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 .
1. The Amendment filed March 20, 2026 in response to the Office Action of September 23, 2025, is acknowledged and has been entered. Claims 146-148, 151-153, 155, and 156 are pending and being examined. Claims 1-145, 149, 150, and 154 are canceled. Claim 146 is amended.
New Rejections
(necessitated by amendments)
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
2. Claims 146-148, 151-153, 155 and 156 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 146 is amended to recite:
A method of treating a patient for cancer, comprising:
(a) counting the number of molecules of a cell surface antigen on cancer cells from a patient, wherein the cancer cells have been identified as antigen-negative by flow cytometry and the counting is done using super-resolution microscopy that is capable of resolving single molecules;
(b) identifying the patient as having cancer cells that individually express the cell surface antigen at between 4 and 200 cell surface antigen molecules per cell; and
(c) administering to said patient a cytotoxic immune cell that targets said cell surface antigen, wherein said cell surface antigen is CD19 or CD19 and CD20.
The claim repeatedly recites a singular “cell surface antigen” is counted and expressed on cancer cells (see bold font above), however the claim ends by reciting the singular cell surface antigen is “CD19 and CD20”. The newly claimed option of “CD19 and CD20” is not a singular cell surface antigen, but rather, a plurality of cell surface antigens. A singular cell surface antigen cannot be simultaneously CD19 and CD20. These are two structurally distinct antigens. Therefore, the claim language is contradictory and is unclear with regards to what antigen is being expressed by the cancer cells and what antigen is being targeted by the cytotoxic immune cell. Clarification is required.
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.
3. Claim(s) 146-148, 151-153, 155, and 156 are rejected under 35 U.S.C. 103 as being unpatentable over Zah et al (Cancer Immunology Research, 2016, 4(6):498-508) and Supplementary Materials and Figures; in view of Weijtens et al (Gene Therapy, 2000, 7:35-42, cited in IDS submitted April 30, 2024); Watanabe et al (Journal of Immunology, 2015, 194:911-920); Rufener et al (Cancer Immunology Research, 2016, 4:509-519); Godin et al (Biophysical Journal, 2014, 107:1777-1784); Dietz et al (ChemPhysChem 2014, 15:671-676); US Patent Application Publication 2017/0370847, Ghadiali, claiming priority to May 2015; Hartley et al (ChemBioChem, 2015, 16:1725-1729); and Mattila et al (Cell Press, 2013, 38:461-474) and Supplementary Information.
Zah teaches loss of antigen expression (antigen escape) is a known problem in treating cancer with CAR T cell therapy (abstract; Introduction; Discussion first paragraph). CD19 CAR T cell therapy has yielded remarkable clinical outcomes in the treatment of acute and chronic B-cell malignancies, however, this treatment strategy’s vulnerability to antigen escape has been highlighted by multiple cases resulting from the emergence of CD19 negative (CD19-) tumor cells (Discussion first paragraph). Zah teaches a solution is to produce and administer CAR T cells that have dual targeting capacity, so that when the expression of one antigen is lost, the CAR T cell can still effectively target the other antigen. Zah teaches CD19 and CD20 are two clinically validated targets for B-cell malignancies (abstract). Zah demonstrates successfully treating mice harboring CD20+/CD19+ (wild type) lymphoma tumors or harboring mixed CD20+/CD19+ and CD20+/CD19- lymphoma tumors by administering T cells expressing bispecific CD19/CD20 CARs.
Zah:
(1) produced lymphoma Raji cells that were either wild type (WT) (CD20+/CD19+) or CD19- (CD20+/CD19-) through CRISPR/Cas9 genome editing of CD19 expression, and confirmed cell surface CD19 negativity in the lymphoma cells by flow cytometry (Figure S3 A) as well as Western blot of cell lysates (Figure S3 B) (see Supplemental Figure S3 below);
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(2) inoculated mice with Raji lymphoma WT cells or a mix of WT (75%) and CD19- cells (25%) (Figure 6);
(3) treated mice with the bispecific CAR T cells, demonstrating that the bispecific CAR T cells were superior (statistically significant) compared to singular CD19 CAR T cells for enhancing survival in the mixed Raji mice (Figure 6); and
(4) obtained biological samples from the mice 10 days and 20 days post-tumor cell injection and used flow cytometry to measure cell surface expression of CD20 and CD19 on cancer cells, wherein the cancer cell population retained CD19 and CD20 expression and the bispecific CAR T cells prevented antigen escape, effectively targeting the CD19- cell subpopulation (Supplementary Materials p. 6-7; Supplementary Figure S7; p. 504, col. 1-2).
Zah demonstrates proof of concept that their bispecific CAR T cells: (1) trigger robust T-cell mediated cytokine production, cytotoxicity, and efficient cell lysis when only one or both CD19 and CD20 antigens is present on the target cell (p. 498, col. 2; Figures 2-4; p. 510-511); and (2) efficiently lyse and eliminate CD19- tumor cells (Figure 5; p. 530, col. 2 to p. 504, col. 1). Therefore, the bispecific CAR T cells effectively lyse tumor cells having no or negative expression of CD19 or CD20. Zah concludes that their in vitro and in vivo data demonstrate their bispecific CD19/CD20 CARs can safeguard against the effects of antigen escape by targeting malignant B cells through CD20 when CD19 expression has been lost (p. 505, col. 2).
