DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant’s election of Group I in the reply filed on 7/31/2025 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Alternative Names
Dipeptidyl peptidase4 is also referred to as DPP4, DPPIV, DPP IV and CD26 here and in the literature. (See also paragraph [0004] of the specification.)
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 deficiencies and the required response to this Office Action are as follows:
A) Specific deficiency - The Incorporation by Reference paragraph required by 37 CFR 1.821(c)(1) is missing or incomplete. See item 1) a) or 1) b) above.
B) Specific deficiency – Nucleotide and/or amino acid sequences appearing in the specification are not identified by sequence identifiers in accordance with 37 CFR 1.821(d). See the last line of [0019], fourth line of [0053], third line of [0119].
Required response – Applicant must 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.
Claim Interpretation
Claim 25 is drawn to “A method of treating cancer in a subject in need thereof”. It reasonably appears this means the subject has cancer because the treatment is of "cancer in a subject”. Therefore, as the specification defines “treating” in [0036] as including “blocking,… or preventing symptoms associated with a condition”, the prevention is not of the cancer per se but of the symptoms thereof. The claim is being interpreted as meaning that the subject being treated already has cancer.
Specification
The disclosure is objected to because of the following informalities: In [0013], there is no period at the end. In [0023], line 1, “inhibits proliferation Hela” does not make sense. Further, in [0023], [0118], the cell line name should be “HeLa” not “Hela” (see NIH, https://osp.od.nih.gov/hela-cells/, Oct. 2022, attached). In [0129], line 1, “In order to assessed whether the mutants still able…” should be “…to assess whether the mutants are still…” In [00128], line 6, a period is missing after the end parenthesis.
Appropriate correction is required.
The use of the term MILLIPORE (end of [00113]), which is a trade name or a mark used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01. See [0129].
Claim Objections
Claims 14 and 25 are objected to because of the following informalities: Claim 14, recites “immunoglobulin” then separately recites “Ig”. As exemplified in claim 6, the first occurrence of “immunoglobulin” should be accompanied the abbreviation “Ig” if the abbreviation will later be used on its own. Claim 25 recites in line 2, “therapeutically amount”, which is incorrect. It appears a word such as “effective” may have been omitted before “amount”. Appropriate correction is required.
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 1, 9, 12, 20 and dependent claims 3, 6-8, 23 and 25 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 is indefinite because it is drawn to a modified CXCL9 polypeptide but in line 3 recites “a corresponding wild type CXCL9”. It is unclear what the word “corresponding” means. For example, does it include other differences from the wild type CXCL9. If it is intended that the modified CXCL9 polypeptide has an insertion at the N-terminus of a wild type CXCL9 polypeptide, then that should be clarified, for example, by deleting “corresponding”. Otherwise, the term “corresponding” should be further defined.
Claim 1 is also indefinite because it is unclear what is meant by the “N-terminus” of wild type CXCL9. The specification defines “wild type” CXCL9 as including “full-length” CXCL9 ([0008]). Human CXCL9 has the sequence of SEQ ID NO:8 ([0043]), which includes a 22 amino acid signal sequence. Therefore, it reasonably appears the N-terminus CXCL9 is position 1 of, for example, SEQ ID NO:8, i.e., the full-length protein. However, the specification also says ([0127]), “Sequence of Gln, Asn or Pro codons were placed immediately after the glycine at position 22 (the cleavage site of the signal peptide) to assure that it is placed at position 0 of the N-terminus CXCL9.” Therefore, it is unclear if the N-terminus refers to the N-terminus of the full-length CXCL9, the mature form thereof, i.e., after cleavage of the signal peptide, or either (see [0156]-[0157]).
Claim 9 recites the limitation "the immunoglobulin or the fragment thereof" in line 2. There is insufficient antecedent basis for this limitation in the claim. It appears this claim may have been intended to depend from claim 6.
