DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Response to Amendments and Arguments
2. Claims 1-3, 42, 43, 48 and 62-83 are pending.
Claims 43 and 48 are withdrawn from consideration as being drawn to non-elected invention.
Claims 1-3 and 78 have been amended.
Claims 1-3, 42 and 62-83 are examined on the merits.
3. The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Withdrawn Grounds of Rejection
Claim Rejections - 35 USC § 112
4. The rejection of claims 1-3, 42 and 62-83 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or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention is withdrawn in light of the amendment to claims 1-3 and 78 deleting the unclear term, “material”, see Amendments to the Claims, pages 2, 4 and 5.
Claim Rejections - 35 USC § 102
5. The rejection of claim(s) 1-3, 42, 66-74 and 83 under 35 U.S.C. 102(a)(1) as being anticipated by Ferrone et al., WO 2016/164408 A1 (published 13 October 2016/ IDS reference B on sheet 1 submitted May 5, 2025) is withdrawn in light of Applicant’s amendment to claim 1, see Amendments to the Claims submitted December 15, 2025 and corresponding Remarks on page 10, 1st paragraph.
Claim Rejections - 35 USC § 103
6. The rejection of claim(s) 1-3, 42 and 62-83 under 35 U.S.C. 103 as being unpatentable over Ferrone et al., WO 2016/164408 A1 (published 13 October 2016/ IDS reference B on sheet 1 submitted May 5, 2025), and further in view of Szatanek et al., (International Journal of Molecule Medicine 36: 11-17, 2015) and Yang et al., (Clinical and Developmental Immunology 2011 (article id 842849) 1-11, published online 30 November 2011) is withdrawn in light of Applicant’s amendment to claim 1, see Amendments to the Claims submitted December 15, 2025 and corresponding Remarks on page 11.
New and Maintained Grounds of Rejection
Claim Rejections - 35 USC § 112
7. 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.
8. Claim 3 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. THIS IS A NEW MATTER REJECTION.
Applicant has amended the wherein clause of claim 3 to recite “the patient is not likely to benefit from treatment with immunotherapy”. This conclusion is drawn once the method of detecting one or more melanoma-cell derived exosomes (MTEX) with antibodies or antigen-binding fragments that specifically bind chondroitin sulphate proteoglycan 4 (CSPG4) epitopes on melanoma exosomes and a finding and evaluation of protein cargo comprising immunosuppressive proteins at a ratio of MTEX to non melanoma-cell derived exosomes (nonMTEX) of 20% to 60% is indicative of a melanoma diagnosis.
Applicant’s disclosure does not support this experimental design. A review of the disclosure does not note a finding of MTEX exosomes and protein cargo comprising immunosuppressive proteins at a ratio of MTEX to nonMTEX of 20% to 60% will render a patient not likely to benefit from immunotherapy. In fact, the specification states “… a finding that the material bound to the antibody, antigen-binding fragment, or ligand comprises CSPG4 identifies the patient as likely to benefit from treatment with immunotherapy”, see page 2, lines 16-18, 28-30; and page 13, #2, 3, 6, 7.
Applicant is requested to identify support for the amendment by page, paragraph, sentence and/or line number or delete the new matter.
9. 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.
10. Claims 73-76 recite the limitation "the one or more tumor cell-derived exosomes…" in lines 1 and 2. There is insufficient antecedent basis for this limitation in the claim.
Claim Rejections - 35 USC § 101
11. 35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
12. The claimed invention continues to be directed to a judicial exception (i.e., a law of nature, a natural phenomenon, or an abstract idea) without significantly more. Claim(s) 1-3, 42 and 62-83 continue to be directed to a judicial exception.
Applicant disagrees with the instant rejection in view of the current amendments submitted December 15, 2025. Applicant asserts “[t]he present claims do not recite a judicial exception”, however does “…integrate a practical application” and an “…includes an inventive concept”, see pages 7-9 of the Remarks submitted December 15, 2025. More specifically, Applicant argues “…the present claims as a whole indicates…the claims are not directed solely to a judicial exception” and “…recite [an] ordered combination of steps”, see page 7, 3rd paragraph (para.) and para. bridging pages 7 and 8.
