p.12, [0050]).
Collectively, Askgene teaches a method of treating cancer in a subject by administering a masked cytokine comprising a protein heterodimer of a first polypeptide with a first half-life extension domain (instant “HL1”) linked to a masking moiety (instant “MM”) via a first linker (“L1”) and a second polypeptide with a second half-life extension domain (instant “HL2”) linked, via a second linker (instant “L2”) to a heterodimeric cytokine (instant “C”) comprised of two subunits covalently joined via a linker; and, wherein the first and second half-life extension domains associate with one another (instant claim 128); wherein the linker joining the two subunits of the cytokine is GGGGSGGGGSGGGGS (i.e., instant SEQ ID NO: 3; instant claim 131).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to combine the teachings of Pat’655 with the teachings of Askgene by using the IL-12 masked cytokine comprising a first polypeptide of H-linker-MM wherein the Fc domain comprises S354C and T366W mutations and a second polypeptide of H-linker-MM wherein the Fc domain comprises Y349C, T366S, L368A, and Y407V mutations; and that comprises a cleavable sinker sequence of ISSGLLSGRS (taught by Pat’655), to arrive at the instantly claimed invention, because the combination of prior art teachings results in a predictable result and benefit of using the masked IL-12 cytokine in a method of treating solid cancer/tumors. One of ordinary skill in the art would have a reasonable expectation of success because both Pat’655 and Askgene teach IL-12 masked cytokines that comprise IgG1 Fc half-life extension domains that comprise “knobs-in-holes” mutations to facilitate interaction of the two Fc polypeptides.
The combination of Pat’655/Askgene does not teach a method of cancer treatment wherein the masked cytokine is a masked IL-12 cytokine that specifically comprises an IL-12p40 polypeptide covalently linked to an IL-12p35 polypeptide via a linker (instant claim 128); or, that the linker joining the IL-12 subunits is instant SEQ ID NO: 3 of GGGSGGGSGGGS (instant claim 131).
Wojno teaches that IL-12 has an important role for cancer and that expression of IL-12 has shown results of tumor regression and anti-tumor responses; but that there remain concerns about using cytokines as drugs due to side effects (p.861, col.2, paras.1-2). Wojno further teaches that these issues are exemplified in multiple trials that utilized IL-12 as a treatment for different cancers, which revealed significant toxicity and limited efficacy (p.862, col.1, para.1). Wojno also teaches that IL-12 is a heterodimer comprised of two subunits: the α subunit IL-12p35 and the β subunit IL-12p40 (p.851, col.2, para.2). Wojno further teaches that the IL-12 heterodimer utilizes a heterodimeric receptor complex for signaling made up of IL-12Rβ1 and IL-12Rβ2 (p.852, Fig.1 and legend).
Chen teaches fusion protein linkers, including flexible linkers, rigid linkers, and in vivo cleavable linkers (title; sections 3.1 – 3.3). Chen teaches that direct fusion of functional domains without a linker may lead to many undesirable outcomes, including misfolding of the fusion proteins, low yield in protein production, or impaired bioactivity (p.2, para.2). Chen teaches that linkers can be used to join protein drugs to carrier proteins such as antibodies in order to extend plasma half-lives and to achieve enhances therapeutic effects by drug targeting to specific types of cells (p.1, para.1). Chen teaches the use of flexible linkers for recombinant fusion proteins; that flexible linkers are useful when the domains require a certain degree of movement or interaction; that flexible linkers are generally composed of small, non-polar or polar amino acids which provides for flexibility and mobility of connecting functional domains; and that the most commonly used flexible linkers consist of stretches of glycine and serine residues (i.e., “GS linkers”) that have the sequence of (Gly-Gly-Gly-Gly-Ser)n (p.4, para.4).
Regarding instant claim 143: It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’655/Askgene with the teachings of Wojno by modifying the method of treating a cancer using a masked IL-12 cytokine as taught by Pat’655, by using an IL-12 cytokine taught by Wojno in order to arrive at the instantly claimed invention, because the combination of prior art elements according to known methods results in a predictable result and a beneficial outcome of reducing toxicity with administration of an IL-12 cytokine for solid cancer treatment (as taught by Askgene/Wojno).
Regarding instant claim 131: It would have further been obvious to one of ordinary skill in the art to use Askgene’s SEQ ID NO: 49 (noncleavable) covalent cytokine domains linker GGGGSGGGGSGGGGS to covalently join the subunits of the IL-12 heterodimer (taught by Pat’655 and Wojno) in order to tether the p40 and p35 subunits (taught by Wojno) to form a fusion protein (taught by Chen), because the combination of prior art elements according to known methods results in a predictable result of forming a biologically active IL-12 heterodimer for receptor stimulation (taught by Wojno) for IL-12 mediated cancer treatment (taught by Wojno). One of ordinary skill in the art would be motivated to do so because Chen teaches that flexible GS linkers of the formula GGGGSn provide a benefit of joining protein domains to facilitate interaction between the domains and one would have a reasonable expectation of success because Askgene’s SEQ ID NO: 49 linker is a GGGGSn linker.
Regarding the first and second half-life extension domains, it would have been prima facie obvious for one of ordinary skill in the art to further combine the teachings of Pat’655/Askgene by using a “knobs-in-holes” carrier comprised of two Fc polypeptides comprising the “knob” mutations and “holes” mutations, in order to arrive at the instantly claimed invention, because the combination of prior art elements taught by Askgene teaches that these mutations facilitate association of the two Fc chains to facilitate dimerization. One would be motivated to do so because this would produce a “carrier” domain that would provide the benefits of extended half-life for a masked cytokine “prodrug” for solid cancer treatment. One of ordinary skill in the art would have a reasonable expectation of success because Askgene teaches the specific amino residue mutations at amino acid positions of the instant claims.
Regarding bioactivation of the masked IL-12 cytokine, Askgene specifically teaches that the cytokine moiety polypeptide can be fused to the carrier directly or through a cleavable or noncleavable linker; and, that the masking moiety can be fused to the cytokine or to the carrier through a cleavable peptide linker (p.12, [0050]); and, that the cytokine is activated by cleavage of the linker joined to the masking moiety. Pat’655/Askgene does not specifically teach an embodiment wherein it is explicitly stated that the cytokine’s linker is noncleavable and that the masking moiety’s linker is cleavable. It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’655/Askgene to make the cytokine’s linker cleavable and the masking moiety’s linker noncleavable, in order to arrive at the instantly claimed invention, because the combination of prior art elements results in a predictable result of cleavage of either the cytokine or the masking moiety in order to facilitate dissociation of the cytokine from the masking moiety for bioactivation. The decision to cleave the cytokine from the “prodrug” would be obvious to try given that the ultimate goal is to produce a cleavage that would release the cytokine from the masking moiety. One of ordinary skill in the art would have a reasonable expectation of success because making the cytokine’s linker cleavable or the masking moiety’s linker cleavable would predictably result in dissociation of the cytokine and masking moiety and would make the cytokine available for receptor binding and activation in order to treat the solid tumor.
Regarding instant claim 129: Merck teaches IL-12p40 amino acid SEQ ID NO: 7 which aligns with instant SEQ ID NO: 60 with 99.7% identity wherein the only variation from applicant’s sequence is that Merck’s SEQ ID NO: 7 retains a cysteine at position 252 (see alignment below):
[AltContent: textbox (Instant SEQ: 60 (IL-12p40) vs Merck (IL-12p40)
[img-media_image3.png])]
Thus, Askgene/Wojno/Chen and Merck do not teach that the IL-12p40 subunit comprises a C252S mutation.
Xencor teaches that IL-12 is a promising cytokine for cancer treatment, but that it has faced hurdles in clinical trials due to toxicity (p.1, [0003]). IL-12/Fc fusion proteins comprising both IL-12p40 and IL-12p35 subunits linked to half-life extension domains for cancer treatment, including several variants wherein the IL-12p40 subunit comprises mutations that modulate receptor affinity and potency and that removes free cysteines that may bond with other free cysteines leading to heterogeneity and undesirable immunogenicity, including an IL-12p40 variant comprising a C252S mutation for the purpose of removing the C252 free cysteine (p.6, [0011]; p.14, [0041]; p.15, [0042 – 0043]; p.151, [00477 – 00479]; p.277 –278, [00881]). Xencor teaches that the C252S mutation can be used alone or in combination with any other IL-12p40 variants (p.278, [00881]). Xencor further teaches that IL-12p40 variants comprising the C252S mutation exhibited improved potency compared to variants without the mutation (p.151, [00479]; p.193, Fig.67A-D).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’655/Askgene/Wojno/ Chen with the teachings of Merck and Xencor by using Merck’s SEQ ID NO: 7 that encodes the IL-12p40 subunit and to modify the IL-12p40 subunit to have a C252S mutation (taught by Xencor), in order to arrive at the instantly claimed invention because the combination of prior art elements results in a predictable result of encoding a IL-12p40 polypeptide that has a C252S mutation. Further, one would be motivated to mutate C252S in order to receive the expected benefit (as taught by Xencor) that the C252S mutation would remove a free cysteine from the IL-12p40 subunit to reduce heterogeneity and immunogenicity, and to improve potency of the IL-12 cytokine (as taught by Xencor) for the IL-12p40 subunit for the masked IL-12 cytokine in a method of treating cancer (as taught by Pat’655/Askgene/Wojno/Chen). One of ordinary skill in the art would have a reasonable expectation of success because the combination of Merck and Xencor teach the amino acid sequence for an improved IL-12p40 cytokine; Pat’655 and Askgene teach masked cytokines; Pat’655 and Wojno teach the use of IL-12 for cancer treatment; and, Pat’655/Askgene/Wojno/Chen and Xencor teach fusion proteins comprising IL-12p40 and IL-12p35 subunits as well as their linkage to half-life extension domains.
[AltContent: textbox (Instant SEQ: 2 (IL-12p35) vs UniProt (IL-12A P29459)
[img-media_image4.png])]Regarding instant claim 130: UP-IL12A teaches P29459: IL-12A, which is the human sequence for the IL-12 p35 subunit and instant SEQ ID NO: 2, at 100% identity (p.5; see alignment below):
UP-IL12A also teaches that IL12A (i.e., IL-12p35) heterodimerizes with IL12B(i.e., IL-12p40) to form the IL-12 cytokine to exert its biological effects through the IL-12 receptor subunits (p.1).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’655/Askgene/Wojno/ Chen with the teachings of UP-IL12A by using UP-IL12A’s mature “P29549 IL-12A” amino acid sequence that encodes the IL-12p35 subunit, in order to arrive at the instantly claimed invention because the combination of prior art elements results in a predictable result of encoding the a human IL-12p35 polypeptide for the masked IL-12 cytokine in a method of treating cancer (as taught by Pat’655/Askgene/Wojno/Chen). One of ordinary skill would have a reasonable expectation of success because Wojno and UP-IL12A both teach that the functional IL-12 cytokine is comprised of the IL-12B/IL-12p40 and IL-12A/IL-12p35 subunits.
Regarding instant claim 132: Instant SEQ ID NO: 64 (as recited in instant claim 132) is an amino acid sequence that encodes for a fusion protein comprising an IL-12p40 subunit joined to an IL-12p35 subunit via the flexible “GS linker” GGGGSGGGGSGGGGS (i.e., instant SEQ ID NOs: 60 (p40) –3 (linker) – 2 (p35); see sequence fusion below):
[AltContent: textbox (Instant SEQ: 64 vs fusion of instant SEQ: 60 (p40)/SEQ:3 (linker)/SEQ: 2 (p35)
[img-media_image5.png])]
The combination of Pat’655, Askgene, Wojno, and Chen teaches a method of using an IL-12 masked cytokine wherein a polypeptide chain is comprised of an Fc half-life extension domain fused to a IL-12 cytokine via a cleavable linker, wherein the IL-12 cytokine is comprised of a p40 subunit linked to the p35 subunit using the flexible fusion protein linker GGGGSGGGGSGGGGS, as described in detail above for claim 128.
The combination of Askgene/Wojno/Chen, Merck, and Xencor teaches the IL-12p40 subunit for the IL-12 cytokine for the masked cytokine as described in detail above for claim 129.
The combination of Askgene/Wojno/Chen and UP-IL12A teaches the IL-12p35 subunit of the IL-12 cytokine for the masked cytokine as described in detail for claim 130 above.
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’655/Askgene/Wojno/ Chen with the teachings of Merck/Xencor and the teachings of UP-IL12A by using the flexible GS linker GGGGSGGGGSGGGGS (as taught by Askgene/Chen) to join the p40 and p35 subunits to produce a functional IL-12 heterodimeric cytokine (as taught by Wojno, Xencor, and UP-IL12A) for the IL-12 cytokine for a masked IL-12 cytokine (as taught by Pat’655/Askgene/Wojno/Chen) for IL-12-mediated cancer treatment (as taught by Askgene/Wojno/Chen and Xencor). Further, one would be motivated to link the p40 and p35 subunits using the flexible GS linker, as described in detail above, to facilitate their interaction (as taught by Chen). One of ordinary skill in the art would have a reasonable expectation of success because the combination of prior art elements results in the predictable result of producing a biologically functional IL-12 cytokine and because the p40, flexible linker, and p35 sequences are all taught by the prior art as described above.
Regarding instant claim 134: Jounce teaches antibodies and fusion proteins, wherein the antibody fragment can be an Fc region that is linked to another protein via a linker; wherein the conjugate a specific embodiment has the formula of a drug-linker-antibody conjugate where the drug is a cytotoxic agent that can be a cytokine (p.65, [0245]; p.65, [0242]); and, wherein the linker is a cleavable or noncleavable linker (p.65; [0245]); and, wherein the conjugate can be used in a method of treating cancer, including solid tumors (p.75, [0285]). Jounce also specifically teaches SEQ ID NO: 3 of SGGSGGGSG which is “GS linker” that comprises instant SEQ ID NO: 14 of GGSGGGSG (p.165; Table of Certain Sequences: SEQ ID NO: 3).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’655/Askgene/Wojno/ Chen with the teachings of Jounce by modifying the first polypeptide comprising a first half-life extension domain (HL1), a first linker (L1), and a masking moiety (MM) as for a masked IL-12 cytokine (as taught by Pat’655/Askgene/Wojno/Chen) by using noncleavable linker SGGSGGGSG, as taught by Jounce, to join the masking moiety to the Fc half-life extension domain, to arrive at the instantly claimed invention, because the combination of prior art elements would result in the predictable result of producing an polypeptide where a masking moiety is linked covalently to an Fc half-life extension domain. One of ordinary skill in the art would be motivated to do so because Chen teaches flexible “GS linkers” are used to join functional proteins to create fusion proteins. One of ordinary skill would also have a reasonable expectation of success because Pat’655, Askgene, Chen, and Jounce teach fusion of functional proteins using flexible GS linkers.
Regarding instant claim 135: CytomX teaches activatable antibodies and methods of manufacturing activatable antibodies that comprise masking moieties (MM), a cleavable moiety (CM), and an antibody or antigen binding fragment thereof (AB) that binds PDL1 for use in cancer treatment, including solid cancers (p.59, col.54, paras.1-3); wherein, the cleavable moiety (CM) is a polypeptide that is cleavable by a protease and activated in the tumor microenvironment (p.164, col.264, para.1); wherein the activatable antibody has the format MM – CM – AB or AB – CM – MM; and, wherein prior to cleavage the MM interferes with binding and when cleaved the MM no longer interferes with binding (p59, col.53, para.1). Further, CytomX specifically teaches multiple cleavable linker sequences comprising instant SEQ ID NO: 44 of ISSGLLSGRS; for example, see CytomX SEQ ID NO: 377, 394, 901-911 (p.50, cols.35 –36 through p.51, col.37; p.158 -- 159, Table 18).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’655/Askgene/Wojno/ Chen with the teachings of CytomX by modifying the IL-12 masked cytokine (as taught by Pat’655/Askgene/Wojno/Chen) to use a cleavable linker comprising ISSGLLSGRS (as taught by CytomX) in order to activate a fusion protein drug for the treatment of solid cancers (as taught by Askgene and CytomX). One of ordinary skill in the art would be motivated to do so because Pat’655, Askgene and CytomX teach activatable drugs conjugated to antigen-binding fragments wherein the drug is activated via cleavage of a protease-cleavable linker in the tumor microenvironment. One of ordinary skill in the art would have a reasonable expectation of success because CytomX teaches various cleavable amino acid sequences comprising the cleavable polypeptide sequence of ISSGLLSGRS.