Claims 12 and 20 are indefinite because it is unclear what is meant by “inducing CD8+ T cells.” It is unclear what they are being induced to do or to become. As a result, the metes and bounds of the claim are not clear. This rejection could be obviated if appropriate by specifying instead that the activity of the CD8+ T cell is potentiated (see [0013]).
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1, 3, 6-9, 12, 23 and 25 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claim 1 is drawn to “A modified CXCL9 polypeptide, comprising an insertion of an additional amino acid at the N-terminus of a .. wild type CXCL9.” There are two issues for claim 1 and dependent claims relating to a lack of written description. The first deals with the location of the insertion being at the N-terminus of the full-length wildtype CXCL9. The second deals with the inclusion of multiple amino acid insertions.
The specification (first paragraph of [0038]) defines a “wild type” CXCL9 polypeptide as being “naturally occurring CXCL9” and “un-modified CXCL9”. “The terms refer to the naturally occurring form of CXCL9 (i.e., an endogenous, non-mutated CLCL9 or full-length CXCL9).” The human form of wild type CXCL9 has the sequence of SEQ ID NO:8 ([0043]). However, the only insertion of an additional amino acid at the N-terminus of a wild type CXCL9 is shown as an insertion at the N-terminus of the mature CXCL9 protein, i.e., after removal of the signal sequence (see, e.g., SEQ ID NO:1-4). The disclosure is drawn to stabilized forms of CXCL9 (e.g., [0006]-[0007]). The claims include the “full-length” CXCL9 as defined by the term “wild type”. That means including the signal sequence. Therefore, “insertion of an additional amino acid at the N-terminus of ..a wild type CXCL9” includes insertion at the actual N-terminus, i.e., before the signal sequence of the full-length polypeptide. Martoglio et al. (Trends Cell Biol. 8:410-, 1998; p. 410, paragraph bridging cols.1-2) explains that signal peptides are short amino acid sequences, typically at the N-terminus, followed by the mature region of the protein and act to designate an extracytoplasmic location for proper routing of the protein in a bacterial or eukaryotic cell (to the plasma membrane of bacteria and endoplasmic reticulum membrane in eukaryotes). The signal peptide is cleaved by a signal peptidase after membrane insertion. Signal peptides are 15 to more than 50 amino acids in length, but have a 6-15 amino acid hydrophobic core and C-terminal region where cleavage occurs (p. 410, col. 2, first full paragraph, and Fig. 1). One skilled in the art would have reasonably expected that an amino acid added to the N-terminus of a full-length protein, i.e., to the N-terminus of the signal sequence, would have been cleaved when the signal sequence was cleaved, leaving the mature protein with its original sequence. As explained by the specification, peptidase DPP4 cleaves CXCL9 and CXCL10 at the proline at position 2 of the mature protein, resulting in non-functional CXCL9 and CXCL10 ([0004]). Note there is no proline in the signal sequence of wild type CXCL9 (SEQ ID NO:8). The cleavage mechanism of DPP4 means that for CXCR3 ligands CXCL9 and CXCL10, the ligand from which a modified signal peptide was cleaved would still have been subject to DPP4 cleavage of the unmodified mature protein and loss of activity because when the modified signal sequence was cleaved, the mature CXCL ligand would still have a proline at position 2. The specification does not disclose encompassed modified CXCL9 polypeptides having an insertion of an additional amino acid at the N-terminus of the corresponding full-length wild type CXCL9, e.g., of SEQ ID NO:8. It does, however, disclose a modified CXCL9 wherein the insertion is at the N-terminus of the mature protein (e.g., after amino acid 22 of SEQ ID NO:8, see SEQ ID NO:1-4), which meets the written description provision of 35 USC 112(a). Also, it is clear if multiple amino acids added at the N-terminus of the mature protein would affect CXCR3 binding or activity thereof.
Further, because claim 1 uses the open language “comprising” before “an insertion of an additional amino acid”, this means one or more additional amino acids. The specification discloses only insertion of a single amino acid in the human CXCL9, resulting in a protein with the sequence of SEQ ID NO:1, the sequence of full-length human CXCL9 wherein position 23 is any naturally occurring amino acid. There is no disclosure of a modified CXCL9 with multiple amino acids inserted immediately after the signal sequence.