Applicant further asserts the present invention reads on a method, wherein “…a comparison of the phenotype and functional properties of melanoma-cell derived exosomes [MTEX] and non melanoma-cell derived exosomes [nonMTEX] show that MTEX carry an abundance of immunosuppressive proteins and inhibit numerous functions of human primary T cells and natural killer cells.”, see page 8, 2nd para. Applicant avers commercially available anti-CSPG4 antibodies failed “…to meet the standards for tumor cell specificity and could not be reliably used for immune capture of MTEX”, see page 8, last para.
Applicant concludes arguments noting, “the step of isolating MTEX bound by the antibodies or antigen-binding fragments thereof which specifically bind distinct and spatially distant CSPG4 epitopes on MTEX is not a conventional method for isolating MTEX. Moreover, the amended claims specifically recite that a finding of protein cargo comprising immunosuppressive proteins, and a ratio of MTEX to nonMTEX of 20% to 60%, identifies the patient as having melanoma. Prior to Applicant's discovery, the ratio of MTEX to nonMTEX could not have been determined without using techniques that involve an inventive step over traditional, known methods in the art; thus, the amended claims engender an inventive
concept.
Therefore, the present claims include elements and combinations of elements that amount to an inventive concept that ensures that the claims as a whole amount to significantly more than any judicial
exception, such that the present claims are also eligible under Step 2B of the patent eligibility analysis.”, see page 9.
Applicant’s arguments and amended claims have been carefully considered, but fail to persuade.
Contrary to Applicant’s assertions, the claims as amended continue to recite a judicial exception. The whole invention still relies on the presence of one or more MTEX within a patient’s sample, wherein detecting CSPG4 epitopes on the MTEX and the analysis of protein cargo at a ratio of MTEX to nonMTEX of 20% to 60% indicates whether or not the patient has melanoma.
The Federal Circuit has noted that “a claim directed to a newly discovered law of nature . . . cannot rely on the novelty of that discovery for the inventive concept necessary for patent eligibility; instead, the application must provide something inventive, beyond mere well-understood, routine, conventional activity.” Id., see Alice Corp. Pty. Ltd. v.
CLS Bank Int’l, 134 S. Ct. 2347, 2355 (2014). Notwithstanding, detecting one or more MTEX in a patient having melanoma with the detection of spatially distinct CSPG4 with antibodies or antigen-binding fragments thereof and protein cargo of the MTEX is not an inventive concept (see prior art rejections, herein), nor does it add additional limitations, nor recite an additional element or a combination of elements that amount to significantly more than the judicial exception. For the reasons set forth herein and of record the rejection is maintained.
Claims 1-3, 42 and 62-83 are drawn to a non-statutory method having a "natural principle" as a limiting element or step without reciting additional elements/steps that integrate the natural principle into the claimed invention such that the natural principle is practically applied, and are sufficient to ensure that the claim amounts to significantly more than the natural principle itself.
In the instant case, the "natural principle" is: detection of one or more MTEX with antibodies or antigen-binding fragments that specifically bind chondroitin sulphate proteoglycan 4 (CSPG4) epitopes on melanoma exosomes is indicative of a melanoma diagnosis, as well as a finding of protein cargo comprising immunosuppressive proteins at a ratio of MTEX to nonMTEX of 20% to 60% is indicative of a melanoma diagnosis.
The analysis as set forth in the 2019 Guidance is as follows:
Step 1: Yes, claims are drawn to a method which is one of the four statutory categories, a process.
Step 2A, prong 1: Yes, the claims recite/describe/set forth a judicial exception. The claims describe the relationship between the presence of CSPG4 epitopes on melanoma exosomes within a patient’s biological sample, as well as a finding of protein cargo comprising immunosuppressive proteins at a ratio of MTEX to nonMTEX of 20% to 60% is indicative of the patient has melanoma.
Step 2A, prong 2: No, the judicial exception is not integrated into a practical application. The claims do not rely on or use the exception here. Once the presence of the CSPG4 epitopes and protein cargo is detected on the patient’s tumor cell derived exosomes by conventional means, i.e. implementing antibodies to detect CSPG4, there are no additional elements or combination of additional elements to apply, rely on, or use the judicial exception in a manner that imposes a meaningful limit on the judicial exception.