As discussed in detail above for instant claim 128, regarding bioactivation of the masked IL-12 cytokine, Askgene specifically teaches that the cytokine moiety polypeptide can be fused to the carrier directly or through a cleavable or noncleavable linker; and, that the masking moiety can be fused to the cytokine or to the carrier through a cleavable peptide linker (p.12, [0050]); and, that the cytokine is activated by cleavage of the linker joined to the masking moiety. Askgene does not specifically teach an embodiment wherein it is explicitly stated that the cytokine’s linker is noncleavable and that the masking moiety’s linker is cleavable. It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Askgene (along with the teachings of CytomX) to make the cytokine’s linker cleavable and the masking moiety’s linker noncleavable, in order to arrive at the instantly claimed invention, because the combination of prior art elements results in a predictable result of cleavage of either the cytokine or the masking moiety in order to facilitate dissociation of the cytokine from the masking moiety for bioactivation. The decision to cleave the cytokine from the “prodrug” would be obvious to try given that the ultimate goal is to produce a cleavage that would release the cytokine from the masking moiety. One of ordinary skill in the art would have a reasonable expectation of success because making the cytokine’s linker cleavable or the masking moiety’s linker cleavable would predictably result in dissociation of the cytokine and masking moiety and would make the cytokine available for receptor binding and activation in order to treat the solid tumor.
[AltContent: textbox (Instant SEQ: 22 vs instant SEQ: 25 (HL1)
[img-media_image6.png])] Regarding instant claims 139-140: In addition to the HL1 and HL2 “knobs-in-holes” mutations taught by Askgene above (i.e., Y349C, T366S, L368A, Y407V, S354C, and T366W), instant SEQ ID NO: 25 of instant claim 139 and SEQ ID NO: 26 of instant claim 140 harbor a N>A mutation compared to Fc SEQ ID NO: 22 (see arrows in alignments below):
[AltContent: textbox (Instant SEQ: 22 vs instant SEQ: 26 (HL2)
[img-media_image7.png])]Thus, the combination of Askgene/Wojno/Chen does not teach an IgG1 Fc sequence (i.e., a backbone sequence) comprising the N>A mutation that, with the “knobs-in-holes” mutation of Askgene, would arrive at a first Fc domain of instant SEQ ID NO: 25 and a second Fc domain of instant SEQ ID NO: 26.
UP-IGG1 teaches the human IgG1 human immunoglobulin gamma-1 heavy chain amino acid sequence P0DOX5 (p.4), which provides the backbone sequence for human IgG1 Fc, except for the “knobs-in-holes” mutations taught by Askgene, and the N>A mutation.
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’655/Askgene/Wojno/ Chen with the teachings of UP-IGG1 by using the human IgG1 Fc taught by UP-IGG1 to encode the human IgG1 Fc half-life extension domains and to modify the first and second half-life extension Fc domains to include the “knobs-in-holes” mutations taught by Pat’655 and Askgene, to arrive at a first Fc half-life extension domain and a second half-life extension domain that arrives at the HL1 and HL2 domains of instant claims 128, 139, and 140 except for the N>A mutations, because the combination of prior art elements teaches the IgG1 sequence and in order to achieve the expected benefit of the “knobs-in-holes”-mediated association between the two half-life extension domains (taught by Askgene) for the IL-12 masked cytokine for cancer treatment (taught by Pat’655/Askgene/Wojno/Chen). One of ordinary skill in the art would have a reasonable expectation of success because Askgene teaches that the half-life extension domains can be derived from human IgG1 Fc regions, UP-IgG1 teaches the human IgG1 Fc amino acid sequence, and Askgene teaches the specific mutations and positions of the “knobs-in-holes” mutations.
Pat’655/Askgene/Wojno/Chen and UP-IgG1 do not teach a human IgG1 Fc sequence that comprises the N>A mutation (as it corresponds to instant SEQ ID NO: 22, and amino acid position 77 of the above alignment).
Wang teaches mutations introduced into Fc domains to eliminate FcγR binding in order to avoid or reduce antigen-independent cytokine release syndrome (CRS; p.2013 – 2014). Wang also teaches that human IgG contains a single conserved site in the CH2 domain of Fc, Asn297, for N-linked glycosylation and that glycans at this site determine the interaction between IgG Fc domain and Fcγ receptors. Wang also teaches that the N297A mutation removes the N-glycosylation, thereby preventing binding to Fcγ receptors (p.2013, col.2, para.2).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’655/Askgene/Wojno/ Chen and UP-IGG1 with the teachings of Wang by modifying the human IgG1 Fc half-life extension domains to comprise the N>A mutation (i.e., N297A of full IgG1 in instant SEQ ID NO: 22) as taught by Wang, in order to arrive at the instantly claimed invention, in order to receive the expected benefit, as taught by Wang, that the N297A mutation prevents binding of human IgG1 Fc to FcγRs to avoid or reduce CRS with administration of the IL-12 masked cytokine that comprises Fc half-life extension domains. One of ordinary skill in the art would have a reasonable expectation of success because UP-IGG1 teaches the human IgG1 sequence and Wang teaches the position of the N>A mutation.
US11053294B2
Claims 128-132, 134-135, 137-140, and 143 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-15 of U.S. Patent No. US11053294B2 (herein referred to as Pat’294); and further in view of Askgene, Wojno, Chen, Merck, Xencor, UP-IL12A, Jounce, CytomX, UP-IGG1, and Wang.
Pat’294 teaches a masked IL-2 cytokine having a first Fc domain and C; and, a second Fc domain and a masking moiety (Pat’294 claims 1-5 and 8-11; instant claim 128).
Pat’294 does not teach a method of treating cancer comprising administering an IL-12 cytokine (instant claims 128) with first and second half-life extension domains comprising “knobs-in-holes” mutations (instant claims 137-138); a method wherein the IL-12 cytokine comprises an amino acid sequence that encodes an IL-12p40 polypeptide of instant SEQ ID NO: 60 (instant claim 129) that is linked to an IL-12035 polypeptide of instant SEQ ID NO: 2 (instant claim 130) via a linker of amino acid instant SEQ ID NO: 3 (instant claim 131); that the IL-12 cytokine comprises an amino acid sequence of SEQ ID NO: 64 (instant claim 132); that the first linker comprises a noncleavable linker of SEQ ID NO: 14 (instant claim 134); that the first and second Fc domains comprise an additional N>A mutation (instant claims 139 and 140, respectively); or, a method wherein the cancer is a solid tumor (instant claim 142).
Askgene teaches novel cytokine muteins and prodrugs and methods of making and using thereof, including using the cytokine muteins and prodrugs in a method of treating cancer or an infectious disease, or stimulating the immune system in a patient (title; abstract). The method of treating cancer includes treatment of solid cancer/tumors, including breast cancer, lung cancer, pancreatic cancer, esophageal cancer, medullary thyroid cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, colorectal cancer, and stomach cancer solid tumors (p.6, [0021]; p.27, [0106]). Specifically, Askgene teaches cytokine prodrugs wherein the cytokines are “masked” and metabolized in vivo, via cleavage, to become activated therapeutics (p.4, [0008]). Askgene teaches that such cytokine prodrugs provide benefits of fewer side effects, better in vivo pharmacokinetic profiles/longer half-life, and better target specificity; and, are thus, more efficacious as compared to prior cytokine therapeutics (p.11, [0046]). Askgene teaches that the prodrugs comprise a cytokine agonist moiety, a carrier moiety that can be an antigen-binding moiety/antibody for binding a target site, and a masking moiety (i.e., “cytokine antagonist”) that can be an extracellular domain (ECD) of a receptor for the cytokine wherein the receptor ECD inhibits the cytokine moiety’s biological functions while the receptor mask is binding to it (p.11, [0046]; p.13, [0057]). Askgene teaches that the prodrugs can be activated at a target site such as a tumor site in a patient by cleavage of the linker and consequent release of the cytokine mask from the prodrug, which exposes the previously masked cytokine moiety and allowing the cytokine to bind to its receptor on a target cell and exert its biological functions on the target cell (p.11, [0046]).
Askgene teaches prodrugs that are pro-inflammatory cytokine prodrugs (p.11, [0047]). Askgene teaches specific embodiments for IL-2 and IL-15 prodrugs, but also teaches that prodrugs for other cytokines, and in particular cytokines that are potent immune regulators and have strong side effects, are also contemplated and can be made according to the same principles as for IL-2 and IL-15 (p.11, [0048]). Askgene teaches IL-2 and IL-15 prodrugs wherein the masking moiety comprises an IL-2 or IL-15 receptor extracellular domain of their IL-2Rβ or IL-2Rγ receptors (p.13 – 14, [0058]). Askgene additionally teaches several linker sequences, including SEQ ID NO: 49 (i.e., GGGGSGGGGSGGGGS).
Askgene teaches that the carrier moiety can an antibody or antigen-binding fragment thereof that is a full-length antibody with two heavy chains and two light chains, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a disulfide linked Fv fragment, a single domain antibody, a nanobody, or a single chain variable fragment (scFv); and that the antigen-binding moiety can provide additional and potentially synergetic therapeutic efficacy to the cytokine (p.14, [0062]). Askgene teaches that the antibody may be of any heavy chain isotype or subtype including IgG1 Fc; and, that the antibody may be human, non-human, chimeric, or humanized (p.9, [0036]). Askgene teaches that the carrier moiety can improve the serum half-life of the cytokine agonist polypeptide and may also target the cytokine agonist polypeptide to a target site in the body, such as a tumor site (p.14, [0061]). Askgene teaches that strategies of forming heterodimers between the two heavy chains/Fc regions are well known: “For example, the two heavy chain polypeptides in the prodrug may form stable heterodimers through "knobs-into-holes" mutations. "Knobs-into-holes" mutations are made to promote the formation of the heterodimers of the antibody heavy chains and are commonly used to make bispecific antibodies. For example, the Fc domain of the antibody may comprise a T366W mutation in the CH3 domain of the "knob chain" and T366S, L368A, and/or Y407V mutations in the CH3 domain of the "hole chain." An additional interchain disulfide bridge between the CH3 domains can also be used, e.g., by introducing a Y349C mutation into the CH3 domain of the "knobs chain" and an E356C or S354C mutation into the CH3 domain of the "hole chain"… In other embodiments, the antibody moiety may comprise Y349C and/or T366W mutations in one of the two CH3 domains, and E356C, T366S, L368A, and/or Y407V mutations in the other CH3 domain. In certain embodiments, the antibody moiety may comprise Y349C and/or T366W mutations in one of the two CH3 domains, and S354C (or E356C), T366S, L368A, and/or Y407V mutations in the other CH3 domain, with the additional Y349C mutation in one CH3 domain and the additional E356C or S354C mutation in the other CH3 domain, forming an interchain disulfide bridge…” (p.15, [0064]).
Askgene teaches that the cytokine moiety and the masking moiety can be fused to the N-terminus or C-terminus of the light chains and/or heavy chains of the antigen-binding moiety (i.e., carrier; p.14, [0063]). Askgene teaches that the cytokine agonist polypeptide can be fused to the C-terminus of one of the heavy chains of an antibody via a (first) linker, and the cytokine’s mask is fused to the C-terminus of the other heavy chain of the antibody through a (second) cleavable peptide linker, wherein the two heavy chains contain mutations that allow the specific pairing of the two different heavy chains (i.e., association of a first half-life extension domain with a second half-life extension domain; p.15, [0065]).
Askgene additionally teaches that the cytokine moiety polypeptide can be fused to the carrier directly or through a cleavable or noncleavable linker; and, that the masking moiety can be fused to the cytokine or to the carrier through a cleavable peptide linker (p.12, [0050]).
Collectively, Askgene teaches a method of treating cancer in a subject by administering a masked cytokine comprising a protein heterodimer of a first polypeptide with a first half-life extension domain (instant “HL1”) linked to a masking moiety (instant “MM”) via a first linker (“L1”) and a second polypeptide with a second half-life extension domain (instant “HL2”) linked, via a second linker (instant “L2”) to a heterodimeric cytokine (instant “C”) comprised of two subunits covalently joined via a linker; and, wherein the first and second half-life extension domains associate with one another (instant claim 128); wherein the linker joining the two subunits of the cytokine is GGGGSGGGGSGGGGS (i.e., instant SEQ ID NO: 3; instant claim 131).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to combine the teachings of Pat’294 with the teachings of Askgene by using the IL-12 masked cytokine comprising a first polypeptide of H-linker-MM wherein the Fc domain comprises S354C and T366W mutations and a second polypeptide of H-linker-MM wherein the Fc domain comprises Y349C, T366S, L368A, and Y407V mutations; and that comprises a cleavable sinker sequence of ISSGLLSGRS (taught by Pat’294), to arrive at the instantly claimed invention, because the combination of prior art teachings results in a predictable result and benefit of using the masked IL-12 cytokine in a method of treating solid cancer/tumors. One of ordinary skill in the art would have a reasonable expectation of success because both Pat’294 and Askgene teach IL-12 masked cytokines that comprise IgG1 Fc half-life extension domains that comprise “knobs-in-holes” mutations to facilitate interaction of the two Fc polypeptides.
The combination of Pat’294/Askgene does not teach a method of cancer treatment wherein the masked cytokine is a masked IL-12 cytokine that specifically comprises an IL-12p40 polypeptide covalently linked to an IL-12p35 polypeptide via a linker (instant claim 128); or, that the linker joining the IL-12 subunits is instant SEQ ID NO: 3 of GGGSGGGSGGGS (instant claim 131).
Wojno teaches that IL-12 has an important role for cancer and that expression of IL-12 has shown results of tumor regression and anti-tumor responses; but that there remain concerns about using cytokines as drugs due to side effects (p.861, col.2, paras.1-2). Wojno further teaches that these issues are exemplified in multiple trials that utilized IL-12 as a treatment for different cancers, which revealed significant toxicity and limited efficacy (p.862, col.1, para.1). Wojno also teaches that IL-12 is a heterodimer comprised of two subunits: the α subunit IL-12p35 and the β subunit IL-12p40 (p.851, col.2, para.2). Wojno further teaches that the IL-12 heterodimer utilizes a heterodimeric receptor complex for signaling made up of IL-12Rβ1 and IL-12Rβ2 (p.852, Fig.1 and legend).
Chen teaches fusion protein linkers, including flexible linkers, rigid linkers, and in vivo cleavable linkers (title; sections 3.1 – 3.3). Chen teaches that direct fusion of functional domains without a linker may lead to many undesirable outcomes, including misfolding of the fusion proteins, low yield in protein production, or impaired bioactivity (p.2, para.2). Chen teaches that linkers can be used to join protein drugs to carrier proteins such as antibodies in order to extend plasma half-lives and to achieve enhances therapeutic effects by drug targeting to specific types of cells (p.1, para.1). Chen teaches the use of flexible linkers for recombinant fusion proteins; that flexible linkers are useful when the domains require a certain degree of movement or interaction; that flexible linkers are generally composed of small, non-polar or polar amino acids which provides for flexibility and mobility of connecting functional domains; and that the most commonly used flexible linkers consist of stretches of glycine and serine residues (i.e., “GS linkers”) that have the sequence of (Gly-Gly-Gly-Gly-Ser)n (p.4, para.4).
Regarding instant claim 143: It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’294/Askgene with the teachings of Wojno by modifying the method of treating a cancer using a masked IL-12 cytokine as taught by Pat’294, by using an IL-12 cytokine taught by Wojno in order to arrive at the instantly claimed invention, because the combination of prior art elements according to known methods results in a predictable result and a beneficial outcome of reducing toxicity with administration of an IL-12 cytokine for solid cancer treatment (as taught by Askgene/Wojno).
Regarding instant claim 131: It would have further been obvious to one of ordinary skill in the art to use Askgene’s SEQ ID NO: 49 (noncleavable) covalent cytokine domains linker GGGGSGGGGSGGGGS to covalently join the subunits of the IL-12 heterodimer (taught by Pat’294 and Wojno) in order to tether the p40 and p35 subunits (taught by Wojno) to form a fusion protein (taught by Chen), because the combination of prior art elements according to known methods results in a predictable result of forming a biologically active IL-12 heterodimer for receptor stimulation (taught by Wojno) for IL-12 mediated cancer treatment (taught by Wojno). One of ordinary skill in the art would be motivated to do so because Chen teaches that flexible GS linkers of the formula GGGGSn provide a benefit of joining protein domains to facilitate interaction between the domains and one would have a reasonable expectation of success because Askgene’s SEQ ID NO: 49 linker is a GGGGSn linker.
Regarding the first and second half-life extension domains, it would have been prima facie obvious for one of ordinary skill in the art to further combine the teachings of Pat’294/Askgene by using a “knobs-in-holes” carrier comprised of two Fc polypeptides comprising the “knob” mutations and “holes” mutations, in order to arrive at the instantly claimed invention, because the combination of prior art elements taught by Askgene teaches that these mutations facilitate association of the two Fc chains to facilitate dimerization. One would be motivated to do so because this would produce a “carrier” domain that would provide the benefits of extended half-life for a masked cytokine “prodrug” for solid cancer treatment. One of ordinary skill in the art would have a reasonable expectation of success because Askgene teaches the specific amino residue mutations at amino acid positions of the instant claims.