Vas-Cath Inc. v. Mahurkar, 19USPQ2d 1111 (Fed. Cir. 1991), clearly states that “applicant must convey with reasonable clarity to those skilled in the art that, as of the filing date sought, he or she was in possession of the invention. The invention is, for purposes of the ‘written description' inquiry, whatever is now claimed.” (See page 1117.) The specification does not “clearly allow persons of ordinary skill in the art to recognize that [he or she] invented what is claimed.” (See Vas-Cath at page 1116).
Therefore, only a modified CXCL9 polypeptide comprising a single insertion of an additional amino acid after the signal peptide, but not the full breadth of the claim meets the written description provision of 35 U.S.C. § 112(a). Applicant is reminded that Vas-Cath makes clear that the written description provision of 35 U.S.C. § 112 is severable from its enablement provision (see page 1115).
Claim 25 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a method of treating cancer in a subject in need thereof by administrating a therapeutically effective amount of a pharmaceutical composition comprising a modified CXCL9 polypeptide comprising a single insertion of an additional amino acid at the N-terminus of the mature protein, that is, immediately after the signal peptide, does not reasonably provide enablement for wherein the modified CXCL9 polypeptide has additionally inserted amino acids or wherein the additional amino acid is at the N-terminus of the full-length CXCL9. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
Claim 25 is drawn to “A method of treating cancer in a subject in need thereof…” There is an issue of enablement with which of the encompassed modified CXCL9 polypeptide can be use in the method. As discussed in the rejection under 35 USC 112(a) above, claim 1, from which claim 25 ultimately depends, is drawn to “A modified CXCL9 polypeptide, comprising an insertion of an additional amino acid at the N-terminus of a .. wild type CXCL9.” This includes insertion at the actual N-terminus of the full-length protein (see [0008] for the definition of “wild type”). Because CXCL9 comprises an N-terminal signal sequence, a 22 amino acid sequence for human CLXCL9 of SEQ ID NO:8 (see also [0127]), the addition may be at the N-terminus of the signal sequence. This would reasonably be expected by the skilled artisan to result in cleavage of the signal peptide with its additional N-terminal amino acid, still leaving the mature CXCL9 which would be subject to DPP4 peptidase cleavage. As discussed in the specification ([0004]), DPP4 cleaves the 2 N-terminal amino acids of the mature protein which results in nonfunctional CXCL9. Further, because claim 1 uses the open language “comprising” before “an insertion of an additional amino acid”, this means ‘one or more additional amino acids’. In order to function, CXCL9 must bind its receptor, CXCR3. Mature human CXCL9 is only 173 amino acids long. It is unpredictable how many amino acids and which ones could be added to the N-terminus while retaining sufficient receptor binding activity for therapeutic use. The specification has working examples only in which a single amino acid is added.
One skilled in the art would not know how to use a nonfunctional CXCL9 and the specification does not provide guidance or direction for such use. Additionally, not only must the CXCL9 be able to bind CXCR3, it must do so with sufficient biophysical properties to promote treatment of cancer. There are no working examples of treatment of cancer with a full-length or mature CXCL9 modified by addition of an N-terminal amino acid, let alone with a nonfunctional CXCL9. In contrast, the specification states in [0006], “There is a need to develop modified CXCL9 polypeptides that will be stable and efficient as an anticancer drug. Further, there is a need to develop a stabilized and modified CXCL9 that will be resistant to DPP4 cleavage.” Therefore, it reasonably appears that in order for a modified CXCL9 protein to be used in a method of treating cancer, it would need an insertion of a single additional amino acid at the N-terminus of the mature protein, that is, after the signal peptide.