Step 2B: There is no inventive concept present in the clams. The steps of analyzing the presence of biomarker(s) or more specifically CSPG4 epitopes on melanoma exosomes and protein cargo comprising immunosuppressive proteins in a biological sample is established by well understood, routine conventional methods, i.e. data gathering necessary to perform the correlation. The claims and the steps within inform one of ordinary skilled in the art the presence of CSPG4 expressing epitopes and protein cargo on tumor-cell derived melanoma exosomes within a patient’s biological sample identifies an individual as affected with melanoma. The claims do not recite additional elements that amount to significantly more than the judicial exception.
The claim(s) recite(s) detecting CSPG4 epitopes and protein cargo on melanoma exosomes from a patient’s biological sample with antibodies to arrive at a melanoma diagnosis. In regard to Step 2B, this judicial exception is not integrated into a practical application because the additional elements and do not add a meaningful limitation to the method because they amount to simply implementing the method using standard immunological techniques. The claim(s) does/do not include additional elements that are sufficient to amount to significantly more than the judicial exception. Accordingly, these claims are not be eligible under step 2A or step 2B.
The claim(s) does/do not include additional elements that are sufficient to amount to significantly more than the judicial exception because assaying for candidate cancer biomarkers and making clinical decisions based on the presence of said biomarkers is routine in the art. This does not add significantly more and is not an inventive concept. Methods for making such determinations were well known in the art, these steps simply tell researchers to engage in well-understood, routine, conventional activity previously engaged in by scientists in the field. Such activities are normally not sufficient to transform an unpatentable law of nature into a patent-eligible application of such law. Detection of complexes comprising candidate cancer biomarkers and binding agents has been observed by applicant but not engineered by applicant. The claims do not add significantly more to the natural phenomenon because the claims do not require a novel reagent, apparatus of incorporate a novel treatment based on the correlation.
A claim that focuses on use of a natural principle must also include additional elements or steps to show that the inventor has practically applied, and added something significant to, the natural principle itself. See Mayo, 101 USPQ2d at 1966. Recited elements such as “detecting” and “finding” based on the natural principle impose no meaningful limit on the performance of the claimed invention. Likewise, as well as equations and formulas based on the natural principle impose no meaningful limit on the performance of the claimed invention.
As set forth the claims do not impose meaningful limits on the performance of the claimed invention.
Patents cannot be obtained on subject matter identified by the courts as being exempted from eligibility (i.e., laws of nature, natural phenomenon, and abstract ideas). Further, the active method steps are conventional and routine in the art for the reasons stated above and the claims do not amount to significantly more than the recited natural principle. The claims do not "practically apply" the natural principle; rather, the claims "simply inform" the natural principle to one performing routine active method steps and do not amount to significantly more than the natural principle itself. Thus, the technology used by the instant claims is well-known in the art and does not contribute significantly more to the judicial exception. See the 2019 Revised Patent Subject Matter Eligibility Guidance and Federal Register https://www.federalregister.gov/documents/2019/10/18/2019-22782/october-2019-patent-eligibility-guidance-update; and FDsys.gov.
Claim Rejections - 35 USC § 102
13. 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)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
14. Claim(s) 1-3, 42, 62-83 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Sharma et al., (Journal of Extracellular Vesicles 7(1435138): 1-14, First published 15 February 2018/ IDS reference AF on sheet 5 submitted May 5, 2025). Sharma discloses “[i]mmunoaffinity-based isolation of melanoma cell-derived exosomes from plasma of patients with melanoma”, see title on page 1; Table 3 on page 8; and Figure 1 on page 3. The plasma samples were processed from venous blood samples, see Patients… and Exosome… segments on page 2. “Plasma samples were pre-clarified by centrifugation first at 2000 g for 10 min at 4º C and then at 10,000g for 30 min. Filtration through 0.22 µm Millipore filter was then performed, exosomes were isolated from the plasma samples “…by mini-Size-Exclusion Chromatography (SEC) as previously described.”, see page 2, Exosome…segment. “[A] method for isolation of morphologically intact, biologically active exosomes from cancer patients’ plasma or other body fluids by mini-size exclusion chromatography (mini SEC).”, see page 2, 2nd col., full paragraph (para.).