Regarding bioactivation of the masked IL-12 cytokine, Askgene specifically teaches that the cytokine moiety polypeptide can be fused to the carrier directly or through a cleavable or noncleavable linker; and, that the masking moiety can be fused to the cytokine or to the carrier through a cleavable peptide linker (p.12, [0050]); and, that the cytokine is activated by cleavage of the linker joined to the masking moiety. Pat’294/Askgene does not specifically teach an embodiment wherein it is explicitly stated that the cytokine’s linker is noncleavable and that the masking moiety’s linker is cleavable. It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’294/Askgene to make the cytokine’s linker cleavable and the masking moiety’s linker noncleavable, in order to arrive at the instantly claimed invention, because the combination of prior art elements results in a predictable result of cleavage of either the cytokine or the masking moiety in order to facilitate dissociation of the cytokine from the masking moiety for bioactivation. The decision to cleave the cytokine from the “prodrug” would be obvious to try given that the ultimate goal is to produce a cleavage that would release the cytokine from the masking moiety. One of ordinary skill in the art would have a reasonable expectation of success because making the cytokine’s linker cleavable or the masking moiety’s linker cleavable would predictably result in dissociation of the cytokine and masking moiety and would make the cytokine available for receptor binding and activation in order to treat the solid tumor.
[AltContent: textbox (Instant SEQ: 60 (IL-12p40) vs Merck (IL-12p40)
[img-media_image3.png])]Regarding instant claim 129: Merck teaches IL-12p40 amino acid SEQ ID NO: 7 which aligns with instant SEQ ID NO: 60 with 99.7% identity wherein the only variation from applicant’s sequence is that Merck’s SEQ ID NO: 7 retains a cysteine at position 252 (see alignment below):
Thus, Askgene/Wojno/Chen and Merck do not teach that the IL-12p40 subunit comprises a C252S mutation.
Xencor teaches that IL-12 is a promising cytokine for cancer treatment, but that it has faced hurdles in clinical trials due to toxicity (p.1, [0003]). IL-12/Fc fusion proteins comprising both IL-12p40 and IL-12p35 subunits linked to half-life extension domains for cancer treatment, including several variants wherein the IL-12p40 subunit comprises mutations that modulate receptor affinity and potency and that removes free cysteines that may bond with other free cysteines leading to heterogeneity and undesirable immunogenicity, including an IL-12p40 variant comprising a C252S mutation for the purpose of removing the C252 free cysteine (p.6, [0011]; p.14, [0041]; p.15, [0042 – 0043]; p.151, [00477 – 00479]; p.277 –278, [00881]). Xencor teaches that the C252S mutation can be used alone or in combination with any other IL-12p40 variants (p.278, [00881]). Xencor further teaches that IL-12p40 variants comprising the C252S mutation exhibited improved potency compared to variants without the mutation (p.151, [00479]; p.193, Fig.67A-D).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’294/Askgene/Wojno/ Chen with the teachings of Merck and Xencor by using Merck’s SEQ ID NO: 7 that encodes the IL-12p40 subunit and to modify the IL-12p40 subunit to have a C252S mutation (taught by Xencor), in order to arrive at the instantly claimed invention because the combination of prior art elements results in a predictable result of encoding a IL-12p40 polypeptide that has a C252S mutation. Further, one would be motivated to mutate C252S in order to receive the expected benefit (as taught by Xencor) that the C252S mutation would remove a free cysteine from the IL-12p40 subunit to reduce heterogeneity and immunogenicity, and to improve potency of the IL-12 cytokine (as taught by Xencor) for the IL-12p40 subunit for the masked IL-12 cytokine in a method of treating cancer (as taught by Pat’294/Askgene/Wojno/Chen). One of ordinary skill in the art would have a reasonable expectation of success because the combination of Merck and Xencor teach the amino acid sequence for an improved IL-12p40 cytokine; Pat’294 and Askgene teach masked cytokines; Pat’294 and Wojno teach the use of IL-12 for cancer treatment; and, Pat’294/Askgene/Wojno/Chen and Xencor teach fusion proteins comprising IL-12p40 and IL-12p35 subunits as well as their linkage to half-life extension domains.
[AltContent: textbox (Instant SEQ: 2 (IL-12p35) vs UniProt (IL-12A P29459)
[img-media_image4.png])]Regarding instant claim 130: UP-IL12A teaches P29459: IL-12A, which is the human sequence for the IL-12 p35 subunit and instant SEQ ID NO: 2, at 100% identity (p.5; see alignment below):
UP-IL12A also teaches that IL12A (i.e., IL-12p35) heterodimerizes with IL12B(i.e., IL-12p40) to form the IL-12 cytokine to exert its biological effects through the IL-12 receptor subunits (p.1).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’294/Askgene/Wojno/ Chen with the teachings of UP-IL12A by using UP-IL12A’s mature “P29549 IL-12A” amino acid sequence that encodes the IL-12p35 subunit, in order to arrive at the instantly claimed invention because the combination of prior art elements results in a predictable result of encoding the a human IL-12p35 polypeptide for the masked IL-12 cytokine in a method of treating cancer (as taught by Pat’294/Askgene/Wojno/Chen). One of ordinary skill would have a reasonable expectation of success because Wojno and UP-IL12A both teach that the functional IL-12 cytokine is comprised of the IL-12B/IL-12p40 and IL-12A/IL-12p35 subunits.
Regarding instant claim 132: Instant SEQ ID NO: 64 (as recited in instant claim 132) is an amino acid sequence that encodes for a fusion protein comprising an IL-12p40 subunit joined to an IL-12p35 subunit via the flexible “GS linker” GGGGSGGGGSGGGGS (i.e., instant SEQ ID NOs: 60 (p40) –3 (linker) – 2 (p35); see sequence fusion below):
[AltContent: textbox (Instant SEQ: 64 vs fusion of instant SEQ: 60 (p40)/SEQ:3 (linker)/SEQ: 2 (p35)
[img-media_image5.png])]
The combination of Pat’294, Askgene, Wojno, and Chen teaches a method of using an IL-12 masked cytokine wherein a polypeptide chain is comprised of an Fc half-life extension domain fused to a IL-12 cytokine via a cleavable linker, wherein the IL-12 cytokine is comprised of a p40 subunit linked to the p35 subunit using the flexible fusion protein linker GGGGSGGGGSGGGGS, as described in detail above for claim 128.
The combination of Askgene/Wojno/Chen, Merck, and Xencor teaches the IL-12p40 subunit for the IL-12 cytokine for the masked cytokine as described in detail above for claim 129.
The combination of Askgene/Wojno/Chen and UP-IL12A teaches the IL-12p35 subunit of the IL-12 cytokine for the masked cytokine as described in detail for claim 130 above.
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’294/Askgene/Wojno/ Chen with the teachings of Merck/Xencor and the teachings of UP-IL12A by using the flexible GS linker GGGGSGGGGSGGGGS (as taught by Askgene/Chen) to join the p40 and p35 subunits to produce a functional IL-12 heterodimeric cytokine (as taught by Wojno, Xencor, and UP-IL12A) for the IL-12 cytokine for a masked IL-12 cytokine (as taught by Pat’294/Askgene/Wojno/Chen) for IL-12-mediated cancer treatment (as taught by Askgene/Wojno/Chen and Xencor). Further, one would be motivated to link the p40 and p35 subunits using the flexible GS linker, as described in detail above, to facilitate their interaction (as taught by Chen). One of ordinary skill in the art would have a reasonable expectation of success because the combination of prior art elements results in the predictable result of producing a biologically functional IL-12 cytokine and because the p40, flexible linker, and p35 sequences are all taught by the prior art as described above.
Regarding instant claim 134: Jounce teaches antibodies and fusion proteins, wherein the antibody fragment can be an Fc region that is linked to another protein via a linker; wherein the conjugate a specific embodiment has the formula of a drug-linker-antibody conjugate where the drug is a cytotoxic agent that can be a cytokine (p.65, [0245]; p.65, [0242]); and, wherein the linker is a cleavable or noncleavable linker (p.65; [0245]); and, wherein the conjugate can be used in a method of treating cancer, including solid tumors (p.75, [0285]). Jounce also specifically teaches SEQ ID NO: 3 of SGGSGGGSG which is “GS linker” that comprises instant SEQ ID NO: 14 of GGSGGGSG (p.165; Table of Certain Sequences: SEQ ID NO: 3).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’294/Askgene/Wojno/ Chen with the teachings of Jounce by modifying the first polypeptide comprising a first half-life extension domain (HL1), a first linker (L1), and a masking moiety (MM) as for a masked IL-12 cytokine (as taught by Pat’294/Askgene/Wojno/Chen) by using noncleavable linker SGGSGGGSG, as taught by Jounce, to join the masking moiety to the Fc half-life extension domain, to arrive at the instantly claimed invention, because the combination of prior art elements would result in the predictable result of producing an polypeptide where a masking moiety is linked covalently to an Fc half-life extension domain. One of ordinary skill in the art would be motivated to do so because Chen teaches flexible “GS linkers” are used to join functional proteins to create fusion proteins. One of ordinary skill would also have a reasonable expectation of success because Pat’294, Askgene, Chen, and Jounce teach fusion of functional proteins using flexible GS linkers.
Regarding instant claim 135: CytomX teaches activatable antibodies and methods of manufacturing activatable antibodies that comprise masking moieties (MM), a cleavable moiety (CM), and an antibody or antigen binding fragment thereof (AB) that binds PDL1 for use in cancer treatment, including solid cancers (p.59, col.54, paras.1-3); wherein, the cleavable moiety (CM) is a polypeptide that is cleavable by a protease and activated in the tumor microenvironment (p.164, col.264, para.1); wherein the activatable antibody has the format MM – CM – AB or AB – CM – MM; and, wherein prior to cleavage the MM interferes with binding and when cleaved the MM no longer interferes with binding (p59, col.53, para.1). Further, CytomX specifically teaches multiple cleavable linker sequences comprising instant SEQ ID NO: 44 of ISSGLLSGRS; for example, see CytomX SEQ ID NO: 377, 394, 901-911 (p.50, cols.35 –36 through p.51, col.37; p.158 -- 159, Table 18).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’294/Askgene/Wojno/ Chen with the teachings of CytomX by modifying the IL-12 masked cytokine (as taught by Pat’294/Askgene/Wojno/Chen) to use a cleavable linker comprising ISSGLLSGRS (as taught by CytomX) in order to activate a fusion protein drug for the treatment of solid cancers (as taught by Askgene and CytomX). One of ordinary skill in the art would be motivated to do so because Pat’294, Askgene and CytomX teach activatable drugs conjugated to antigen-binding fragments wherein the drug is activated via cleavage of a protease-cleavable linker in the tumor microenvironment. One of ordinary skill in the art would have a reasonable expectation of success because CytomX teaches various cleavable amino acid sequences comprising the cleavable polypeptide sequence of ISSGLLSGRS.
As discussed in detail above for instant claim 128, regarding bioactivation of the masked IL-12 cytokine, Askgene specifically teaches that the cytokine moiety polypeptide can be fused to the carrier directly or through a cleavable or noncleavable linker; and, that the masking moiety can be fused to the cytokine or to the carrier through a cleavable peptide linker (p.12, [0050]); and, that the cytokine is activated by cleavage of the linker joined to the masking moiety. Askgene does not specifically teach an embodiment wherein it is explicitly stated that the cytokine’s linker is noncleavable and that the masking moiety’s linker is cleavable. It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Askgene (along with the teachings of CytomX) to make the cytokine’s linker cleavable and the masking moiety’s linker noncleavable, in order to arrive at the instantly claimed invention, because the combination of prior art elements results in a predictable result of cleavage of either the cytokine or the masking moiety in order to facilitate dissociation of the cytokine from the masking moiety for bioactivation. The decision to cleave the cytokine from the “prodrug” would be obvious to try given that the ultimate goal is to produce a cleavage that would release the cytokine from the masking moiety. One of ordinary skill in the art would have a reasonable expectation of success because making the cytokine’s linker cleavable or the masking moiety’s linker cleavable would predictably result in dissociation of the cytokine and masking moiety and would make the cytokine available for receptor binding and activation in order to treat the solid tumor.
[AltContent: textbox (Instant SEQ: 22 vs instant SEQ: 25 (HL1)
[img-media_image6.png])] Regarding instant claims 139-140: In addition to the HL1 and HL2 “knobs-in-holes” mutations taught by Askgene above (i.e., Y349C, T366S, L368A, Y407V, S354C, and T366W), instant SEQ ID NO: 25 of instant claim 139 and SEQ ID NO: 26 of instant claim 140 harbor a N>A mutation compared to Fc SEQ ID NO: 22 (see arrows in alignments below):
[AltContent: textbox (Instant SEQ: 22 vs instant SEQ: 26 (HL2)
[img-media_image7.png])]Thus, the combination of Askgene/Wojno/Chen does not teach an IgG1 Fc sequence (i.e., a backbone sequence) comprising the N>A mutation that, with the “knobs-in-holes” mutation of Askgene, would arrive at a first Fc domain of instant SEQ ID NO: 25 and a second Fc domain of instant SEQ ID NO: 26.
UP-IGG1 teaches the human IgG1 human immunoglobulin gamma-1 heavy chain amino acid sequence P0DOX5 (p.4), which provides the backbone sequence for human IgG1 Fc, except for the “knobs-in-holes” mutations taught by Askgene, and the N>A mutation.
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’294/Askgene/Wojno/ Chen with the teachings of UP-IGG1 by using the human IgG1 Fc taught by UP-IGG1 to encode the human IgG1 Fc half-life extension domains and to modify the first and second half-life extension Fc domains to include the “knobs-in-holes” mutations taught by Pat’294 and Askgene, to arrive at a first Fc half-life extension domain and a second half-life extension domain that arrives at the HL1 and HL2 domains of instant claims 128, 139, and 140 except for the N>A mutations, because the combination of prior art elements teaches the IgG1 sequence and in order to achieve the expected benefit of the “knobs-in-holes”-mediated association between the two half-life extension domains (taught by Askgene) for the IL-12 masked cytokine for cancer treatment (taught by Pat’294/Askgene/Wojno/Chen). One of ordinary skill in the art would have a reasonable expectation of success because Askgene teaches that the half-life extension domains can be derived from human IgG1 Fc regions, UP-IgG1 teaches the human IgG1 Fc amino acid sequence, and Askgene teaches the specific mutations and positions of the “knobs-in-holes” mutations.
Pat’294/Askgene/Wojno/Chen and UP-IgG1 do not teach a human IgG1 Fc sequence that comprises the N>A mutation (as it corresponds to instant SEQ ID NO: 22, and amino acid position 77 of the above alignment).
Wang teaches mutations introduced into Fc domains to eliminate FcγR binding in order to avoid or reduce antigen-independent cytokine release syndrome (CRS; p.2013 – 2014). Wang also teaches that human IgG contains a single conserved site in the CH2 domain of Fc, Asn297, for N-linked glycosylation and that glycans at this site determine the interaction between IgG Fc domain and Fcγ receptors. Wang also teaches that the N297A mutation removes the N-glycosylation, thereby preventing binding to Fcγ receptors (p.2013, col.2, para.2).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’294/Askgene/Wojno/ Chen and UP-IGG1 with the teachings of Wang by modifying the human IgG1 Fc half-life extension domains to comprise the N>A mutation (i.e., N297A of full IgG1 in instant SEQ ID NO: 22) as taught by Wang, in order to arrive at the instantly claimed invention, in order to receive the expected benefit, as taught by Wang, that the N297A mutation prevents binding of human IgG1 Fc to FcγRs to avoid or reduce CRS with administration of the IL-12 masked cytokine that comprises Fc half-life extension domains. One of ordinary skill in the art would have a reasonable expectation of success because UP-IGG1 teaches the human IgG1 sequence and Wang teaches the position of the N>A mutation.
US11827686B2
Claims 128-132, 134-135, 137-140, and 143 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-9 of U.S. Patent No. US11827686B2 (herein referred to as Pat’686); and further in view of Askgene, Wojno, Chen, Merck, Xencor, UP-IL12A, Jounce, CytomX, UP-IGG1, and Wang.
Pat’686 teaches a masked IL-2 cytokine having a first Fc domain and C; and, a second Fc domain and a masking moiety (Pat’686 claim 1; instant claim 128).
Pat’686 does not teach a method of treating cancer comprising administering an IL-12 cytokine (instant claims 128) with first and second half-life extension domains comprising “knobs-in-holes” mutations (instant claims 137-138); a method wherein the IL-12 cytokine comprises an amino acid sequence that encodes an IL-12p40 polypeptide of instant SEQ ID NO: 60 (instant claim 129) that is linked to an IL-12035 polypeptide of instant SEQ ID NO: 2 (instant claim 130) via a linker of amino acid instant SEQ ID NO: 3 (instant claim 131); that the IL-12 cytokine comprises an amino acid sequence of SEQ ID NO: 64 (instant claim 132); that the first linker comprises a noncleavable linker of SEQ ID NO: 14 (instant claim 134); that the first and second Fc domains comprise an additional N>A mutation (instant claims 139 and 140, respectively); or, a method wherein the cancer is a solid tumor (instant claim 142).