Priority
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 120. The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
The disclosure of the prior-filed application, Application No. US 63/041,940, fails to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application. This application does not disclose insertion of an additional amino acid at the N-terminus of a wild type CXCL9. Therefore, the benefit of priority for claims 1, 3 and 5-9, 12, 23 and 25 is extended only to US 63/150,629 (filed 2/18/2021).
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim(s) 1, 3, 6, 8, 12, 14, 17, 20 and 23 is/are rejected under 35 U.S.C. 102(a)(2) as being anticipated by US 2022/0017585 A1 (Yamamoto).
Yamamoto teaches a CXCR3 ligand having resistance to DPP IV and activity to cause migration of cells expressing CXCR3 (claims 1-2). Claim 16 teaches a method of conferring resistance to DPP IV on a parent CXCR3 ligand in which the 2nd amino acid from the N-terminus is P, wherein the method comprising further adding Q, E, D or P to the N-terminus. The CXCR3 ligand that is made to have resistance to DPP IV is a CXCL9 variant ([0328], [0334] and [0337]). It would reasonably be expected to bind CXCR3 because that is an inherent activity of CXCL9. Yamamoto also teaches wherein the CXCR3 ligand is fused to an antibody Fc region via a linker, as wells as a pharmaceutical composition of the modified ligand ([0120]).
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 1, 3, 5-9, 12, 14-17, 20, 23 and 25 is/are rejected under 35 U.S.C. 103 as being unpatentable over US 2022/0017585 A1 (Yamamoto) as applied to claims 1, 3, 6, 8, 12, 14, 17, 20 and 23 above, and further in view of Metzemaekers et al. (Int. J. Mol. Sci. 18:1513, 2017), Bronger et al. (Canc. Metast. Rev. 38:417-430, Sept. 2019, cited in the IDS filed 6/30/2023), Karin et al. (Cytokine, 109:24-28, 2018), US 7,083,784 (US ‘784) and GenBank Database, Accession No. NP_002407 (version NP_0024071, C-X-C motif chemokine 9 precursor [Homo sapiens], 30 Dec. 2018).
Yamamoto teaches a CXCR3 ligand having resistance to DPP IV (DPP4) and activity to cause migration of cells expressing CXCR3 (claims 1-2). Claim 16 teaches a method of conferring resistance to DPP IV on a parent CXCR3 ligand in which the 2nd amino acid from the N-terminus is P, wherein the method comprising further adding Q, E, D or P to the N-terminus. The CXCR3 ligand that is made to have resistance to DPP IV is a CXCL9 variant ([0328], [0334] and [0337]). Figure 1B shows insertion of an amino acid (b) to disrupt the DPP IV cleavage site of CXCL10. Fig. 4B shows insertion of an amino acid (b) to disrupt the DPP IV cleavage site of CXCL11. Both these sites are at the N-terminus of the mature protein, i.e., immediately after the signal peptide sequence. Yamamoto also teaches wherein the CXCR3 ligand is fused to an antibody Fc region via a linker, as wells as a pharmaceutical composition of the modified ligand ([0120]). Yamamoto does not teach insertion of an amino acid at the N-terminus of CXCL9 resulting in an amino acid sequence of one of SEQ ID NO:1-4 or wherein the modified CXCL9 polypeptide is linked to an Ig molecule. An activity of the modified CXCL9 binding CXCR3 and/or inducing CD8+ T cells is not explicitly taught. Yamamoto does not teach treatment of cancer by administration of the modified CXCL9 polypeptide.