“Monoclonal antibodies (mAbs) 763.74 and 225.28 specific for distinct and spatially distant epitopes of CSPG4 uniquely expressed on melanoma cells were generated and characterized”, see the Abstract on page 1; and page 3, 1st column (col.), 2nd paragraph. With the aid of mab 763.74, melanoma-derived exosomes (MTEX) were separated from non-tumour cell-derived exosomes (non-MTEX) and the protein cargo of both fractions was evaluated by quantitative flow cytometry, see Abstract on page 1; page 2, col. 2, last sentence in 2nd para.; and page 3, Figure 1 (herein) and Immune…segment spanning to page 4. “Anti-CSPG4 mAbs were labelled for flow cytometry using a Lightning-Link APC Antibody Labelling Kit purchased from Novus Biologicals.”, see page 3, 2nd col., 1st para.; and page 5, Figure 2 (herein).
Absent evidence to the contrary, the mAbs 763.74 and 225.28 recognize first and second epitopes on CSPG4 that do not share any overlapping amino acid residues, which are distinct and spatially distant epitopes of CSPG4 uniquely expressed on melanoma.
“The ability to isolate MTEX, to separate them from non-tumour-derived vesicles and to probe their cargo for [melanoma-associated antigen] MAA or other molecular or genetic markers of the tumour will facilitate testing of the role of MTEX as liquid biopsies in melanoma.”, see paragraph spanning 12 and 13. “[Tumour-derived exosomes] TEX have been reported to mediate immune suppression and to carry a variety of immunosuppressive molecules and factors known to interfere with immune cell functions.”, see page 9, 1st col.
With the analysis of the protein cargo comprising immunosuppressive proteins, inherently the ratio of MTEX to nonMTEX of 20% to 60%. Absent evidence to the contrary, the bead comprises agarose and the MTEX with the immunosuppressive proteins may be Fas ligand (FasL), transforming growth factor-beta (TGF-beta), TNF superfamily member 10 (TRAIL) and/or programmed death-ligand 1 (PD-L1). Furthermore, absent evidence to the contrary, the MTEX has a size of from about 30 to about 15 nanometers, further comprising one or more enzymes, growth factors, oncogenes, signaling immunoregulatory proteins.
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Claim Rejections - 35 USC § 103
15. 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.
16. Claim(s) 1-3, 42 and 62-83 is/are rejected under 35 U.S.C. 103 as being unpatentable over Sharma et al., (Journal of Extracellular Vesicles 7(1435138): 1-14, First published 15 February 2018/ IDS reference AF on sheet 5 submitted May 5, 2025), and further in view of Yang et al., (Clinical and Developmental Immunology 2011 (article id 842849) 1-11, published online 30 November 2011). Sharma teaches “[i]mmunoaffinity-based isolation of melanoma cell-derived exosomes from plasma of patients with melanoma”, see title on page 1; Table 3 on page 8; and Figure 1 on page 3. The plasma samples were processed from venous blood samples, see Patients… and Exosome… segments on page 2. “Plasma samples were pre-clarified by centrifugation first at 2000 g for 10 min at 4º C and then at 10,000g for 30 min. Filtration through 0.22 µm Millipore filter was then performed, exosomes were isolated from the plasma samples “…by mini-Size-Exclusion Chromatography (SEC) as previously described.”, see page 2, Exosome…segment. “[A] method for isolation of morphologically intact, biologically active exosomes from cancer patients’ plasma or other body fluids by mini-size exclusion chromatography (mini SEC).”, see page 2, 2nd col., full paragraph (para.).
“Monoclonal antibodies (mAbs) 763.74 and 225.28 specific for distinct and spatially distant epitopes of CSPG4 uniquely expressed on melanoma cells were generated and characterized”, see the Abstract on page 1; and page 3, 1st column (col.), 2nd paragraph. With the aid of mab 763.74, melanoma-derived exosomes (MTEX) were separated from non-tumour cell-derived exosomes (non-MTEX) and the protein cargo of both fractions was evaluated by quantitative flow cytometry, see Abstract on page 1; page 2, col. 2, last sentence in 2nd para.; and page 3, Figure 1 (herein) and Immune…segment spanning to page 4. “Anti-CSPG4 mAbs were labelled for flow cytometry using a Lightning-Link APC Antibody Labelling Kit purchased from Novus Biologicals.”, see page 3, 2nd col., 1st para.; and page 5, Figure 2 (herein).