Askgene teaches novel cytokine muteins and prodrugs and methods of making and using thereof, including using the cytokine muteins and prodrugs in a method of treating cancer or an infectious disease, or stimulating the immune system in a patient (title; abstract). The method of treating cancer includes treatment of solid cancer/tumors, including breast cancer, lung cancer, pancreatic cancer, esophageal cancer, medullary thyroid cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, colorectal cancer, and stomach cancer solid tumors (p.6, [0021]; p.27, [0106]). Specifically, Askgene teaches cytokine prodrugs wherein the cytokines are “masked” and metabolized in vivo, via cleavage, to become activated therapeutics (p.4, [0008]). Askgene teaches that such cytokine prodrugs provide benefits of fewer side effects, better in vivo pharmacokinetic profiles/longer half-life, and better target specificity; and, are thus, more efficacious as compared to prior cytokine therapeutics (p.11, [0046]). Askgene teaches that the prodrugs comprise a cytokine agonist moiety, a carrier moiety that can be an antigen-binding moiety/antibody for binding a target site, and a masking moiety (i.e., “cytokine antagonist”) that can be an extracellular domain (ECD) of a receptor for the cytokine wherein the receptor ECD inhibits the cytokine moiety’s biological functions while the receptor mask is binding to it (p.11, [0046]; p.13, [0057]). Askgene teaches that the prodrugs can be activated at a target site such as a tumor site in a patient by cleavage of the linker and consequent release of the cytokine mask from the prodrug, which exposes the previously masked cytokine moiety and allowing the cytokine to bind to its receptor on a target cell and exert its biological functions on the target cell (p.11, [0046]).
Askgene teaches prodrugs that are pro-inflammatory cytokine prodrugs (p.11, [0047]). Askgene teaches specific embodiments for IL-2 and IL-15 prodrugs, but also teaches that prodrugs for other cytokines, and in particular cytokines that are potent immune regulators and have strong side effects, are also contemplated and can be made according to the same principles as for IL-2 and IL-15 (p.11, [0048]). Askgene teaches IL-2 and IL-15 prodrugs wherein the masking moiety comprises an IL-2 or IL-15 receptor extracellular domain of their IL-2Rβ or IL-2Rγ receptors (p.13 – 14, [0058]). Askgene additionally teaches several linker sequences, including SEQ ID NO: 49 (i.e., GGGGSGGGGSGGGGS).
Askgene teaches that the carrier moiety can an antibody or antigen-binding fragment thereof that is a full-length antibody with two heavy chains and two light chains, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a disulfide linked Fv fragment, a single domain antibody, a nanobody, or a single chain variable fragment (scFv); and that the antigen-binding moiety can provide additional and potentially synergetic therapeutic efficacy to the cytokine (p.14, [0062]). Askgene teaches that the antibody may be of any heavy chain isotype or subtype including IgG1 Fc; and, that the antibody may be human, non-human, chimeric, or humanized (p.9, [0036]). Askgene teaches that the carrier moiety can improve the serum half-life of the cytokine agonist polypeptide and may also target the cytokine agonist polypeptide to a target site in the body, such as a tumor site (p.14, [0061]). Askgene teaches that strategies of forming heterodimers between the two heavy chains/Fc regions are well known: “For example, the two heavy chain polypeptides in the prodrug may form stable heterodimers through "knobs-into-holes" mutations. "Knobs-into-holes" mutations are made to promote the formation of the heterodimers of the antibody heavy chains and are commonly used to make bispecific antibodies. For example, the Fc domain of the antibody may comprise a T366W mutation in the CH3 domain of the "knob chain" and T366S, L368A, and/or Y407V mutations in the CH3 domain of the "hole chain." An additional interchain disulfide bridge between the CH3 domains can also be used, e.g., by introducing a Y349C mutation into the CH3 domain of the "knobs chain" and an E356C or S354C mutation into the CH3 domain of the "hole chain"… In other embodiments, the antibody moiety may comprise Y349C and/or T366W mutations in one of the two CH3 domains, and E356C, T366S, L368A, and/or Y407V mutations in the other CH3 domain. In certain embodiments, the antibody moiety may comprise Y349C and/or T366W mutations in one of the two CH3 domains, and S354C (or E356C), T366S, L368A, and/or Y407V mutations in the other CH3 domain, with the additional Y349C mutation in one CH3 domain and the additional E356C or S354C mutation in the other CH3 domain, forming an interchain disulfide bridge…” (p.15, [0064]).
Askgene teaches that the cytokine moiety and the masking moiety can be fused to the N-terminus or C-terminus of the light chains and/or heavy chains of the antigen-binding moiety (i.e., carrier; p.14, [0063]). Askgene teaches that the cytokine agonist polypeptide can be fused to the C-terminus of one of the heavy chains of an antibody via a (first) linker, and the cytokine’s mask is fused to the C-terminus of the other heavy chain of the antibody through a (second) cleavable peptide linker, wherein the two heavy chains contain mutations that allow the specific pairing of the two different heavy chains (i.e., association of a first half-life extension domain with a second half-life extension domain; p.15, [0065]).
Askgene additionally teaches that the cytokine moiety polypeptide can be fused to the carrier directly or through a cleavable or noncleavable linker; and, that the masking moiety can be fused to the cytokine or to the carrier through a cleavable peptide linker (p.12, [0050]).
Collectively, Askgene teaches a method of treating cancer in a subject by administering a masked cytokine comprising a protein heterodimer of a first polypeptide with a first half-life extension domain (instant “HL1”) linked to a masking moiety (instant “MM”) via a first linker (“L1”) and a second polypeptide with a second half-life extension domain (instant “HL2”) linked, via a second linker (instant “L2”) to a heterodimeric cytokine (instant “C”) comprised of two subunits covalently joined via a linker; and, wherein the first and second half-life extension domains associate with one another (instant claim 128); wherein the linker joining the two subunits of the cytokine is GGGGSGGGGSGGGGS (i.e., instant SEQ ID NO: 3; instant claim 131).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to combine the teachings of Pat’686 with the teachings of Askgene by using the IL-12 masked cytokine comprising a first polypeptide of H-linker-MM wherein the Fc domain comprises S354C and T366W mutations and a second polypeptide of H-linker-MM wherein the Fc domain comprises Y349C, T366S, L368A, and Y407V mutations; and that comprises a cleavable sinker sequence of ISSGLLSGRS (taught by Pat’686), to arrive at the instantly claimed invention, because the combination of prior art teachings results in a predictable result and benefit of using the masked IL-12 cytokine in a method of treating solid cancer/tumors. One of ordinary skill in the art would have a reasonable expectation of success because both Pat’686 and Askgene teach IL-12 masked cytokines that comprise IgG1 Fc half-life extension domains that comprise “knobs-in-holes” mutations to facilitate interaction of the two Fc polypeptides.
The combination of Pat’686/Askgene does not teach a method of cancer treatment wherein the masked cytokine is a masked IL-12 cytokine that specifically comprises an IL-12p40 polypeptide covalently linked to an IL-12p35 polypeptide via a linker (instant claim 128); or, that the linker joining the IL-12 subunits is instant SEQ ID NO: 3 of GGGSGGGSGGGS (instant claim 131).
Wojno teaches that IL-12 has an important role for cancer and that expression of IL-12 has shown results of tumor regression and anti-tumor responses; but that there remain concerns about using cytokines as drugs due to side effects (p.861, col.2, paras.1-2). Wojno further teaches that these issues are exemplified in multiple trials that utilized IL-12 as a treatment for different cancers, which revealed significant toxicity and limited efficacy (p.862, col.1, para.1). Wojno also teaches that IL-12 is a heterodimer comprised of two subunits: the α subunit IL-12p35 and the β subunit IL-12p40 (p.851, col.2, para.2). Wojno further teaches that the IL-12 heterodimer utilizes a heterodimeric receptor complex for signaling made up of IL-12Rβ1 and IL-12Rβ2 (p.852, Fig.1 and legend).
Chen teaches fusion protein linkers, including flexible linkers, rigid linkers, and in vivo cleavable linkers (title; sections 3.1 – 3.3). Chen teaches that direct fusion of functional domains without a linker may lead to many undesirable outcomes, including misfolding of the fusion proteins, low yield in protein production, or impaired bioactivity (p.2, para.2). Chen teaches that linkers can be used to join protein drugs to carrier proteins such as antibodies in order to extend plasma half-lives and to achieve enhances therapeutic effects by drug targeting to specific types of cells (p.1, para.1). Chen teaches the use of flexible linkers for recombinant fusion proteins; that flexible linkers are useful when the domains require a certain degree of movement or interaction; that flexible linkers are generally composed of small, non-polar or polar amino acids which provides for flexibility and mobility of connecting functional domains; and that the most commonly used flexible linkers consist of stretches of glycine and serine residues (i.e., “GS linkers”) that have the sequence of (Gly-Gly-Gly-Gly-Ser)n (p.4, para.4).
Regarding instant claim 143: It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’686/Askgene with the teachings of Wojno by modifying the method of treating a cancer using a masked IL-12 cytokine as taught by Pat’686, by using an IL-12 cytokine taught by Wojno in order to arrive at the instantly claimed invention, because the combination of prior art elements according to known methods results in a predictable result and a beneficial outcome of reducing toxicity with administration of an IL-12 cytokine for solid cancer treatment (as taught by Askgene/Wojno).
Regarding instant claim 131: It would have further been obvious to one of ordinary skill in the art to use Askgene’s SEQ ID NO: 49 (noncleavable) covalent cytokine domains linker GGGGSGGGGSGGGGS to covalently join the subunits of the IL-12 heterodimer (taught by Pat’686 and Wojno) in order to tether the p40 and p35 subunits (taught by Wojno) to form a fusion protein (taught by Chen), because the combination of prior art elements according to known methods results in a predictable result of forming a biologically active IL-12 heterodimer for receptor stimulation (taught by Wojno) for IL-12 mediated cancer treatment (taught by Wojno). One of ordinary skill in the art would be motivated to do so because Chen teaches that flexible GS linkers of the formula GGGGSn provide a benefit of joining protein domains to facilitate interaction between the domains and one would have a reasonable expectation of success because Askgene’s SEQ ID NO: 49 linker is a GGGGSn linker.
Regarding the first and second half-life extension domains, it would have been prima facie obvious for one of ordinary skill in the art to further combine the teachings of Pat’686/Askgene by using a “knobs-in-holes” carrier comprised of two Fc polypeptides comprising the “knob” mutations and “holes” mutations, in order to arrive at the instantly claimed invention, because the combination of prior art elements taught by Askgene teaches that these mutations facilitate association of the two Fc chains to facilitate dimerization. One would be motivated to do so because this would produce a “carrier” domain that would provide the benefits of extended half-life for a masked cytokine “prodrug” for solid cancer treatment. One of ordinary skill in the art would have a reasonable expectation of success because Askgene teaches the specific amino residue mutations at amino acid positions of the instant claims.
Regarding bioactivation of the masked IL-12 cytokine, Askgene specifically teaches that the cytokine moiety polypeptide can be fused to the carrier directly or through a cleavable or noncleavable linker; and, that the masking moiety can be fused to the cytokine or to the carrier through a cleavable peptide linker (p.12, [0050]); and, that the cytokine is activated by cleavage of the linker joined to the masking moiety. Pat’686/Askgene does not specifically teach an embodiment wherein it is explicitly stated that the cytokine’s linker is noncleavable and that the masking moiety’s linker is cleavable. It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’686/Askgene to make the cytokine’s linker cleavable and the masking moiety’s linker noncleavable, in order to arrive at the instantly claimed invention, because the combination of prior art elements results in a predictable result of cleavage of either the cytokine or the masking moiety in order to facilitate dissociation of the cytokine from the masking moiety for bioactivation. The decision to cleave the cytokine from the “prodrug” would be obvious to try given that the ultimate goal is to produce a cleavage that would release the cytokine from the masking moiety. One of ordinary skill in the art would have a reasonable expectation of success because making the cytokine’s linker cleavable or the masking moiety’s linker cleavable would predictably result in dissociation of the cytokine and masking moiety and would make the cytokine available for receptor binding and activation in order to treat the solid tumor.
[AltContent: textbox (Instant SEQ: 60 (IL-12p40) vs Merck (IL-12p40)
[img-media_image3.png])]Regarding instant claim 129: Merck teaches IL-12p40 amino acid SEQ ID NO: 7 which aligns with instant SEQ ID NO: 60 with 99.7% identity wherein the only variation from applicant’s sequence is that Merck’s SEQ ID NO: 7 retains a cysteine at position 252 (see alignment below):
Thus, Askgene/Wojno/Chen and Merck do not teach that the IL-12p40 subunit comprises a C252S mutation.
Xencor teaches that IL-12 is a promising cytokine for cancer treatment, but that it has faced hurdles in clinical trials due to toxicity (p.1, [0003]). IL-12/Fc fusion proteins comprising both IL-12p40 and IL-12p35 subunits linked to half-life extension domains for cancer treatment, including several variants wherein the IL-12p40 subunit comprises mutations that modulate receptor affinity and potency and that removes free cysteines that may bond with other free cysteines leading to heterogeneity and undesirable immunogenicity, including an IL-12p40 variant comprising a C252S mutation for the purpose of removing the C252 free cysteine (p.6, [0011]; p.14, [0041]; p.15, [0042 – 0043]; p.151, [00477 – 00479]; p.277 –278, [00881]). Xencor teaches that the C252S mutation can be used alone or in combination with any other IL-12p40 variants (p.278, [00881]). Xencor further teaches that IL-12p40 variants comprising the C252S mutation exhibited improved potency compared to variants without the mutation (p.151, [00479]; p.193, Fig.67A-D).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’686/Askgene/Wojno/ Chen with the teachings of Merck and Xencor by using Merck’s SEQ ID NO: 7 that encodes the IL-12p40 subunit and to modify the IL-12p40 subunit to have a C252S mutation (taught by Xencor), in order to arrive at the instantly claimed invention because the combination of prior art elements results in a predictable result of encoding a IL-12p40 polypeptide that has a C252S mutation. Further, one would be motivated to mutate C252S in order to receive the expected benefit (as taught by Xencor) that the C252S mutation would remove a free cysteine from the IL-12p40 subunit to reduce heterogeneity and immunogenicity, and to improve potency of the IL-12 cytokine (as taught by Xencor) for the IL-12p40 subunit for the masked IL-12 cytokine in a method of treating cancer (as taught by Pat’686/Askgene/Wojno/Chen). One of ordinary skill in the art would have a reasonable expectation of success because the combination of Merck and Xencor teach the amino acid sequence for an improved IL-12p40 cytokine; Pat’686 and Askgene teach masked cytokines; Pat’686 and Wojno teach the use of IL-12 for cancer treatment; and, Pat’686/Askgene/Wojno/Chen and Xencor teach fusion proteins comprising IL-12p40 and IL-12p35 subunits as well as their linkage to half-life extension domains.
[AltContent: textbox (Instant SEQ: 2 (IL-12p35) vs UniProt (IL-12A P29459)
[img-media_image4.png])]Regarding instant claim 130: UP-IL12A teaches P29459: IL-12A, which is the human sequence for the IL-12 p35 subunit and instant SEQ ID NO: 2, at 100% identity (p.5; see alignment below):
UP-IL12A also teaches that IL12A (i.e., IL-12p35) heterodimerizes with IL12B(i.e., IL-12p40) to form the IL-12 cytokine to exert its biological effects through the IL-12 receptor subunits (p.1).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’686/Askgene/Wojno/ Chen with the teachings of UP-IL12A by using UP-IL12A’s mature “P29549 IL-12A” amino acid sequence that encodes the IL-12p35 subunit, in order to arrive at the instantly claimed invention because the combination of prior art elements results in a predictable result of encoding the a human IL-12p35 polypeptide for the masked IL-12 cytokine in a method of treating cancer (as taught by Pat’686/Askgene/Wojno/Chen). One of ordinary skill would have a reasonable expectation of success because Wojno and UP-IL12A both teach that the functional IL-12 cytokine is comprised of the IL-12B/IL-12p40 and IL-12A/IL-12p35 subunits.
Regarding instant claim 132: Instant SEQ ID NO: 64 (as recited in instant claim 132) is an amino acid sequence that encodes for a fusion protein comprising an IL-12p40 subunit joined to an IL-12p35 subunit via the flexible “GS linker” GGGGSGGGGSGGGGS (i.e., instant SEQ ID NOs: 60 (p40) –3 (linker) – 2 (p35); see sequence fusion below):
[AltContent: textbox (Instant SEQ: 64 vs fusion of instant SEQ: 60 (p40)/SEQ:3 (linker)/SEQ: 2 (p35)
[img-media_image5.png])]
The combination of Pat’686, Askgene, Wojno, and Chen teaches a method of using an IL-12 masked cytokine wherein a polypeptide chain is comprised of an Fc half-life extension domain fused to a IL-12 cytokine via a cleavable linker, wherein the IL-12 cytokine is comprised of a p40 subunit linked to the p35 subunit using the flexible fusion protein linker GGGGSGGGGSGGGGS, as described in detail above for claim 128.