Metzemaekers et al. teaches on p. 3, second paragraph:
An enzyme that has been shown to provoke NH2-terminal processing of various chemokines including CXCL9, CXCL10 and CXCL11 is dipeptidyl peptidase IV or CD26 [50,52,53]. In addition to its enzymatic activity as a serine protease, the multifunctional or “moonlighting” protein CD26 functions as a receptor, costimulator for T cell activation, adhesion molecule and has been associated with apoptosis [54–57]. The membrane-bound enzyme is expressed on cells of different origins, including certain immune cells, whereas soluble proteolytically active CD26 exists in several body fluids such as plasma and seminal fluid. CD26 preferentially removes the two most NH2-terminal amino acids from substrates whose penultimate position is occupied by a (hydroxy) proline or alanine residue. Pro is present at this position in a number of chemokine sequences. The NH2-terminal chemokine domain is responsible for GPCR binding and activation and, consequently, limited proteolysis by CD26 (but also by other enzymes) may have drastic effects on the biological functioning of a chemokine [50–52]. It turned out that the biological effect of CD26-mediated cleavage is highly complex and depends on the chemokine ligand involved. For all three CXCR3 agonists, it was previously demonstrated that processing by CD26 results in drastic loss of receptor signaling and impaired capacity to direct lymphocyte chemotaxis, while leaving the angiostatic properties of these chemokines unaffected [53]. For human CXCL10 and CXCL11, the corresponding CD26-truncated isoforms CXCL10 (3–77) and CXCL11 (3–73) were previously isolated from natural sources, including conditioned medium from MG-63-osteosarcoma cells, fibroblasts and keratinocytes [22,58–61].
Bronger et al. taught that the CXCR3 and CXCR1 ligands, CXCL9-11 and CX3CL1, respectively, are mainly responsible for tumor-suppressive lymphocyte (TIL) infiltration into the tumor microenvironment (second paragraph of Abstract). CXCR3-expressing cells include Tregs, CD4+ and CD8+ T cells, dendritic cells NK and NKT cells (p. 419, col. 2, second paragraph). “Raising the intratumoral concentration of intact and functional CXCR3 chemokines, e.g., by inhibition of their proteolytic inactivation, might thus kill two birds with one stone: tumor-suppressive immune cells would be attracted to the tumor site, and CXCR3-positive tumor cells would be chemotactically prevented from escaping the primary cancer. This idea is supported by both preclinical and clinical findings…. Moreover, in human cancers, overexpression of CXCL9 and CXCL10 is associated with a higher number of tumor-infiltrating lymphocyte and improved survival, e.g., in breast, ovarian, colon, lung, and several other cancers [67, 76-83].” (p. 421, col. 1, second paragraph) CXCR3 chemokines also contribute to antitumor activity of multiple current therapeutics such as immune checkpoint inhibitors of the PD-1/PD-L1 axis, as well as CDK4/6 inhibitors and poly[ADP-ribose] polymerase 1 inhibitors (p. 421, paragraph bridging cols. 1-2). CXCR3 chemokine inactivation presents a cancer treatment resistance mechanism, “which renders inhibitors of CXCR3-chemokine cleaving proteases feasible adjuvants to all these therapies.” (p. 421, col. 2, end of first paragraph) In line with this, dipeptidyl peptidase 4 (DPP4) removes N-terminal amino acids from CXCR3 chemokines (p. 423, last paragraph). It has already been shown that (p. 425, col. 1, last paragraph), “In syngeneic models of melanoma and colorectal cancer (B16F10 and CT26 models, respectively), inhibition of DPP4 by sitagliptin or DPP4 knockout led to enhanced T cell infiltration, impaired tumor growth and less metastatic spread [19].” Also, in a xenograft and fully immunocompetent model of hepatocellular carcinoma, inhibition of DPP4 impaired tumor growth through enhanced CXCR3-mediated NK and T cell infiltration (p. 425, col. 1, last paragraph). It is concluded that, “proteolytic cleavage of the tumor-suppressive CXCR3 and CX3CR1 chemokines impairs their functions and, on top of this, in feedback loops, the chemokines may even lead to increased expression of the proteases targeting themselves.”