Absent evidence to the contrary, the mAbs 763.74 and 225.28 recognize first and second epitopes on CSPG4 that do not share any overlapping amino acid residues, which are distinct and spatially distant epitopes of CSPG4 uniquely expressed on melanoma.
“The ability to isolate MTEX, to separate them from non-tumour-derived vesicles and to probe their cargo for [melanoma-associated antigen] MAA or other molecular or genetic markers of the tumour will facilitate testing of the role of MTEX as liquid biopsies in melanoma.”, see paragraph spanning 12 and 13. “[Tumour-derived exosomes] TEX have been reported to mediate immune suppression and to carry a variety of immunosuppressive molecules and factors known to interfere with immune cell functions.”, see page 9, 1st col.
With the analysis of the protein cargo comprising immunosuppressive proteins, inherently the ratio of MTEX to nonMTEX of 20% to 60%. Absent evidence to the contrary, the bead comprises agarose and the MTEX has a size of from about 30 to about 15 nanometers, further comprising one or more enzymes, growth factors, oncogenes, signaling immunoregulatory proteins.
Sharma does not explicitly teach the MTEX the protein cargo immunosuppressive proteins may be Fas ligand (FasL), transforming growth factor-beta (TGF-beta), TNF superfamily member 10 (TRAIL) and/or programmed death-ligand 1 (PD-L1).
However, Yang teaches tumor-derived exosomes contain not only tumor antigens, but cytoplasmic enzymes, metalloproteinases, heat shock protein 70 (HSP70) and immunosuppressive proteins such as Fas ligand (FasL), TNF-related apoptosis-inducing ligand (TRAIL) and transforming growth factor beta (TGF-b), see page 1, last paragraph in 2nd column; page 2, 2nd column, 1st paragraph (para.); page 4, 1st column, segment 3.1, 1st para.; and page 5, 2nd column, 1st full para.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to recognize that the tumor cell-derived exosomes express additional molecules because it is well known in the art, see the entireties of both, Yang and Sharma.
One of ordinary skill in the art would have been motivated to do so with a reasonable expectation of success exemplified in Yang that “[e]xosomes are…recognized as important mediators of cell-to-cell communication”, hence exosome expressed molecules are able to interact with target cells, affect tumorigenesis and have clinical relevance, see entire Yang document and in particular, page 2, 1st column and page 6, section 4.
17. Claim(s) 1-3, 42 and 62-83 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ferrone et al., WO 2016/164408 A1 (published 13 October 2016/ IDS reference B on sheet 1 submitted May 5, 2025), and further in view of Sharma et al., (Journal of Extracellular Vesicles 7(1435138): 1-14, First published 15 February 2018/ IDS reference AF on sheet 5 submitted May 5, 2025) and Yang et al., (Clinical and Developmental Immunology 2011 (article id 842849) 1-11, published online 30 November 2011). Ferrone teaches an assay or method of detecting chondroitin sulfate proteoglycan [4] (CSPG4) expressed by melanoma cancer cells with an antibody or antigen-binding portion thereof, see page 1, section 0004; page 3, section 0014; and page 31, section 00118. Test samples which include exosomes are plasma, blood and tumor samples, see page 35, section 00133; and page 41, section 00155. The exosomes expressed on melanoma are regarded as melanoma-cell derived exosomes. Absent evidence to the contrary, the tumor cell-derived exosomes have a size of from about 30 nm to about 150 nm. “In some embodiments, the sample comprising exosomes can be a sample obtained from a subject. In some embodiments, the CSPG4+ cells can be melanoma cells.”, see page 34, section 00131. “[T]he assay or method can further comprise a step of isolating exosomes from the sample.”, see page 5, section 0017. The test sample can be treated by centrifugation, see page 41, section 00157.