The combination of Askgene/Wojno/Chen, Merck, and Xencor teaches the IL-12p40 subunit for the IL-12 cytokine for the masked cytokine as described in detail above for claim 129.
The combination of Askgene/Wojno/Chen and UP-IL12A teaches the IL-12p35 subunit of the IL-12 cytokine for the masked cytokine as described in detail for claim 130 above.
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’686/Askgene/Wojno/ Chen with the teachings of Merck/Xencor and the teachings of UP-IL12A by using the flexible GS linker GGGGSGGGGSGGGGS (as taught by Askgene/Chen) to join the p40 and p35 subunits to produce a functional IL-12 heterodimeric cytokine (as taught by Wojno, Xencor, and UP-IL12A) for the IL-12 cytokine for a masked IL-12 cytokine (as taught by Pat’686/Askgene/Wojno/Chen) for IL-12-mediated cancer treatment (as taught by Askgene/Wojno/Chen and Xencor). Further, one would be motivated to link the p40 and p35 subunits using the flexible GS linker, as described in detail above, to facilitate their interaction (as taught by Chen). One of ordinary skill in the art would have a reasonable expectation of success because the combination of prior art elements results in the predictable result of producing a biologically functional IL-12 cytokine and because the p40, flexible linker, and p35 sequences are all taught by the prior art as described above.
Regarding instant claim 134: Jounce teaches antibodies and fusion proteins, wherein the antibody fragment can be an Fc region that is linked to another protein via a linker; wherein the conjugate a specific embodiment has the formula of a drug-linker-antibody conjugate where the drug is a cytotoxic agent that can be a cytokine (p.65, [0245]; p.65, [0242]); and, wherein the linker is a cleavable or noncleavable linker (p.65; [0245]); and, wherein the conjugate can be used in a method of treating cancer, including solid tumors (p.75, [0285]). Jounce also specifically teaches SEQ ID NO: 3 of SGGSGGGSG which is “GS linker” that comprises instant SEQ ID NO: 14 of GGSGGGSG (p.165; Table of Certain Sequences: SEQ ID NO: 3).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’686/Askgene/Wojno/ Chen with the teachings of Jounce by modifying the first polypeptide comprising a first half-life extension domain (HL1), a first linker (L1), and a masking moiety (MM) as for a masked IL-12 cytokine (as taught by Pat’686/Askgene/Wojno/Chen) by using noncleavable linker SGGSGGGSG, as taught by Jounce, to join the masking moiety to the Fc half-life extension domain, to arrive at the instantly claimed invention, because the combination of prior art elements would result in the predictable result of producing an polypeptide where a masking moiety is linked covalently to an Fc half-life extension domain. One of ordinary skill in the art would be motivated to do so because Chen teaches flexible “GS linkers” are used to join functional proteins to create fusion proteins. One of ordinary skill would also have a reasonable expectation of success because Pat’686, Askgene, Chen, and Jounce teach fusion of functional proteins using flexible GS linkers.
Regarding instant claim 135: CytomX teaches activatable antibodies and methods of manufacturing activatable antibodies that comprise masking moieties (MM), a cleavable moiety (CM), and an antibody or antigen binding fragment thereof (AB) that binds PDL1 for use in cancer treatment, including solid cancers (p.59, col.54, paras.1-3); wherein, the cleavable moiety (CM) is a polypeptide that is cleavable by a protease and activated in the tumor microenvironment (p.164, col.264, para.1); wherein the activatable antibody has the format MM – CM – AB or AB – CM – MM; and, wherein prior to cleavage the MM interferes with binding and when cleaved the MM no longer interferes with binding (p59, col.53, para.1). Further, CytomX specifically teaches multiple cleavable linker sequences comprising instant SEQ ID NO: 44 of ISSGLLSGRS; for example, see CytomX SEQ ID NO: 377, 394, 901-911 (p.50, cols.35 –36 through p.51, col.37; p.158 -- 159, Table 18).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’686/Askgene/Wojno/ Chen with the teachings of CytomX by modifying the IL-12 masked cytokine (as taught by Pat’686/Askgene/Wojno/Chen) to use a cleavable linker comprising ISSGLLSGRS (as taught by CytomX) in order to activate a fusion protein drug for the treatment of solid cancers (as taught by Askgene and CytomX). One of ordinary skill in the art would be motivated to do so because Pat’686, Askgene and CytomX teach activatable drugs conjugated to antigen-binding fragments wherein the drug is activated via cleavage of a protease-cleavable linker in the tumor microenvironment. One of ordinary skill in the art would have a reasonable expectation of success because CytomX teaches various cleavable amino acid sequences comprising the cleavable polypeptide sequence of ISSGLLSGRS.
As discussed in detail above for instant claim 128, regarding bioactivation of the masked IL-12 cytokine, Askgene specifically teaches that the cytokine moiety polypeptide can be fused to the carrier directly or through a cleavable or noncleavable linker; and, that the masking moiety can be fused to the cytokine or to the carrier through a cleavable peptide linker (p.12, [0050]); and, that the cytokine is activated by cleavage of the linker joined to the masking moiety. Askgene does not specifically teach an embodiment wherein it is explicitly stated that the cytokine’s linker is noncleavable and that the masking moiety’s linker is cleavable. It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Askgene (along with the teachings of CytomX) to make the cytokine’s linker cleavable and the masking moiety’s linker noncleavable, in order to arrive at the instantly claimed invention, because the combination of prior art elements results in a predictable result of cleavage of either the cytokine or the masking moiety in order to facilitate dissociation of the cytokine from the masking moiety for bioactivation. The decision to cleave the cytokine from the “prodrug” would be obvious to try given that the ultimate goal is to produce a cleavage that would release the cytokine from the masking moiety. One of ordinary skill in the art would have a reasonable expectation of success because making the cytokine’s linker cleavable or the masking moiety’s linker cleavable would predictably result in dissociation of the cytokine and masking moiety and would make the cytokine available for receptor binding and activation in order to treat the solid tumor.
[AltContent: textbox (Instant SEQ: 22 vs instant SEQ: 25 (HL1)
[img-media_image6.png])] Regarding instant claims 139-140: In addition to the HL1 and HL2 “knobs-in-holes” mutations taught by Askgene above (i.e., Y349C, T366S, L368A, Y407V, S354C, and T366W), instant SEQ ID NO: 25 of instant claim 139 and SEQ ID NO: 26 of instant claim 140 harbor a N>A mutation compared to Fc SEQ ID NO: 22 (see arrows in alignments below):
[AltContent: textbox (Instant SEQ: 22 vs instant SEQ: 26 (HL2)
[img-media_image7.png])]Thus, the combination of Askgene/Wojno/Chen does not teach an IgG1 Fc sequence (i.e., a backbone sequence) comprising the N>A mutation that, with the “knobs-in-holes” mutation of Askgene, would arrive at a first Fc domain of instant SEQ ID NO: 25 and a second Fc domain of instant SEQ ID NO: 26.
UP-IGG1 teaches the human IgG1 human immunoglobulin gamma-1 heavy chain amino acid sequence P0DOX5 (p.4), which provides the backbone sequence for human IgG1 Fc, except for the “knobs-in-holes” mutations taught by Askgene, and the N>A mutation.
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’686/Askgene/Wojno/ Chen with the teachings of UP-IGG1 by using the human IgG1 Fc taught by UP-IGG1 to encode the human IgG1 Fc half-life extension domains and to modify the first and second half-life extension Fc domains to include the “knobs-in-holes” mutations taught by Pat’686 and Askgene, to arrive at a first Fc half-life extension domain and a second half-life extension domain that arrives at the HL1 and HL2 domains of instant claims 128, 139, and 140 except for the N>A mutations, because the combination of prior art elements teaches the IgG1 sequence and in order to achieve the expected benefit of the “knobs-in-holes”-mediated association between the two half-life extension domains (taught by Askgene) for the IL-12 masked cytokine for cancer treatment (taught by Pat’686/Askgene/Wojno/Chen). One of ordinary skill in the art would have a reasonable expectation of success because Askgene teaches that the half-life extension domains can be derived from human IgG1 Fc regions, UP-IgG1 teaches the human IgG1 Fc amino acid sequence, and Askgene teaches the specific mutations and positions of the “knobs-in-holes” mutations.
Pat’686/Askgene/Wojno/Chen and UP-IgG1 do not teach a human IgG1 Fc sequence that comprises the N>A mutation (as it corresponds to instant SEQ ID NO: 22, and amino acid position 77 of the above alignment).
Wang teaches mutations introduced into Fc domains to eliminate FcγR binding in order to avoid or reduce antigen-independent cytokine release syndrome (CRS; p.2013 – 2014). Wang also teaches that human IgG contains a single conserved site in the CH2 domain of Fc, Asn297, for N-linked glycosylation and that glycans at this site determine the interaction between IgG Fc domain and Fcγ receptors. Wang also teaches that the N297A mutation removes the N-glycosylation, thereby preventing binding to Fcγ receptors (p.2013, col.2, para.2).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’686/Askgene/Wojno/ Chen and UP-IGG1 with the teachings of Wang by modifying the human IgG1 Fc half-life extension domains to comprise the N>A mutation (i.e., N297A of full IgG1 in instant SEQ ID NO: 22) as taught by Wang, in order to arrive at the instantly claimed invention, in order to receive the expected benefit, as taught by Wang, that the N297A mutation prevents binding of human IgG1 Fc to FcγRs to avoid or reduce CRS with administration of the IL-12 masked cytokine that comprises Fc half-life extension domains. One of ordinary skill in the art would have a reasonable expectation of success because UP-IGG1 teaches the human IgG1 sequence and Wang teaches the position of the N>A mutation.
US11866476B2
Claims 128-132, 134-135, 137-140, and 143 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-10 of U.S. Patent No. US11866476B2 (herein referred to as Pat’476); and further in view of Askgene, Wojno, Chen, Merck, Xencor, UP-IL12A, Jounce, CytomX, UP-IGG1, and Wang.
Pat’476 teaches a masked IL-2 cytokine having a first Fc domain and C; and, a second Fc domain and a masking moiety (Pat’476 claim 1; instant claim 128).
Pat’476 does not teach a method of treating cancer comprising administering an IL-12 cytokine (instant claims 128) with first and second half-life extension domains comprising “knobs-in-holes” mutations (instant claims 137-138); a method wherein the IL-12 cytokine comprises an amino acid sequence that encodes an IL-12p40 polypeptide of instant SEQ ID NO: 60 (instant claim 129) that is linked to an IL-12035 polypeptide of instant SEQ ID NO: 2 (instant claim 130) via a linker of amino acid instant SEQ ID NO: 3 (instant claim 131); that the IL-12 cytokine comprises an amino acid sequence of SEQ ID NO: 64 (instant claim 132); that the first linker comprises a noncleavable linker of SEQ ID NO: 14 (instant claim 134); that the first and second Fc domains comprise an additional N>A mutation (instant claims 139 and 140, respectively); or, a method wherein the cancer is a solid tumor (instant claim 142).
Askgene teaches novel cytokine muteins and prodrugs and methods of making and using thereof, including using the cytokine muteins and prodrugs in a method of treating cancer or an infectious disease, or stimulating the immune system in a patient (title; abstract). The method of treating cancer includes treatment of solid cancer/tumors, including breast cancer, lung cancer, pancreatic cancer, esophageal cancer, medullary thyroid cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, colorectal cancer, and stomach cancer solid tumors (p.6, [0021]; p.27, [0106]). Specifically, Askgene teaches cytokine prodrugs wherein the cytokines are “masked” and metabolized in vivo, via cleavage, to become activated therapeutics (p.4, [0008]). Askgene teaches that such cytokine prodrugs provide benefits of fewer side effects, better in vivo pharmacokinetic profiles/longer half-life, and better target specificity; and, are thus, more efficacious as compared to prior cytokine therapeutics (p.11, [0046]). Askgene teaches that the prodrugs comprise a cytokine agonist moiety, a carrier moiety that can be an antigen-binding moiety/antibody for binding a target site, and a masking moiety (i.e., “cytokine antagonist”) that can be an extracellular domain (ECD) of a receptor for the cytokine wherein the receptor ECD inhibits the cytokine moiety’s biological functions while the receptor mask is binding to it (p.11, [0046]; p.13, [0057]). Askgene teaches that the prodrugs can be activated at a target site such as a tumor site in a patient by cleavage of the linker and consequent release of the cytokine mask from the prodrug, which exposes the previously masked cytokine moiety and allowing the cytokine to bind to its receptor on a target cell and exert its biological functions on the target cell (p.11, [0046]).
Askgene teaches prodrugs that are pro-inflammatory cytokine prodrugs (p.11, [0047]). Askgene teaches specific embodiments for IL-2 and IL-15 prodrugs, but also teaches that prodrugs for other cytokines, and in particular cytokines that are potent immune regulators and have strong side effects, are also contemplated and can be made according to the same principles as for IL-2 and IL-15 (p.11, [0048]). Askgene teaches IL-2 and IL-15 prodrugs wherein the masking moiety comprises an IL-2 or IL-15 receptor extracellular domain of their IL-2Rβ or IL-2Rγ receptors (p.13 – 14, [0058]). Askgene additionally teaches several linker sequences, including SEQ ID NO: 49 (i.e., GGGGSGGGGSGGGGS).
Askgene teaches that the carrier moiety can an antibody or antigen-binding fragment thereof that is a full-length antibody with two heavy chains and two light chains, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a disulfide linked Fv fragment, a single domain antibody, a nanobody, or a single chain variable fragment (scFv); and that the antigen-binding moiety can provide additional and potentially synergetic therapeutic efficacy to the cytokine (p.14, [0062]). Askgene teaches that the antibody may be of any heavy chain isotype or subtype including IgG1 Fc; and, that the antibody may be human, non-human, chimeric, or humanized (p.9, [0036]). Askgene teaches that the carrier moiety can improve the serum half-life of the cytokine agonist polypeptide and may also target the cytokine agonist polypeptide to a target site in the body, such as a tumor site (p.14, [0061]). Askgene teaches that strategies of forming heterodimers between the two heavy chains/Fc regions are well known: “For example, the two heavy chain polypeptides in the prodrug may form stable heterodimers through "knobs-into-holes" mutations. "Knobs-into-holes" mutations are made to promote the formation of the heterodimers of the antibody heavy chains and are commonly used to make bispecific antibodies. For example, the Fc domain of the antibody may comprise a T366W mutation in the CH3 domain of the "knob chain" and T366S, L368A, and/or Y407V mutations in the CH3 domain of the "hole chain." An additional interchain disulfide bridge between the CH3 domains can also be used, e.g., by introducing a Y349C mutation into the CH3 domain of the "knobs chain" and an E356C or S354C mutation into the CH3 domain of the "hole chain"… In other embodiments, the antibody moiety may comprise Y349C and/or T366W mutations in one of the two CH3 domains, and E356C, T366S, L368A, and/or Y407V mutations in the other CH3 domain. In certain embodiments, the antibody moiety may comprise Y349C and/or T366W mutations in one of the two CH3 domains, and S354C (or E356C), T366S, L368A, and/or Y407V mutations in the other CH3 domain, with the additional Y349C mutation in one CH3 domain and the additional E356C or S354C mutation in the other CH3 domain, forming an interchain disulfide bridge…” (p.15, [0064]).
Askgene teaches that the cytokine moiety and the masking moiety can be fused to the N-terminus or C-terminus of the light chains and/or heavy chains of the antigen-binding moiety (i.e., carrier; p.14, [0063]). Askgene teaches that the cytokine agonist polypeptide can be fused to the C-terminus of one of the heavy chains of an antibody via a (first) linker, and the cytokine’s mask is fused to the C-terminus of the other heavy chain of the antibody through a (second) cleavable peptide linker, wherein the two heavy chains contain mutations that allow the specific pairing of the two different heavy chains (i.e., association of a first half-life extension domain with a second half-life extension domain; p.15, [0065]).
Askgene additionally teaches that the cytokine moiety polypeptide can be fused to the carrier directly or through a cleavable or noncleavable linker; and, that the masking moiety can be fused to the cytokine or to the carrier through a cleavable peptide linker (p.12, [0050]).
Collectively, Askgene teaches a method of treating cancer in a subject by administering a masked cytokine comprising a protein heterodimer of a first polypeptide with a first half-life extension domain (instant “HL1”) linked to a masking moiety (instant “MM”) via a first linker (“L1”) and a second polypeptide with a second half-life extension domain (instant “HL2”) linked, via a second linker (instant “L2”) to a heterodimeric cytokine (instant “C”) comprised of two subunits covalently joined via a linker; and, wherein the first and second half-life extension domains associate with one another (instant claim 128); wherein the linker joining the two subunits of the cytokine is GGGGSGGGGSGGGGS (i.e., instant SEQ ID NO: 3; instant claim 131).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to combine the teachings of Pat’476 with the teachings of Askgene by using the IL-12 masked cytokine comprising a first polypeptide of H-linker-MM wherein the Fc domain comprises S354C and T366W mutations and a second polypeptide of H-linker-MM wherein the Fc domain comprises Y349C, T366S, L368A, and Y407V mutations; and that comprises a cleavable sinker sequence of ISSGLLSGRS (taught by Pat’476), to arrive at the instantly claimed invention, because the combination of prior art teachings results in a predictable result and benefit of using the masked IL-12 cytokine in a method of treating solid cancer/tumors. One of ordinary skill in the art would have a reasonable expectation of success because both Pat’476 and Askgene teach IL-12 masked cytokines that comprise IgG1 Fc half-life extension domains that comprise “knobs-in-holes” mutations to facilitate interaction of the two Fc polypeptides.