Karin et al. teaches that chemokine receptor CXCR3-A binds CXCL9, CXCL10 and CXCL11 and is expressed primarily on activated T lymphocytes and NK cells (p. 25, col. 2, second and start of third paragraph). CXCL11 binds a different site on CXCR3 than do CXCL9 and CXCL10 (p. 25, col.2, third paragraph). It is further taught (paragraph bridging pp. 25-26) that chemokines have very short in vivo half-lifes and as a result need to be stabilized for effective therapeutic use. To accomplish this chemokine-Ig based fusion proteins were made. The researchers had already shown that generation of an Ig based fusion protein of chemokine CCL1 (CCL1-Ig) increased in vivo half-life time [32]. This has also been shown for an Ig stabilized cytokine, IL-31 [64]. “We therefor suggest using CXCL10-Ig for cancer therapy.” The reasoning was that CXCL10 induced effector Th1 cells and has been associated with recruitment of CXCR3+ CD8+ T cells to a tumor site and Granzyme B production by these T cells, leading to potentiation of antitumor activity (Fig. 1 and p. 26, col. 2, first full paragraph). Further, CXCL10-Ig had been shown in mice engrafted with myeloma tumor cells to increase effector CD4, CD8 and NK cells at the tumor site, reduce Tregs and “significantly reduce accumulation of myeloma [65]”. Separately it has been shown that inhibition of DPP4 increased CXCL10 levels and suppressed experimental melanoma (p.26, col. 2, first full paragraph).
US 7,083,784 teaches IgG hinge-Fc regions from human having the amino acid sequence of SEQ ID NO:83 (col. 6, lines 61-63, and Fig. 2; which is identical to instant SEQ ID NO:5). The prior art discloses fusion with the IgG hinge-Fc increased in vivo half-life of the non-IgG Fc protein of the fusion (col. 3, lines 6-11), Additionally, modified IgG-Fc fusions are taught with increased in vivo half-lives (e.g., col. 31, lines 56-64, and claims 1-4) and wherein the IgG-Fc is linked to the bioactive protein, which linkage may be by a linker sequence (col. 34, lines 59-67, and col. 37, lines 10-14).
GenBank Database, Accession No. NP_002407 teaches the sequence of C-X-C motif chemokine 9 precursor from human, also known as CXCL9, Humig and MIG (CDS section). The signal peptide is amino acids 1-22 (sig_peptide).
It would have been obvious to the artisan of ordinary skill before the effective filing date of the instant invention wherein CXCL9 had a human sequence as shown in GenBank Database Accession No. NP_002407 because of the role the chemokine appears to play in human cancers (e.g., Bronger et al. and Karin et al.). As taught by Yamamoto, inserting an amino acid in the N-terminus of the mature CXCR3 ligands results in resistance to peptidase DPP4, thereby maintaining CXCR3 activity. As Yamamoto also taught wherein the inserted amino acid may be glutamine, asparagine or proline (see claims), modified human CXCL9 polypeptides would have the sequence of one of instant SEQ ID NO:1-4. The use of such a modified CXCL9 polypeptide in a pharmaceutical composition for the treatment of cancer would have been obvious because activation of CXCR3 is needed for such treatment based on administration of its ligand(s). The prior art supports the negative effect of DPP4 by cleavage of CXCR3 ligands, resulting in inactive ligands; therefore, use of the modified, noncleavable DPP4 ligand(s) would allow treatment of cancer. There would have been a reasonable expectation of success in view of the teachings of Bronger et al. that in human cancers, overexpression of CXCL9 and CXCL10 is associated with a higher number of tumor-infiltrating lymphocyte and improved survival, e.g., in breast ovarian, colon, lung, and several other cancers. It further would have been obvious wherein the modified CXCL9 polypeptide was linked to an IgG-Fc as taught generally by US ‘784 to increase the half-life of the CXCL9, including wherein the IgG-Fc was the human hinge-CH2-CH3 sequence of SEQ ID NO:11 of US ‘784 so as to reduce the chance of a negative immunogenic reaction if administered in a human. A CXCL9-Ig fusion is supported by Karin et al., which