“In one aspect, described herein is an assay comprising contacting a sample comprising exosomes obtained from a subject with an antibody or antigen-binding portion thereof as described herein; and detecting the presence or intensity of a signal which indicates the presence or level of CSPG4 in the sample; … In some embodiments, the assay can further comprise a step of isolating the exosomes. In some embodiments, the step of isolating the exosomes is performed prior to contacting the sample with the antibody or antigen-binding portion thereof. In some embodiments, an increase in the CSPG4 level relative to a reference level indicates the subject has a higher risk of having or developing CSPG4+ cancer. In some embodiments, the increase in the CSPG4 level relative to a reference level indicates the subject has CSPG4+ cancer.
In one aspect, described herein is a method of isolating exosomes originating from CSPG4+ cells, the method comprising contacting a sample comprising exosomes with an antibody or antigen- binding portion thereof of as described herein; and isolating exosomes bound to an antibody or antigen- binding portion thereof. In some embodiments, the antibody or antigen-binding portion thereof can be detectably labeled and/or epitope-tagged. In some embodiments, the antibody or antigen-binding portion thereof is conjugated to a solid support.”, see sections 0021 and 0022 spanning pages 6 and 7.
“[D]escribed herein is a method of isolating exosomes originating from CSPG4+ cells, the method comprising contacting a sample comprising exosomes with an antibody or antigen- binding portion thereof as described herein or an antibody or antigen-binding portion thereof that is specific for CSPG4; and isolating exosomes
bound to the antibody or antigen-binding portion thereof. Antibodies and antigen-binding portions thereof may be labeled by any suitable means, such as affixing fluorescent moieties, radioactive labels, forming chemical conjugates, biotinylation, adding epitope tags, or any other moiety that facilitates detection and/or isolation. After exosomes have bound to one or more antibodies or antigen-binding portions thereof, the bound complex comprising the exosomes bound to the antibodies or antigen-binding portions thereof may be isolated. In some embodiments, the method further comprises separating bound exosomes from the unbound exosomes (and/or unbound portions of the sample) in order to isolate the exosomes originating from CSPG4+ cells. In some embodiments, the antibody or antigen-binding portion thereof is detectably labeled and/or epito-tagged. In some embodiments, the antibody or antigen-binding portion thereof is affixed (e.g. conjugated) to a solid support.”, see page 34, section 00129.
“In other embodiments, the detection antibody is labeled with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. In some embodiments, a detectable label can be a fluorescent dye molecule, or fluorophore including,…biotin”, see page 39, section 00145.
Methods for isolating bound complexes may include immunoprecipitation, ELISA, immunodetection, affinity purification, or detection of the label of the antibody and/or antigen-binding portion thereof. Detecting binding of the antibody and/or antigen-binding portion thereof and exosome can be performed with antibodies to the antibody and/or antigen-binding portion thereof, antibodies to the exosome, or antibodies that have been generated to recognize the bound complex.”, see page 34, sections 00129-00130.
“Immunochemical methods require the use of an antibody reagent specific for the target molecule (e.g., the antigen or in the embodiments described herein, a CSPG4 polypeptide. In some embodiments, the assays, methods, and/or systems described herein can comprise: an anti-CSPG4 antibody reagent. In some embodiments, the antibody reagent can be detectably labeled. In some embodiments, the antibody reagent can be attached to a solid support (e.g., bound to a solid support). In some embodiments, the solid support can comprise a particle (including, but not limited to an agarose or latex bead or particle or a magnetic particle), a bead,…The solid support can include many different materials including,…polysaccharides,”, see section 00137 spanning pages 35 and 36.
“In one embodiment, an assay, method, and/or system as described herein can comprise an ELISA. In an exemplary embodiment, a first antibody reagent can be immobilized on a solid support (usually a polystyrene micro titer plate). The solid support can be contacted with a sample obtained from a subject, and the antibody reagent will bind ("capture") antigens for which it is specific (e.g., CSPG4). The solid support can then be contacted with a second labeled antibody reagent (e.g., a detection antibody reagent). The detection antibody reagent can, e.g., comprise a detectable signal, be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bio- conjugation. The presence of a signal indicates that both the first antibody reagent immobilized on the support and the second "detection" antibody reagent have bound to an antigen, i.e., the presence of a signal indicated the presence of a CSPG4 molecule. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of CSPG4 polypeptides in the sample.”, see page 36, section 00138.