The combination of Pat’476/Askgene does not teach a method of cancer treatment wherein the masked cytokine is a masked IL-12 cytokine that specifically comprises an IL-12p40 polypeptide covalently linked to an IL-12p35 polypeptide via a linker (instant claim 128); or, that the linker joining the IL-12 subunits is instant SEQ ID NO: 3 of GGGSGGGSGGGS (instant claim 131).
Wojno teaches that IL-12 has an important role for cancer and that expression of IL-12 has shown results of tumor regression and anti-tumor responses; but that there remain concerns about using cytokines as drugs due to side effects (p.861, col.2, paras.1-2). Wojno further teaches that these issues are exemplified in multiple trials that utilized IL-12 as a treatment for different cancers, which revealed significant toxicity and limited efficacy (p.862, col.1, para.1). Wojno also teaches that IL-12 is a heterodimer comprised of two subunits: the α subunit IL-12p35 and the β subunit IL-12p40 (p.851, col.2, para.2). Wojno further teaches that the IL-12 heterodimer utilizes a heterodimeric receptor complex for signaling made up of IL-12Rβ1 and IL-12Rβ2 (p.852, Fig.1 and legend).
Chen teaches fusion protein linkers, including flexible linkers, rigid linkers, and in vivo cleavable linkers (title; sections 3.1 – 3.3). Chen teaches that direct fusion of functional domains without a linker may lead to many undesirable outcomes, including misfolding of the fusion proteins, low yield in protein production, or impaired bioactivity (p.2, para.2). Chen teaches that linkers can be used to join protein drugs to carrier proteins such as antibodies in order to extend plasma half-lives and to achieve enhances therapeutic effects by drug targeting to specific types of cells (p.1, para.1). Chen teaches the use of flexible linkers for recombinant fusion proteins; that flexible linkers are useful when the domains require a certain degree of movement or interaction; that flexible linkers are generally composed of small, non-polar or polar amino acids which provides for flexibility and mobility of connecting functional domains; and that the most commonly used flexible linkers consist of stretches of glycine and serine residues (i.e., “GS linkers”) that have the sequence of (Gly-Gly-Gly-Gly-Ser)n (p.4, para.4).
Regarding instant claim 143: It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’476/Askgene with the teachings of Wojno by modifying the method of treating a cancer using a masked IL-12 cytokine as taught by Pat’476, by using an IL-12 cytokine taught by Wojno in order to arrive at the instantly claimed invention, because the combination of prior art elements according to known methods results in a predictable result and a beneficial outcome of reducing toxicity with administration of an IL-12 cytokine for solid cancer treatment (as taught by Askgene/Wojno).
Regarding instant claim 131: It would have further been obvious to one of ordinary skill in the art to use Askgene’s SEQ ID NO: 49 (noncleavable) covalent cytokine domains linker GGGGSGGGGSGGGGS to covalently join the subunits of the IL-12 heterodimer (taught by Pat’476 and Wojno) in order to tether the p40 and p35 subunits (taught by Wojno) to form a fusion protein (taught by Chen), because the combination of prior art elements according to known methods results in a predictable result of forming a biologically active IL-12 heterodimer for receptor stimulation (taught by Wojno) for IL-12 mediated cancer treatment (taught by Wojno). One of ordinary skill in the art would be motivated to do so because Chen teaches that flexible GS linkers of the formula GGGGSn provide a benefit of joining protein domains to facilitate interaction between the domains and one would have a reasonable expectation of success because Askgene’s SEQ ID NO: 49 linker is a GGGGSn linker.
Regarding the first and second half-life extension domains, it would have been prima facie obvious for one of ordinary skill in the art to further combine the teachings of Pat’476/Askgene by using a “knobs-in-holes” carrier comprised of two Fc polypeptides comprising the “knob” mutations and “holes” mutations, in order to arrive at the instantly claimed invention, because the combination of prior art elements taught by Askgene teaches that these mutations facilitate association of the two Fc chains to facilitate dimerization. One would be motivated to do so because this would produce a “carrier” domain that would provide the benefits of extended half-life for a masked cytokine “prodrug” for solid cancer treatment. One of ordinary skill in the art would have a reasonable expectation of success because Askgene teaches the specific amino residue mutations at amino acid positions of the instant claims.
Regarding bioactivation of the masked IL-12 cytokine, Askgene specifically teaches that the cytokine moiety polypeptide can be fused to the carrier directly or through a cleavable or noncleavable linker; and, that the masking moiety can be fused to the cytokine or to the carrier through a cleavable peptide linker (p.12, [0050]); and, that the cytokine is activated by cleavage of the linker joined to the masking moiety. Pat’476/Askgene does not specifically teach an embodiment wherein it is explicitly stated that the cytokine’s linker is noncleavable and that the masking moiety’s linker is cleavable. It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’476/Askgene to make the cytokine’s linker cleavable and the masking moiety’s linker noncleavable, in order to arrive at the instantly claimed invention, because the combination of prior art elements results in a predictable result of cleavage of either the cytokine or the masking moiety in order to facilitate dissociation of the cytokine from the masking moiety for bioactivation. The decision to cleave the cytokine from the “prodrug” would be obvious to try given that the ultimate goal is to produce a cleavage that would release the cytokine from the masking moiety. One of ordinary skill in the art would have a reasonable expectation of success because making the cytokine’s linker cleavable or the masking moiety’s linker cleavable would predictably result in dissociation of the cytokine and masking moiety and would make the cytokine available for receptor binding and activation in order to treat the solid tumor.
[AltContent: textbox (Instant SEQ: 60 (IL-12p40) vs Merck (IL-12p40)
[img-media_image3.png])]Regarding instant claim 129: Merck teaches IL-12p40 amino acid SEQ ID NO: 7 which aligns with instant SEQ ID NO: 60 with 99.7% identity wherein the only variation from applicant’s sequence is that Merck’s SEQ ID NO: 7 retains a cysteine at position 252 (see alignment below):
Thus, Askgene/Wojno/Chen and Merck do not teach that the IL-12p40 subunit comprises a C252S mutation.
Xencor teaches that IL-12 is a promising cytokine for cancer treatment, but that it has faced hurdles in clinical trials due to toxicity (p.1, [0003]). IL-12/Fc fusion proteins comprising both IL-12p40 and IL-12p35 subunits linked to half-life extension domains for cancer treatment, including several variants wherein the IL-12p40 subunit comprises mutations that modulate receptor affinity and potency and that removes free cysteines that may bond with other free cysteines leading to heterogeneity and undesirable immunogenicity, including an IL-12p40 variant comprising a C252S mutation for the purpose of removing the C252 free cysteine (p.6, [0011]; p.14, [0041]; p.15, [0042 – 0043]; p.151, [00477 – 00479]; p.277 –278, [00881]). Xencor teaches that the C252S mutation can be used alone or in combination with any other IL-12p40 variants (p.278, [00881]). Xencor further teaches that IL-12p40 variants comprising the C252S mutation exhibited improved potency compared to variants without the mutation (p.151, [00479]; p.193, Fig.67A-D).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’476/Askgene/Wojno/ Chen with the teachings of Merck and Xencor by using Merck’s SEQ ID NO: 7 that encodes the IL-12p40 subunit and to modify the IL-12p40 subunit to have a C252S mutation (taught by Xencor), in order to arrive at the instantly claimed invention because the combination of prior art elements results in a predictable result of encoding a IL-12p40 polypeptide that has a C252S mutation. Further, one would be motivated to mutate C252S in order to receive the expected benefit (as taught by Xencor) that the C252S mutation would remove a free cysteine from the IL-12p40 subunit to reduce heterogeneity and immunogenicity, and to improve potency of the IL-12 cytokine (as taught by Xencor) for the IL-12p40 subunit for the masked IL-12 cytokine in a method of treating cancer (as taught by Pat’476/Askgene/Wojno/Chen). One of ordinary skill in the art would have a reasonable expectation of success because the combination of Merck and Xencor teach the amino acid sequence for an improved IL-12p40 cytokine; Pat’476 and Askgene teach masked cytokines; Pat’476 and Wojno teach the use of IL-12 for cancer treatment; and, Pat’476/Askgene/Wojno/Chen and Xencor teach fusion proteins comprising IL-12p40 and IL-12p35 subunits as well as their linkage to half-life extension domains.
[AltContent: textbox (Instant SEQ: 2 (IL-12p35) vs UniProt (IL-12A P29459)
[img-media_image4.png])]Regarding instant claim 130: UP-IL12A teaches P29459: IL-12A, which is the human sequence for the IL-12 p35 subunit and instant SEQ ID NO: 2, at 100% identity (p.5; see alignment below):
UP-IL12A also teaches that IL12A (i.e., IL-12p35) heterodimerizes with IL12B(i.e., IL-12p40) to form the IL-12 cytokine to exert its biological effects through the IL-12 receptor subunits (p.1).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’476/Askgene/Wojno/ Chen with the teachings of UP-IL12A by using UP-IL12A’s mature “P29549 IL-12A” amino acid sequence that encodes the IL-12p35 subunit, in order to arrive at the instantly claimed invention because the combination of prior art elements results in a predictable result of encoding the a human IL-12p35 polypeptide for the masked IL-12 cytokine in a method of treating cancer (as taught by Pat’476/Askgene/Wojno/Chen). One of ordinary skill would have a reasonable expectation of success because Wojno and UP-IL12A both teach that the functional IL-12 cytokine is comprised of the IL-12B/IL-12p40 and IL-12A/IL-12p35 subunits.
Regarding instant claim 132: Instant SEQ ID NO: 64 (as recited in instant claim 132) is an amino acid sequence that encodes for a fusion protein comprising an IL-12p40 subunit joined to an IL-12p35 subunit via the flexible “GS linker” GGGGSGGGGSGGGGS (i.e., instant SEQ ID NOs: 60 (p40) –3 (linker) – 2 (p35); see sequence fusion below):
[AltContent: textbox (Instant SEQ: 64 vs fusion of instant SEQ: 60 (p40)/SEQ:3 (linker)/SEQ: 2 (p35)
[img-media_image5.png])]
The combination of Pat’476, Askgene, Wojno, and Chen teaches a method of using an IL-12 masked cytokine wherein a polypeptide chain is comprised of an Fc half-life extension domain fused to a IL-12 cytokine via a cleavable linker, wherein the IL-12 cytokine is comprised of a p40 subunit linked to the p35 subunit using the flexible fusion protein linker GGGGSGGGGSGGGGS, as described in detail above for claim 128.
The combination of Askgene/Wojno/Chen, Merck, and Xencor teaches the IL-12p40 subunit for the IL-12 cytokine for the masked cytokine as described in detail above for claim 129.
The combination of Askgene/Wojno/Chen and UP-IL12A teaches the IL-12p35 subunit of the IL-12 cytokine for the masked cytokine as described in detail for claim 130 above.
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’476/Askgene/Wojno/ Chen with the teachings of Merck/Xencor and the teachings of UP-IL12A by using the flexible GS linker GGGGSGGGGSGGGGS (as taught by Askgene/Chen) to join the p40 and p35 subunits to produce a functional IL-12 heterodimeric cytokine (as taught by Wojno, Xencor, and UP-IL12A) for the IL-12 cytokine for a masked IL-12 cytokine (as taught by Pat’476/Askgene/Wojno/Chen) for IL-12-mediated cancer treatment (as taught by Askgene/Wojno/Chen and Xencor). Further, one would be motivated to link the p40 and p35 subunits using the flexible GS linker, as described in detail above, to facilitate their interaction (as taught by Chen). One of ordinary skill in the art would have a reasonable expectation of success because the combination of prior art elements results in the predictable result of producing a biologically functional IL-12 cytokine and because the p40, flexible linker, and p35 sequences are all taught by the prior art as described above.
Regarding instant claim 134: Jounce teaches antibodies and fusion proteins, wherein the antibody fragment can be an Fc region that is linked to another protein via a linker; wherein the conjugate a specific embodiment has the formula of a drug-linker-antibody conjugate where the drug is a cytotoxic agent that can be a cytokine (p.65, [0245]; p.65, [0242]); and, wherein the linker is a cleavable or noncleavable linker (p.65; [0245]); and, wherein the conjugate can be used in a method of treating cancer, including solid tumors (p.75, [0285]). Jounce also specifically teaches SEQ ID NO: 3 of SGGSGGGSG which is “GS linker” that comprises instant SEQ ID NO: 14 of GGSGGGSG (p.165; Table of Certain Sequences: SEQ ID NO: 3).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’476/Askgene/Wojno/ Chen with the teachings of Jounce by modifying the first polypeptide comprising a first half-life extension domain (HL1), a first linker (L1), and a masking moiety (MM) as for a masked IL-12 cytokine (as taught by Pat’476/Askgene/Wojno/Chen) by using noncleavable linker SGGSGGGSG, as taught by Jounce, to join the masking moiety to the Fc half-life extension domain, to arrive at the instantly claimed invention, because the combination of prior art elements would result in the predictable result of producing an polypeptide where a masking moiety is linked covalently to an Fc half-life extension domain. One of ordinary skill in the art would be motivated to do so because Chen teaches flexible “GS linkers” are used to join functional proteins to create fusion proteins. One of ordinary skill would also have a reasonable expectation of success because Pat’476, Askgene, Chen, and Jounce teach fusion of functional proteins using flexible GS linkers.
Regarding instant claim 135: CytomX teaches activatable antibodies and methods of manufacturing activatable antibodies that comprise masking moieties (MM), a cleavable moiety (CM), and an antibody or antigen binding fragment thereof (AB) that binds PDL1 for use in cancer treatment, including solid cancers (p.59, col.54, paras.1-3); wherein, the cleavable moiety (CM) is a polypeptide that is cleavable by a protease and activated in the tumor microenvironment (p.164, col.264, para.1); wherein the activatable antibody has the format MM – CM – AB or AB – CM – MM; and, wherein prior to cleavage the MM interferes with binding and when cleaved the MM no longer interferes with binding (p59, col.53, para.1). Further, CytomX specifically teaches multiple cleavable linker sequences comprising instant SEQ ID NO: 44 of ISSGLLSGRS; for example, see CytomX SEQ ID NO: 377, 394, 901-911 (p.50, cols.35 –36 through p.51, col.37; p.158 -- 159, Table 18).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’476/Askgene/Wojno/ Chen with the teachings of CytomX by modifying the IL-12 masked cytokine (as taught by Pat’476/Askgene/Wojno/Chen) to use a cleavable linker comprising ISSGLLSGRS (as taught by CytomX) in order to activate a fusion protein drug for the treatment of solid cancers (as taught by Askgene and CytomX). One of ordinary skill in the art would be motivated to do so because Pat’476, Askgene and CytomX teach activatable drugs conjugated to antigen-binding fragments wherein the drug is activated via cleavage of a protease-cleavable linker in the tumor microenvironment. One of ordinary skill in the art would have a reasonable expectation of success because CytomX teaches various cleavable amino acid sequences comprising the cleavable polypeptide sequence of ISSGLLSGRS.
As discussed in detail above for instant claim 128, regarding bioactivation of the masked IL-12 cytokine, Askgene specifically teaches that the cytokine moiety polypeptide can be fused to the carrier directly or through a cleavable or noncleavable linker; and, that the masking moiety can be fused to the cytokine or to the carrier through a cleavable peptide linker (p.12, [0050]); and, that the cytokine is activated by cleavage of the linker joined to the masking moiety. Askgene does not specifically teach an embodiment wherein it is explicitly stated that the cytokine’s linker is noncleavable and that the masking moiety’s linker is cleavable. It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Askgene (along with the teachings of CytomX) to make the cytokine’s linker cleavable and the masking moiety’s linker noncleavable, in order to arrive at the instantly claimed invention, because the combination of prior art elements results in a predictable result of cleavage of either the cytokine or the masking moiety in order to facilitate dissociation of the cytokine from the masking moiety for bioactivation. The decision to cleave the cytokine from the “prodrug” would be obvious to try given that the ultimate goal is to produce a cleavage that would release the cytokine from the masking moiety. One of ordinary skill in the art would have a reasonable expectation of success because making the cytokine’s linker cleavable or the masking moiety’s linker cleavable would predictably result in dissociation of the cytokine and masking moiety and would make the cytokine available for receptor binding and activation in order to treat the solid tumor.