taught the advantages of having a CCL1-Ig with increased in vivo half-life time and which suggested treatment of cancer with a CXCL10-Ig fusion and pointed to the prior art results showing CXCL10-Ig increased effector CD4, CD8 and NK cells at the tumor site in mice engrafted with myeloma tumor cells. Use of CXCL9 in this context would have been obvious and desirable because of the teachings of Bronger et al. that CXCR3 and CXCR1 ligands, CXCL9-11 and CX3CL1, respectively, are mainly responsible for tumor-suppressive lymphocyte (TIL) infiltration into the tumor microenvironment and that CXCR3-expressing cells include Tregs, CD4+ and CD8+ T cells, dendritic cells NK and NKT cells (p. 419, col. 2, second paragraph). This is also obvious in view of the suggestion by Bronger et al. (p. 421, col. 1, second paragraph) of “Raising the intratumoral concentration of intact and functional CXCR3 chemokines, e.g., by inhibition of their proteolytic inactivation, might thus kill two birds with one stone: tumor-suppressive immune cells would be attracted to the tumor site, and CXCR3-positive tumor cells would be chemotactically prevented from escaping the primary cancer. This idea is supported by both preclinical and clinical findings….” Also as taught by US ‘784, it would have been obvious wherein the linkage between the Ig-Fc and bioactive protein (CXCL9 in this case) was a linker sequence. Increase in half-life would reasonably have been expected to enhance the positive antitumor effects of CXCR3 because Karin et al. and Bronger et al. respectively suggested increased activation of CXCR3 by a CXCL-Ig fusion and inhibition of DPP4, both of which function to increase time that the CXCR3 ligand can bind and activate its receptor, for treatment of cancers.
Claim(s) 14-17 and 20 is/are rejected under 35 U.S.C. 103 as being unpatentable over Zohar et al. (J. Con. Invest. 124(5):2009-2022, 2014, cited in the IDS filed 6/20/25) in view of Karin et al. (Cytokine, 109:24-28, 2018).
Zohar et al. teaches (p. 2009, col. 1, second paragraph and col. 2, first full paragraph) the G protein-coupled receptor (GPCR) CXCR3 binds ligands CXCL9, -10 and -11. CXCL11 binds to CXCR3 at a different site than CXCL9 and -10. It was shown that CXCL11 and CXCL10 have different effects, with CXCL10 polarizing effector Th1 immune cells and CXCL11 polarizing native T cells into IL-10hi Tregs (p. 2009, col. 2, end). Zohar et al. made fusion proteins of murine CXCL11 or CXCL10 linked to murine IgG1 Fc (hinge-CH2-CH3; CXCL11-ig and CXCL10-Ig), which preserved the biological properties of each chemokine, stabilized and prolonged in vivo half-life (p. 2014, col. 2, first full paragraph, and p. 2020, col. 1, last paragraph). Each fusion maintained CXCR3 binding and the ability to attract CXCR3+ T cells, while anti-cytokine specific antibodies inhibited this migration (p. 2014, col. 2, first full paragraph). The fusions were tested on an EAE murine model of inflammatory autoimmune disease (p. 2014, last paragraph through p. 2018, col. 1, second paragraph). Different activities of different ligands on the same receptor are discussed (p. 2018, col. 1, third paragraph, and col. 2, fourth paragraph), with the conclusion that, “Whereas CXCL10 (and possibly CXCL9) drives Th1/Th17 polarization by signaling via STAT4 (and perhaps STAT5), CXCL11 not only competes with these proinflammatory activities, but can directly activate, via STAT3, mTOR, and STAT6 pathways, leading to enhanced polarization of FOXP3–IL-10hi- and IL-4hi-producing Tregs.” The advantage of using a cytokine-Ig fusion is described as likely extending in vivo half-life while maintaining biological activity, as well as being able to use low doses due to the extended half-life (p. 2019, col. 1, last paragraph). It is concluded (p. 2019, col. 2, end of first full paragraph), “Whether these findings with CXCR3 and its distinct ligands can be extended to other different autoimmune diseases, as well as to other chronic inflammatory processes, remains to be investigated.” Zohar et al. does not teach a CXCL9-immunoglobulin conjugate.