“In some embodiments, antibodies can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin. Other detection systems can also be used, for example, a biotin-streptavidin system. In this system, the antibodies immunoreactive (i. e. specific for) with the biomarker of interest is biotinylated. Quantity of biotinylated antibody bound to the biomarker is determined using a streptavidin -peroxidase conjugate and a chromagenic substrate. Such streptavidin peroxidase detection kits are commercially available,” see section 00150 on page 40.
“In some embodiments, the antibody or antigen-binding portion thereof described herein can be bispecific. Bispecific agents comprise a molecule which is able to physically contact and inhibit at least CSPG4 and another antigen simultaneously. As used herein, the term "bispecific agent" refers to a polypeptide that comprises a first polypeptide domain which has a binding site that has binding specificity for a first target, and a second polypeptide domain which has a binding site that has binding specificity for a second target, i.e., the agent has specificity for two targets, e.g., CSPG4 and a second antigen. The first target and the second target are not the same (i.e., are different targets (e.g., proteins)). In some embodiments, the different targets can be co-expressed on the same cell. In some embodiments, a bispecific polypeptide agent can bind targets present on a single cell (heterophilic binding in cis), and/or bind one target on one cell and the other on another cell (heterophilic binding in trans). Accordingly, a bispecific polypeptide agent as described herein can selectively and specifically bind to a cell that expresses the first target and the second target. Bispecific antibody reagents comprising antigen-binding portions of antibodies specific for two different antigens, e.g., CSPG4 and a second antigen, can be readily constructed by one of skill in the art. Generally, sequences encoding the antigen-binding domain of a first antibody characterized and known to bind a desired epitope on one antigen can be joined, either directly, or through any of a variety of linkers as known to the ordinarily skilled artisan, to sequences encoding the antigen-binding domain of a second antibody characterized and known to bind a desired epitope on a second antigen.”, see section 0014 spanning pages 10 and 11.
Ferrone does not teach the taught method, wherein prior to contacting the sample with the antibodies or antigen-binding fragments thereof, the sample is subjected to differential centrifugation, ultrafiltration through a filter having a pore size of about 0.2mm and then size exclusion chromatography and arriving at the ratio of MTEX to nonMTEX of 20% to 60%.
Nor, does Ferrone teach the MTEX comprise one or more enzymes, growth factors, oncogenes, signaling immunoregulatory proteins and immunosuppressive proteins, wherein the MTEX the protein cargo immunosuppressive proteins may be Fas ligand (FasL), transforming growth factor-beta (TGF-beta), TNF superfamily member 10 (TRAIL) and/or programmed death-ligand 1 (PD-L1).
Nor, does Ferrone teach the method wherein the antibodies or antigen-binding fragments thereof specifically bind a first epitope on CSPG4 and a second epitope on CSPG4, wherein the first and second epitopes on CSPG4 are different from one another.
However, Sharma teaches “[i]mmunoaffinity-based isolation of melanoma cell-derived exosomes from plasma of patients with melanoma”, see title on page 1; Table 3 on page 8; and Figure 1 on page 3. Plasma sample were processed from venous blood samples with melanoma patients, see Patients… and Exosome… segments on page 2. “Plasma samples were pre-clarified by centrifugation first at 2000 g for 10 min at 4º C and then at 10,000g for 30 min. Filtration through 0.22 µm Millipore filter was then performed, exosomes were isolated from the plasma samples “…by mini-Size-Exclusion Chromatography (SEC) as previously described.”, see page 2, Exosome…segment. “[A] method for isolation of morphologically intact, biologically active exosomes from cancer patients’ plasma or other body fluids by mini-size exclusion chromatography (mini SEC).”, see page 2, 2nd col., full paragraph (para.).
“Monoclonal antibodies (mAbs) 763.74 and 225.28 specific for distinct and spatially distant epitopes of CSPG4 uniquely expressed on melanoma cells were generated and characterized”, see the Abstract on page 1; and page 3, 1st column (col.), 2nd paragraph. With the aid of mab 763.74, melanoma-derived exosomes (MTEX) were separated from non-tumour cell-derived exosomes (non-MTEX) and the protein cargo of both fractions was evaluated by quantitative flow cytometry, see Abstract on page 1; page 2, col. 2, last sentence in 2nd para.; and page 3, Figure 1 (herein) and Immune…segment spanning to page 4. “Anti-CSPG4 mAbs were labelled for flow cytometry using a Lightning-Link APC Antibody Labelling Kit purchased from Novus Biologicals.”, see page 3, 2nd col., 1st para.; and page 5, Figure 2 (herein).