[AltContent: textbox (Instant SEQ: 22 vs instant SEQ: 25 (HL1)
[img-media_image6.png])] Regarding instant claims 139-140: In addition to the HL1 and HL2 “knobs-in-holes” mutations taught by Askgene above (i.e., Y349C, T366S, L368A, Y407V, S354C, and T366W), instant SEQ ID NO: 25 of instant claim 139 and SEQ ID NO: 26 of instant claim 140 harbor a N>A mutation compared to Fc SEQ ID NO: 22 (see arrows in alignments below):
[AltContent: textbox (Instant SEQ: 22 vs instant SEQ: 26 (HL2)
[img-media_image7.png])]Thus, the combination of Askgene/Wojno/Chen does not teach an IgG1 Fc sequence (i.e., a backbone sequence) comprising the N>A mutation that, with the “knobs-in-holes” mutation of Askgene, would arrive at a first Fc domain of instant SEQ ID NO: 25 and a second Fc domain of instant SEQ ID NO: 26.
UP-IGG1 teaches the human IgG1 human immunoglobulin gamma-1 heavy chain amino acid sequence P0DOX5 (p.4), which provides the backbone sequence for human IgG1 Fc, except for the “knobs-in-holes” mutations taught by Askgene, and the N>A mutation.
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’476/Askgene/Wojno/ Chen with the teachings of UP-IGG1 by using the human IgG1 Fc taught by UP-IGG1 to encode the human IgG1 Fc half-life extension domains and to modify the first and second half-life extension Fc domains to include the “knobs-in-holes” mutations taught by Pat’476 and Askgene, to arrive at a first Fc half-life extension domain and a second half-life extension domain that arrives at the HL1 and HL2 domains of instant claims 128, 139, and 140 except for the N>A mutations, because the combination of prior art elements teaches the IgG1 sequence and in order to achieve the expected benefit of the “knobs-in-holes”-mediated association between the two half-life extension domains (taught by Askgene) for the IL-12 masked cytokine for cancer treatment (taught by Pat’476/Askgene/Wojno/Chen). One of ordinary skill in the art would have a reasonable expectation of success because Askgene teaches that the half-life extension domains can be derived from human IgG1 Fc regions, UP-IgG1 teaches the human IgG1 Fc amino acid sequence, and Askgene teaches the specific mutations and positions of the “knobs-in-holes” mutations.
Pat’476/Askgene/Wojno/Chen and UP-IgG1 do not teach a human IgG1 Fc sequence that comprises the N>A mutation (as it corresponds to instant SEQ ID NO: 22, and amino acid position 77 of the above alignment).
Wang teaches mutations introduced into Fc domains to eliminate FcγR binding in order to avoid or reduce antigen-independent cytokine release syndrome (CRS; p.2013 – 2014). Wang also teaches that human IgG contains a single conserved site in the CH2 domain of Fc, Asn297, for N-linked glycosylation and that glycans at this site determine the interaction between IgG Fc domain and Fcγ receptors. Wang also teaches that the N297A mutation removes the N-glycosylation, thereby preventing binding to Fcγ receptors (p.2013, col.2, para.2).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Pat’476/Askgene/Wojno/ Chen and UP-IGG1 with the teachings of Wang by modifying the human IgG1 Fc half-life extension domains to comprise the N>A mutation (i.e., N297A of full IgG1 in instant SEQ ID NO: 22) as taught by Wang, in order to arrive at the instantly claimed invention, in order to receive the expected benefit, as taught by Wang, that the N297A mutation prevents binding of human IgG1 Fc to FcγRs to avoid or reduce CRS with administration of the IL-12 masked cytokine that comprises Fc half-life extension domains. One of ordinary skill in the art would have a reasonable expectation of success because UP-IGG1 teaches the human IgG1 sequence and Wang teaches the position of the N>A mutation.
Copending Application 17/995,163
Claims 128-132, 134-135, 137-140, and 143 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 3, 9, 11, 16, 19, 24, 27, 30, 33, 44, 57, 81-82, 124, 126, and 150-151 of copending Application No. 17/995,163 (herein referred to as App’163); and further in view of Askgene, Wojno, Chen, Merck, Xencor, UP-IL12A, Jounce, CytomX, UP-IGG1, and Wang.
App’163 teaches a masked IL-2 cytokine having a first Fc domain and C; and, a second Fc domain and a masking moiety (App’163 claim 1; instant claim 128).
App’163 does not teach a method of treating cancer comprising administering an IL-12 cytokine (instant claims 128) with first and second half-life extension domains comprising “knobs-in-holes” mutations (instant claims 137-138); a method wherein the IL-12 cytokine comprises an amino acid sequence that encodes an IL-12p40 polypeptide of instant SEQ ID NO: 60 (instant claim 129) that is linked to an IL-12035 polypeptide of instant SEQ ID NO: 2 (instant claim 130) via a linker of amino acid instant SEQ ID NO: 3 (instant claim 131); that the IL-12 cytokine comprises an amino acid sequence of SEQ ID NO: 64 (instant claim 132); that the first linker comprises a noncleavable linker of SEQ ID NO: 14 (instant claim 134); that the first and second Fc domains comprise an additional N>A mutation (instant claims 139 and 140, respectively); or, a method wherein the cancer is a solid tumor (instant claim 142).
Askgene teaches novel cytokine muteins and prodrugs and methods of making and using thereof, including using the cytokine muteins and prodrugs in a method of treating cancer or an infectious disease, or stimulating the immune system in a patient (title; abstract). The method of treating cancer includes treatment of solid cancer/tumors, including breast cancer, lung cancer, pancreatic cancer, esophageal cancer, medullary thyroid cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, colorectal cancer, and stomach cancer solid tumors (p.6, [0021]; p.27, [0106]). Specifically, Askgene teaches cytokine prodrugs wherein the cytokines are “masked” and metabolized in vivo, via cleavage, to become activated therapeutics (p.4, [0008]). Askgene teaches that such cytokine prodrugs provide benefits of fewer side effects, better in vivo pharmacokinetic profiles/longer half-life, and better target specificity; and, are thus, more efficacious as compared to prior cytokine therapeutics (p.11, [0046]). Askgene teaches that the prodrugs comprise a cytokine agonist moiety, a carrier moiety that can be an antigen-binding moiety/antibody for binding a target site, and a masking moiety (i.e., “cytokine antagonist”) that can be an extracellular domain (ECD) of a receptor for the cytokine wherein the receptor ECD inhibits the cytokine moiety’s biological functions while the receptor mask is binding to it (p.11, [0046]; p.13, [0057]). Askgene teaches that the prodrugs can be activated at a target site such as a tumor site in a patient by cleavage of the linker and consequent release of the cytokine mask from the prodrug, which exposes the previously masked cytokine moiety and allowing the cytokine to bind to its receptor on a target cell and exert its biological functions on the target cell (p.11, [0046]).
Askgene teaches prodrugs that are pro-inflammatory cytokine prodrugs (p.11, [0047]). Askgene teaches specific embodiments for IL-2 and IL-15 prodrugs, but also teaches that prodrugs for other cytokines, and in particular cytokines that are potent immune regulators and have strong side effects, are also contemplated and can be made according to the same principles as for IL-2 and IL-15 (p.11, [0048]). Askgene teaches IL-2 and IL-15 prodrugs wherein the masking moiety comprises an IL-2 or IL-15 receptor extracellular domain of their IL-2Rβ or IL-2Rγ receptors (p.13 – 14, [0058]). Askgene additionally teaches several linker sequences, including SEQ ID NO: 49 (i.e., GGGGSGGGGSGGGGS).
Askgene teaches that the carrier moiety can an antibody or antigen-binding fragment thereof that is a full-length antibody with two heavy chains and two light chains, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a disulfide linked Fv fragment, a single domain antibody, a nanobody, or a single chain variable fragment (scFv); and that the antigen-binding moiety can provide additional and potentially synergetic therapeutic efficacy to the cytokine (p.14, [0062]). Askgene teaches that the antibody may be of any heavy chain isotype or subtype including IgG1 Fc; and, that the antibody may be human, non-human, chimeric, or humanized (p.9, [0036]). Askgene teaches that the carrier moiety can improve the serum half-life of the cytokine agonist polypeptide and may also target the cytokine agonist polypeptide to a target site in the body, such as a tumor site (p.14, [0061]). Askgene teaches that strategies of forming heterodimers between the two heavy chains/Fc regions are well known: “For example, the two heavy chain polypeptides in the prodrug may form stable heterodimers through "knobs-into-holes" mutations. "Knobs-into-holes" mutations are made to promote the formation of the heterodimers of the antibody heavy chains and are commonly used to make bispecific antibodies. For example, the Fc domain of the antibody may comprise a T366W mutation in the CH3 domain of the "knob chain" and T366S, L368A, and/or Y407V mutations in the CH3 domain of the "hole chain." An additional interchain disulfide bridge between the CH3 domains can also be used, e.g., by introducing a Y349C mutation into the CH3 domain of the "knobs chain" and an E356C or S354C mutation into the CH3 domain of the "hole chain"… In other embodiments, the antibody moiety may comprise Y349C and/or T366W mutations in one of the two CH3 domains, and E356C, T366S, L368A, and/or Y407V mutations in the other CH3 domain. In certain embodiments, the antibody moiety may comprise Y349C and/or T366W mutations in one of the two CH3 domains, and S354C (or E356C), T366S, L368A, and/or Y407V mutations in the other CH3 domain, with the additional Y349C mutation in one CH3 domain and the additional E356C or S354C mutation in the other CH3 domain, forming an interchain disulfide bridge…” (p.15, [0064]).
Askgene teaches that the cytokine moiety and the masking moiety can be fused to the N-terminus or C-terminus of the light chains and/or heavy chains of the antigen-binding moiety (i.e., carrier; p.14, [0063]). Askgene teaches that the cytokine agonist polypeptide can be fused to the C-terminus of one of the heavy chains of an antibody via a (first) linker, and the cytokine’s mask is fused to the C-terminus of the other heavy chain of the antibody through a (second) cleavable peptide linker, wherein the two heavy chains contain mutations that allow the specific pairing of the two different heavy chains (i.e., association of a first half-life extension domain with a second half-life extension domain; p.15, [0065]).
Askgene additionally teaches that the cytokine moiety polypeptide can be fused to the carrier directly or through a cleavable or noncleavable linker; and, that the masking moiety can be fused to the cytokine or to the carrier through a cleavable peptide linker (p.12, [0050]).
Collectively, Askgene teaches a method of treating cancer in a subject by administering a masked cytokine comprising a protein heterodimer of a first polypeptide with a first half-life extension domain (instant “HL1”) linked to a masking moiety (instant “MM”) via a first linker (“L1”) and a second polypeptide with a second half-life extension domain (instant “HL2”) linked, via a second linker (instant “L2”) to a heterodimeric cytokine (instant “C”) comprised of two subunits covalently joined via a linker; and, wherein the first and second half-life extension domains associate with one another (instant claim 128); wherein the linker joining the two subunits of the cytokine is GGGGSGGGGSGGGGS (i.e., instant SEQ ID NO: 3; instant claim 131).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to combine the teachings of App’163 with the teachings of Askgene by using the IL-12 masked cytokine comprising a first polypeptide of H-linker-MM wherein the Fc domain comprises S354C and T366W mutations and a second polypeptide of H-linker-MM wherein the Fc domain comprises Y349C, T366S, L368A, and Y407V mutations; and that comprises a cleavable sinker sequence of ISSGLLSGRS (taught by App’163), to arrive at the instantly claimed invention, because the combination of prior art teachings results in a predictable result and benefit of using the masked IL-12 cytokine in a method of treating solid cancer/tumors. One of ordinary skill in the art would have a reasonable expectation of success because both App’163 and Askgene teach IL-12 masked cytokines that comprise IgG1 Fc half-life extension domains that comprise “knobs-in-holes” mutations to facilitate interaction of the two Fc polypeptides.
The combination of App’163/Askgene does not teach a method of cancer treatment wherein the masked cytokine is a masked IL-12 cytokine that specifically comprises an IL-12p40 polypeptide covalently linked to an IL-12p35 polypeptide via a linker (instant claim 128); or, that the linker joining the IL-12 subunits is instant SEQ ID NO: 3 of GGGSGGGSGGGS (instant claim 131).
Wojno teaches that IL-12 has an important role for cancer and that expression of IL-12 has shown results of tumor regression and anti-tumor responses; but that there remain concerns about using cytokines as drugs due to side effects (p.861, col.2, paras.1-2). Wojno further teaches that these issues are exemplified in multiple trials that utilized IL-12 as a treatment for different cancers, which revealed significant toxicity and limited efficacy (p.862, col.1, para.1). Wojno also teaches that IL-12 is a heterodimer comprised of two subunits: the α subunit IL-12p35 and the β subunit IL-12p40 (p.851, col.2, para.2). Wojno further teaches that the IL-12 heterodimer utilizes a heterodimeric receptor complex for signaling made up of IL-12Rβ1 and IL-12Rβ2 (p.852, Fig.1 and legend).
Chen teaches fusion protein linkers, including flexible linkers, rigid linkers, and in vivo cleavable linkers (title; sections 3.1 – 3.3). Chen teaches that direct fusion of functional domains without a linker may lead to many undesirable outcomes, including misfolding of the fusion proteins, low yield in protein production, or impaired bioactivity (p.2, para.2). Chen teaches that linkers can be used to join protein drugs to carrier proteins such as antibodies in order to extend plasma half-lives and to achieve enhances therapeutic effects by drug targeting to specific types of cells (p.1, para.1). Chen teaches the use of flexible linkers for recombinant fusion proteins; that flexible linkers are useful when the domains require a certain degree of movement or interaction; that flexible linkers are generally composed of small, non-polar or polar amino acids which provides for flexibility and mobility of connecting functional domains; and that the most commonly used flexible linkers consist of stretches of glycine and serine residues (i.e., “GS linkers”) that have the sequence of (Gly-Gly-Gly-Gly-Ser)n (p.4, para.4).
Regarding instant claim 143: It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of App’163/Askgene with the teachings of Wojno by modifying the method of treating a cancer using a masked IL-12 cytokine as taught by App’163, by using an IL-12 cytokine taught by Wojno in order to arrive at the instantly claimed invention, because the combination of prior art elements according to known methods results in a predictable result and a beneficial outcome of reducing toxicity with administration of an IL-12 cytokine for solid cancer treatment (as taught by Askgene/Wojno).
Regarding instant claim 131: It would have further been obvious to one of ordinary skill in the art to use Askgene’s SEQ ID NO: 49 (noncleavable) covalent cytokine domains linker GGGGSGGGGSGGGGS to covalently join the subunits of the IL-12 heterodimer (taught by App’163 and Wojno) in order to tether the p40 and p35 subunits (taught by Wojno) to form a fusion protein (taught by Chen), because the combination of prior art elements according to known methods results in a predictable result of forming a biologically active IL-12 heterodimer for receptor stimulation (taught by Wojno) for IL-12 mediated cancer treatment (taught by Wojno). One of ordinary skill in the art would be motivated to do so because Chen teaches that flexible GS linkers of the formula GGGGSn provide a benefit of joining protein domains to facilitate interaction between the domains and one would have a reasonable expectation of success because Askgene’s SEQ ID NO: 49 linker is a GGGGSn linker.
Regarding the first and second half-life extension domains, it would have been prima facie obvious for one of ordinary skill in the art to further combine the teachings of App’163/Askgene by using a “knobs-in-holes” carrier comprised of two Fc polypeptides comprising the “knob” mutations and “holes” mutations, in order to arrive at the instantly claimed invention, because the combination of prior art elements taught by Askgene teaches that these mutations facilitate association of the two Fc chains to facilitate dimerization. One would be motivated to do so because this would produce a “carrier” domain that would provide the benefits of extended half-life for a masked cytokine “prodrug” for solid cancer treatment. One of ordinary skill in the art would have a reasonable expectation of success because Askgene teaches the specific amino residue mutations at amino acid positions of the instant claims.
Regarding bioactivation of the masked IL-12 cytokine, Askgene specifically teaches that the cytokine moiety polypeptide can be fused to the carrier directly or through a cleavable or noncleavable linker; and, that the masking moiety can be fused to the cytokine or to the carrier through a cleavable peptide linker (p.12, [0050]); and, that the cytokine is activated by cleavage of the linker joined to the masking moiety. App’163/Askgene does not specifically teach an embodiment wherein it is explicitly stated that the cytokine’s linker is noncleavable and that the masking moiety’s linker is cleavable. It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of App’163/Askgene to make the cytokine’s linker cleavable and the masking moiety’s linker noncleavable, in order to arrive at the instantly claimed invention, because the combination of prior art elements results in a predictable result of cleavage of either the cytokine or the masking moiety in order to facilitate dissociation of the cytokine from the masking moiety for bioactivation. The decision to cleave the cytokine from the “prodrug” would be obvious to try given that the ultimate goal is to produce a cleavage that would release the cytokine from the masking moiety. One of ordinary skill in the art would have a reasonable expectation of success because making the cytokine’s linker cleavable or the masking moiety’s linker cleavable would predictably result in dissociation of the cytokine and masking moiety and would make the cytokine available for receptor binding and activation in order to treat the solid tumor.