Karin et al. teaches that chemokine receptor CXCR3-A binds CXCL9, CXCL10 and CXCL11 and is expressed primarily on activated T lymphocytes and NK cells (p. 25, col. 2, second and start of third paragraph). CXCL11 binds a different site on CXCR3 than do CXCL9 and CXCL10 (p. 25, col.2, third paragraph). It is further taught (paragraph bridging pp. 25-26) that chemokines have very short in vivo half-lifes and as a result need to be stabilized for effective therapeutic use. To accomplish this chemokine-Ig based fusion proteins were made. The researchers had already shown that generation of an Ig based fusion protein of chemokine CCL1 (CCL1-Ig) increased in vivo half-life time [32]. This has also been shown for an Ig stabilized cytokine, IL-31 [64]. “We therefor suggest using CXCL10-Ig for cancer therapy.” The reasoning was that CXCL10 induced effector Th1 cells and has been associated with recruitment of CXCR3+ CD8+ T cells to a tumor site and Granzyme B production by these T cells, leading to potentiation of antitumor activity (Fig. 1 and p. 26, col. 2, first full paragraph). Further, CXCL10-Ig had been shown in mice engrafted with myeloma tumor cells to increase effector CD4, CD8 and NK cells at the tumor site, reduce Tregs and “significantly reduce accumulation of myeloma [65]”. Separately it has been shown that inhibition of DPP4 increased CXCL10 levels and suppressed experimental melanoma (p.26, col. 2, first full paragraph).
It would have been obvious to have a fusion protein comprising CXCL9 conjugated to an immunoglobulin molecule or fragment thereof because both Zohar et al. and Karin et al. taught functional Ig fusion of cytokines and of chemokines that had longer half-lives. It would have been obvious to substitute one known element for another, e.g., one CXCL for another. As stated above Zohar et al. acknowledges that further investigation is necessary to determine if their findings with CXCR3 and its distinct ligands can be applied to other autoimmune diseases. Further, Karin et al. suggest CXCL10-Ig may be used for treatment of cancers. The prior art provides motivation to make and use a CXCL9-Ig fusion for investigation of these possibilities. Further, because ultimately most therapeutics are designed for human use, it would have been obvious wherein the Ig, e.g., hinge-CH2-CH3, were of human origin to reduce the chance of undesirable immune response in humans. It further would have been desirable to have a linker between the chemokine and Ig, such as a glycine- and serine-containing linker, in order to increase flexibility between the two components to reduce the chance of steric hindrance during receptor binding. Because the experiments of Zohar et al. and Karin et al. support CXCR3 binding by the CXCL11-Ig and CXCL10-Ig fusion, one of ordinary skill in the art would reasonably have expected a CXCL9-Ig fusion to also bind CXCR3.
Prior Art
The prior art made of record and not relied upon is considered pertinent to Applicant's disclosure.
Chow et al. (Immun. 50:1498-1512, 2019, cited in the IDS filed 6/30/25) teach that CXCL9 activity is critical to anti-PD-1 antibody antitumor activity. This was shown both by using CXCL9-/- mice as well as inhibiting CXCL9 activity with an anti-CXCL9 antibody (p. 1504, col. 1, first and second full paragraphs). It has also been found that CXCL9 expression correlated with clinical response to anti-PD-1 alone and with anti-CTLA-4 immunotherapy in melanoma patients (p. 1506, col. 1, first full paragraph).
Barreira de Silva et al. (Nat. Immunol 16:850-858, 2015, cited in the IDS filed 6/30/25) teach that mice null for DPP4 have lower tumor volume compared to wildtype mice in a B16F10 melanoma cell cancer model (Fig. 1). DPP4 inhibition by sitagliptin enhanced CXCR3-mediated immunity to CT26 tumors in mice (Fig. 3). This reference completely ignores the activity of DPP4 on CXCL9 and its possible contribution to the antitumor effect of DPP4.
Conclusion
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Claire Kaufman
/Claire Kaufman/
Primary Examiner, Art Unit 1674
September 26, 2025
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