Absent evidence to the contrary, the mAbs 763.74 and 225.28 recognize first and second epitopes on CSPG4 that do not share any overlapping amino acid residues, which are distinct and spatially distant epitopes of CSPG4 uniquely expressed on melanoma.
“The ability to isolate MTEX, to separate them from non-tumour-derived vesicles and to probe their cargo for [melanoma-associated antigen] MAA or other molecular or genetic markers of the tumour will facilitate testing of the role of MTEX as liquid biopsies in melanoma.”, see paragraph spanning 12 and 13. “[Tumour-derived exosomes] TEX have been reported to mediate immune suppression and to carry a variety of immunosuppressive molecules and factors known to interfere with immune cell functions.”, see page 9, 1st col.
With the analysis of the protein cargo comprising immunosuppressive proteins, inherently the ratio of MTEX to nonMTEX of 20% to 60%.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to isolate the sample containing EVs utilizing differential centrifugation, ultrafiltration with a filter having a pore size of 0.2 mm and size exclusion chromatography because these techniques are well known in the art, see Sharma in its entirety. And Ferrone does teach centrifugation should is requisite as EV isolation from body fluids allows a complete assessment of the EVs, see page 41, section 00157.
One of ordinary skill in the art would have been motivated to do so with a reasonable expectation of success exemplified in all prior art documents, isolated and purified exosomes yield prognostic and diagnostic information, see all documents in their entireties.
Moreover, Yang teaches tumor-derived exosomes contain not only tumor antigens, but cytoplasmic enzymes, metalloproteinases, heat shock protein 70 (HSP70) and immunosuppressive proteins such as Fas ligand (FasL), TNF-related apoptosis-inducing ligand (TRAIL) and transforming growth factor beta (TGF-b), see page 1, last paragraph in 2nd column; page 2, 2nd column, 1st paragraph (para.); page 4, 1st column, segment 3.1, 1st para.; and page 5, 2nd column, 1st full para.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to recognize that the tumor cell-derived exosomes express additional molecules because it is well known in the art, see the entireties of both, Yang and Ferrone.
One of ordinary skill in the art would have been motivated to do so with a reasonable expectation of success exemplified in Yang that “[e]xosomes are…recognized as important mediators of cell-to-cell communication”, hence exosome expressed molecules are able to interact with target cells, affect tumorigenesis and have clinical relevance, see entire Yang document and in particular, page 2, 1st column and page 6, section 4.
Additionally, Ferrone teaches an assay, wherein two different targets or proteins can be assayed with distinct antibodies and antigen-binding portion thereof and detecting the fluorescence of a bound fluorescently labeled antibody utilizing the proper wavelength, see section 0041 spanning pages 10 and 11; sections 00128 bridging pages 33 and 34; page 39, section 00145; sections 00188-00206 spanning pages 49-54; and numbered paragraphs 1-10 spanning pages 61 and 62.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to utilize different and distinct CSPG4 antibody reagents that bind two different CSPG4 epitopes and do not share any overlapping amino acid residues because Ferrone teaches different CSPG4 antibodies able to detect these variant CSPG4 amino acid residues, see section 00128 bridging pages 33 and 34.
One of ordinary skill in the art would have been motivated to do so with a reasonable expectation of success exemplified in Sharma, as well as Ferrone that antibodies are manufactured with different specificities (i.e., different combining sites for different antigens) utilizing the CDRs taught in Ferrone and specific for CSPG4 epitopes, see page 49, section 00188, see Sharma and Ferrone in their entirety.
Conclusion
18. 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.
19. Any inquiry concerning this communication or earlier communications from the Examiner should be directed to ALANA HARRIS DENT whose telephone number is (571)272-0831. The Examiner works a flexible schedule, however she can generally be reached on 8AM-8PM, Monday through Friday.
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ALANA HARRIS DENT
Primary Examiner
Art Unit 1643
24 February 2026
/Alana Harris Dent/Primary Examiner, Art Unit 1643