[AltContent: textbox (Instant SEQ: 60 (IL-12p40) vs Merck (IL-12p40)
[img-media_image3.png])]Regarding instant claim 129: Merck teaches IL-12p40 amino acid SEQ ID NO: 7 which aligns with instant SEQ ID NO: 60 with 99.7% identity wherein the only variation from applicant’s sequence is that Merck’s SEQ ID NO: 7 retains a cysteine at position 252 (see alignment below):
Thus, Askgene/Wojno/Chen and Merck do not teach that the IL-12p40 subunit comprises a C252S mutation.
Xencor teaches that IL-12 is a promising cytokine for cancer treatment, but that it has faced hurdles in clinical trials due to toxicity (p.1, [0003]). IL-12/Fc fusion proteins comprising both IL-12p40 and IL-12p35 subunits linked to half-life extension domains for cancer treatment, including several variants wherein the IL-12p40 subunit comprises mutations that modulate receptor affinity and potency and that removes free cysteines that may bond with other free cysteines leading to heterogeneity and undesirable immunogenicity, including an IL-12p40 variant comprising a C252S mutation for the purpose of removing the C252 free cysteine (p.6, [0011]; p.14, [0041]; p.15, [0042 – 0043]; p.151, [00477 – 00479]; p.277 –278, [00881]). Xencor teaches that the C252S mutation can be used alone or in combination with any other IL-12p40 variants (p.278, [00881]). Xencor further teaches that IL-12p40 variants comprising the C252S mutation exhibited improved potency compared to variants without the mutation (p.151, [00479]; p.193, Fig.67A-D).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of App’163/Askgene/Wojno/ Chen with the teachings of Merck and Xencor by using Merck’s SEQ ID NO: 7 that encodes the IL-12p40 subunit and to modify the IL-12p40 subunit to have a C252S mutation (taught by Xencor), in order to arrive at the instantly claimed invention because the combination of prior art elements results in a predictable result of encoding a IL-12p40 polypeptide that has a C252S mutation. Further, one would be motivated to mutate C252S in order to receive the expected benefit (as taught by Xencor) that the C252S mutation would remove a free cysteine from the IL-12p40 subunit to reduce heterogeneity and immunogenicity, and to improve potency of the IL-12 cytokine (as taught by Xencor) for the IL-12p40 subunit for the masked IL-12 cytokine in a method of treating cancer (as taught by App’163/Askgene/Wojno/Chen). One of ordinary skill in the art would have a reasonable expectation of success because the combination of Merck and Xencor teach the amino acid sequence for an improved IL-12p40 cytokine; App’163 and Askgene teach masked cytokines; App’163 and Wojno teach the use of IL-12 for cancer treatment; and, App’163/Askgene/Wojno/Chen and Xencor teach fusion proteins comprising IL-12p40 and IL-12p35 subunits as well as their linkage to half-life extension domains.
[AltContent: textbox (Instant SEQ: 2 (IL-12p35) vs UniProt (IL-12A P29459)
[img-media_image4.png])]Regarding instant claim 130: UP-IL12A teaches P29459: IL-12A, which is the human sequence for the IL-12 p35 subunit and instant SEQ ID NO: 2, at 100% identity (p.5; see alignment below):
UP-IL12A also teaches that IL12A (i.e., IL-12p35) heterodimerizes with IL12B(i.e., IL-12p40) to form the IL-12 cytokine to exert its biological effects through the IL-12 receptor subunits (p.1).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of App’163/Askgene/Wojno/ Chen with the teachings of UP-IL12A by using UP-IL12A’s mature “P29549 IL-12A” amino acid sequence that encodes the IL-12p35 subunit, in order to arrive at the instantly claimed invention because the combination of prior art elements results in a predictable result of encoding the a human IL-12p35 polypeptide for the masked IL-12 cytokine in a method of treating cancer (as taught by App’163/Askgene/Wojno/Chen). One of ordinary skill would have a reasonable expectation of success because Wojno and UP-IL12A both teach that the functional IL-12 cytokine is comprised of the IL-12B/IL-12p40 and IL-12A/IL-12p35 subunits.
Regarding instant claim 132: Instant SEQ ID NO: 64 (as recited in instant claim 132) is an amino acid sequence that encodes for a fusion protein comprising an IL-12p40 subunit joined to an IL-12p35 subunit via the flexible “GS linker” GGGGSGGGGSGGGGS (i.e., instant SEQ ID NOs: 60 (p40) –3 (linker) – 2 (p35); see sequence fusion below):
[AltContent: textbox (Instant SEQ: 64 vs fusion of instant SEQ: 60 (p40)/SEQ:3 (linker)/SEQ: 2 (p35)
[img-media_image5.png])]
The combination of App’163, Askgene, Wojno, and Chen teaches a method of using an IL-12 masked cytokine wherein a polypeptide chain is comprised of an Fc half-life extension domain fused to a IL-12 cytokine via a cleavable linker, wherein the IL-12 cytokine is comprised of a p40 subunit linked to the p35 subunit using the flexible fusion protein linker GGGGSGGGGSGGGGS, as described in detail above for claim 128.
The combination of Askgene/Wojno/Chen, Merck, and Xencor teaches the IL-12p40 subunit for the IL-12 cytokine for the masked cytokine as described in detail above for claim 129.
The combination of Askgene/Wojno/Chen and UP-IL12A teaches the IL-12p35 subunit of the IL-12 cytokine for the masked cytokine as described in detail for claim 130 above.
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of App’163/Askgene/Wojno/ Chen with the teachings of Merck/Xencor and the teachings of UP-IL12A by using the flexible GS linker GGGGSGGGGSGGGGS (as taught by Askgene/Chen) to join the p40 and p35 subunits to produce a functional IL-12 heterodimeric cytokine (as taught by Wojno, Xencor, and UP-IL12A) for the IL-12 cytokine for a masked IL-12 cytokine (as taught by App’163/Askgene/Wojno/Chen) for IL-12-mediated cancer treatment (as taught by Askgene/Wojno/Chen and Xencor). Further, one would be motivated to link the p40 and p35 subunits using the flexible GS linker, as described in detail above, to facilitate their interaction (as taught by Chen). One of ordinary skill in the art would have a reasonable expectation of success because the combination of prior art elements results in the predictable result of producing a biologically functional IL-12 cytokine and because the p40, flexible linker, and p35 sequences are all taught by the prior art as described above.
Regarding instant claim 134: Jounce teaches antibodies and fusion proteins, wherein the antibody fragment can be an Fc region that is linked to another protein via a linker; wherein the conjugate a specific embodiment has the formula of a drug-linker-antibody conjugate where the drug is a cytotoxic agent that can be a cytokine (p.65, [0245]; p.65, [0242]); and, wherein the linker is a cleavable or noncleavable linker (p.65; [0245]); and, wherein the conjugate can be used in a method of treating cancer, including solid tumors (p.75, [0285]). Jounce also specifically teaches SEQ ID NO: 3 of SGGSGGGSG which is “GS linker” that comprises instant SEQ ID NO: 14 of GGSGGGSG (p.165; Table of Certain Sequences: SEQ ID NO: 3).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of App’163/Askgene/Wojno/ Chen with the teachings of Jounce by modifying the first polypeptide comprising a first half-life extension domain (HL1), a first linker (L1), and a masking moiety (MM) as for a masked IL-12 cytokine (as taught by App’163/Askgene/Wojno/Chen) by using noncleavable linker SGGSGGGSG, as taught by Jounce, to join the masking moiety to the Fc half-life extension domain, to arrive at the instantly claimed invention, because the combination of prior art elements would result in the predictable result of producing an polypeptide where a masking moiety is linked covalently to an Fc half-life extension domain. One of ordinary skill in the art would be motivated to do so because Chen teaches flexible “GS linkers” are used to join functional proteins to create fusion proteins. One of ordinary skill would also have a reasonable expectation of success because App’163, Askgene, Chen, and Jounce teach fusion of functional proteins using flexible GS linkers.
Regarding instant claim 135: CytomX teaches activatable antibodies and methods of manufacturing activatable antibodies that comprise masking moieties (MM), a cleavable moiety (CM), and an antibody or antigen binding fragment thereof (AB) that binds PDL1 for use in cancer treatment, including solid cancers (p.59, col.54, paras.1-3); wherein, the cleavable moiety (CM) is a polypeptide that is cleavable by a protease and activated in the tumor microenvironment (p.164, col.264, para.1); wherein the activatable antibody has the format MM – CM – AB or AB – CM – MM; and, wherein prior to cleavage the MM interferes with binding and when cleaved the MM no longer interferes with binding (p59, col.53, para.1). Further, CytomX specifically teaches multiple cleavable linker sequences comprising instant SEQ ID NO: 44 of ISSGLLSGRS; for example, see CytomX SEQ ID NO: 377, 394, 901-911 (p.50, cols.35 –36 through p.51, col.37; p.158 -- 159, Table 18).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of App’163/Askgene/Wojno/ Chen with the teachings of CytomX by modifying the IL-12 masked cytokine (as taught by App’163/Askgene/Wojno/Chen) to use a cleavable linker comprising ISSGLLSGRS (as taught by CytomX) in order to activate a fusion protein drug for the treatment of solid cancers (as taught by Askgene and CytomX). One of ordinary skill in the art would be motivated to do so because App’163, Askgene and CytomX teach activatable drugs conjugated to antigen-binding fragments wherein the drug is activated via cleavage of a protease-cleavable linker in the tumor microenvironment. One of ordinary skill in the art would have a reasonable expectation of success because CytomX teaches various cleavable amino acid sequences comprising the cleavable polypeptide sequence of ISSGLLSGRS.
As discussed in detail above for instant claim 128, regarding bioactivation of the masked IL-12 cytokine, Askgene specifically teaches that the cytokine moiety polypeptide can be fused to the carrier directly or through a cleavable or noncleavable linker; and, that the masking moiety can be fused to the cytokine or to the carrier through a cleavable peptide linker (p.12, [0050]); and, that the cytokine is activated by cleavage of the linker joined to the masking moiety. Askgene does not specifically teach an embodiment wherein it is explicitly stated that the cytokine’s linker is noncleavable and that the masking moiety’s linker is cleavable. It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of Askgene (along with the teachings of CytomX) to make the cytokine’s linker cleavable and the masking moiety’s linker noncleavable, in order to arrive at the instantly claimed invention, because the combination of prior art elements results in a predictable result of cleavage of either the cytokine or the masking moiety in order to facilitate dissociation of the cytokine from the masking moiety for bioactivation. The decision to cleave the cytokine from the “prodrug” would be obvious to try given that the ultimate goal is to produce a cleavage that would release the cytokine from the masking moiety. One of ordinary skill in the art would have a reasonable expectation of success because making the cytokine’s linker cleavable or the masking moiety’s linker cleavable would predictably result in dissociation of the cytokine and masking moiety and would make the cytokine available for receptor binding and activation in order to treat the solid tumor.
[AltContent: textbox (Instant SEQ: 22 vs instant SEQ: 26 (HL2)
[img-media_image7.png])][AltContent: textbox (Instant SEQ: 22 vs instant SEQ: 25 (HL1)
[img-media_image6.png])] Regarding instant claims 139-140: In addition to the HL1 and HL2 “knobs-in-holes” mutations taught by Askgene above (i.e., Y349C, T366S, L368A, Y407V, S354C, and T366W), instant SEQ ID NO: 25 of instant claim 139 and SEQ ID NO: 26 of instant claim 140 harbor a N>A mutation compared to Fc SEQ ID NO: 22 (see arrows in alignments below):
Thus, the combination of Askgene/Wojno/Chen does not teach an IgG1 Fc sequence (i.e., a backbone sequence) comprising the N>A mutation that, with the “knobs-in-holes” mutation of Askgene, would arrive at a first Fc domain of instant SEQ ID NO: 25 and a second Fc domain of instant SEQ ID NO: 26.
UP-IGG1 teaches the human IgG1 human immunoglobulin gamma-1 heavy chain amino acid sequence P0DOX5 (p.4), which provides the backbone sequence for human IgG1 Fc, except for the “knobs-in-holes” mutations taught by Askgene, and the N>A mutation.
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of App’163/Askgene/Wojno/ Chen with the teachings of UP-IGG1 by using the human IgG1 Fc taught by UP-IGG1 to encode the human IgG1 Fc half-life extension domains and to modify the first and second half-life extension Fc domains to include the “knobs-in-holes” mutations taught by App’163 and Askgene, to arrive at a first Fc half-life extension domain and a second half-life extension domain that arrives at the HL1 and HL2 domains of instant claims 128, 139, and 140 except for the N>A mutations, because the combination of prior art elements teaches the IgG1 sequence and in order to achieve the expected benefit of the “knobs-in-holes”-mediated association between the two half-life extension domains (taught by Askgene) for the IL-12 masked cytokine for cancer treatment (taught by App’163/Askgene/Wojno/Chen). One of ordinary skill in the art would have a reasonable expectation of success because Askgene teaches that the half-life extension domains can be derived from human IgG1 Fc regions, UP-IgG1 teaches the human IgG1 Fc amino acid sequence, and Askgene teaches the specific mutations and positions of the “knobs-in-holes” mutations.
App’163/Askgene/Wojno/Chen and UP-IgG1 do not teach a human IgG1 Fc sequence that comprises the N>A mutation (as it corresponds to instant SEQ ID NO: 22, and amino acid position 77 of the above alignment).
Wang teaches mutations introduced into Fc domains to eliminate FcγR binding in order to avoid or reduce antigen-independent cytokine release syndrome (CRS; p.2013 – 2014). Wang also teaches that human IgG contains a single conserved site in the CH2 domain of Fc, Asn297, for N-linked glycosylation and that glycans at this site determine the interaction between IgG Fc domain and Fcγ receptors. Wang also teaches that the N297A mutation removes the N-glycosylation, thereby preventing binding to Fcγ receptors (p.2013, col.2, para.2).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date to further combine the teachings of App’163/Askgene/Wojno/ Chen and UP-IGG1 with the teachings of Wang by modifying the human IgG1 Fc half-life extension domains to comprise the N>A mutation (i.e., N297A of full IgG1 in instant SEQ ID NO: 22) as taught by Wang, in order to arrive at the instantly claimed invention, in order to receive the expected benefit, as taught by Wang, that the N297A mutation prevents binding of human IgG1 Fc to FcγRs to avoid or reduce CRS with administration of the IL-12 masked cytokine that comprises Fc half-life extension domains. One of ordinary skill in the art would have a reasonable expectation of success because UP-IGG1 teaches the human IgG1 sequence and Wang teaches the position of the N>A mutation.
This is a provisional non-statutory double patenting rejection.
Response to Arguments
Applicant provides documentation supporting that the Karow reference used in the prior art rejections of the non-final action submitted 08/06/2025 does not meet prior art criteria for further examination. See “Assignee showing of ownership per 37 CFR 3.73” document submitted by applicant on 11/05/2025. Thus, the provided documentation overcomes the use of prior art rejections over the Karow reference on the basis of prior art exception 102(b)(2)(C).
Allowable Subject Matter
The masking moiety of instant SEQ ID NO: 65 is free of the prior art. The closest prior art for instant SEQ ID NO: 65 is provided by UniProt. Q99665 -- Human IL-12Rβ2. 05/01/1997, p.1-8 (herein referred to as UniProt-MM), which encodes the human IL-12 receptor subunit beta-2 at 99-7% identity. Neither UniProt-MM nor prior art available prior to applicant effective filing date teaches the C242S mutation nor a motivation for including this mutation in the IL-12Rβ2 protein (see alignment below):
[AltContent: textbox (Instant SEQ: 65 MM (IL-12R β2) vs UniProt Q99665 (human IL-12Rβ2)
[img-media_image9.png])]
Thus, instant SEQ ID NO: 84, which encodes the first polypeptide that comprises the masking moiety of SEQ ID NO: 65 is also free of the prior art.
The cleavable linker of instant SEQ ID NO: 53 is also free of the prior art. The closest prior art is provided by SEQ ID NO: 505 of Moore, et al. – WO2016118629A1 (publication date: 07/28/2016; effective filing date: 01/20/2015; herein referred to as Moore), which has 82.3% identity to instant SEQ ID NO: 53 (see alignment below):
[AltContent: textbox (Instant SEQ: 53 vs Moore SEQ ID NO: 505
[img-media_image10.png])]
Thus, instant SEQ ID NO: 94, which encodes the second polypeptide that comprises linker SEQ ID NO: 53 is also free of the prior art.
As allowable subject matter has been indicated, applicant's reply must either comply with all formal requirements or specifically traverse each requirement not complied with. See 37 CFR 1.111(b) and MPEP § 707.07(a).
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/JAMI MICHELLE GURLEY/Examiner, Art Unit 1647
/JOANNE HAMA/Supervisory Patent Examiner, Art Unit 1647