Although Zah treated mice harboring CD19- lymphoma cells that were determined to be CD19 cell surface negative by flow cytometry (Supplemental Figure S3 A), Zah does not teach counting the number of CD20 and/or CD19 molecules on the cell surface using super-resolution microscopy that is capable of resolving single molecules and identifying the cells as expressing CD19 on the cell surface between 4 and 200 antigens per cell and treating subjects having tumor cells identified as such.
Weijtens, like Zah, also recognizes that tumor cells expressing low levels of tumor antigens can escape immune recognition by adoptively transferred T lymphocytes (p. 36, col. 1). Weijtens teaches quantifying tumor antigen on the surface of tumor cells using flow cytometry, in an effort to identify CAR T cell therapeutic efficiency as correlated to tumor antigen density on cells and to CAR expression on T cells. Weijtens demonstrated that flow cytometry identified cells as negative for tumor antigen expression, however, alternative assays identified the cells as having very low antigen expression levels below the detection limit of flow cytometry, therefore the cells identified as “negative” for tumor antigen expression by Weijtens are actually expressing very low levels of tumor antigen below the limit of detection of their flow cytometry (Figure 1; p. 40, col. 1). Weijtens successfully demonstrated that CAR T cells, including those engineered to express high levels of CAR, can recognize and lyse cells expressing very low levels of tumor antigen that were “negative” according to flow cytometry (Figures 4 and 6). Weijtens concludes that CAR T cells can be engineered to lyse tumor cells with a wide range of tumor antigen densities and prevent tumor cells from escaping immune recognition and destruction (abstract; p. 40, col. 2 last paragraph to p. 41, col. 1).
Watanabe, like Zah, teaches CD20 is a therapeutic target for B cell malignancies in CAR T cell therapy (p. 912, col. 1). Watanabe recognizes tumor cells express CD20 target antigen at low levels and these tumor cells escape treatment with anti-CD20 monoclonal antibody therapies, becoming mAb-refractory, CD20-down-regulated B cell tumors (p. 912, col. 1). Watanabe teach making and administering CAR T cell therapy to target CD20 that can effectively lyse tumor cells expressing down-regulated or low cell surface CD20 antigen levels (p. 912, col. 1). Watanabe exemplifies successfully:
(a) utilizing flow cytometry to quantify CD20 antigen molecules on the cell surface of tumor cells from CD20-transduced cell lines (CEMs) and B cell lymphoma patient cell lines (RRBL1 and WILL2), as well as patient primary chronic lymphocytic leukemia (CLL) cells obtained before anti-CD20 antibody therapy, during, and after therapy becoming refractory;
(b) identifying “very low CEM” (VL-CEM) cells as expressing ~240 CD20 molecules/cell, “low CEM” (L-CEM) as expressing ~576 CD20 molecules/cell, “medium CEM” (M-CEM) as expressing 26,990 molecules/cell, and “high CEM” (H-CEM) as expressing 142,722 molecules/cell (Figure 2A; Table I); identifying RRBL1 cells as expressing 15,376 CD20 molecules/cell, WILL2 expressing 4,571 molecules/cell (Table I), and identifying patient CLLs as expressing a mix of low and nearly negative CD20-expressing CLLs during anti-CD20 antibody therapy (Figure 5B), and patient CLLs as negative for CD20 expression after anti-CD20 antibody therapy and refractory to therapy (Figure 5C);
(c) assessing the activity of high affinity anti-CD20 CAR T cells against tumor cells expressing low levels of CD20 antigen and:
(i) determining that anti-CD20 CAR T cells are able to lyse VL-CEMS having the lowest level of CD20 expression, and were able to partially eradicate VL-CEMs (p. 915, col. 2, third paragraph);
(ii) determining that L-CEMS are able to induce activation and proliferation of CD20 CAR T cells and are lysed by CAR T cells (p. 916, col. 2; Figure 2D); and
(iii) determining that anti-CD20 CAR T cells are able lyse low CD20-expressing RRBL1 and WILL2 cells efficiently (p. 917, col. 1; Figure 4);
(iv) determining that anti-CD20 CAR T cells are able lyse primary patient CLL cell populations that are either low/nearly negative or negative for CD20 expression by flow cytometry, and that were isolated from patients refractory to anti-CD20 antibody therapy (Figure 5C); and
(v) determining that anti-CD20 CAR T cells are activated and express cytokine IFNɣ after stimulation with the refractory patient CD20-negative post-treatment CLL cells (Figure 5D; p. 918, col. 1, first paragraph).
Clinically, Watanabe demonstrates that anti-CD20 monoclonal antibody therapy resulted in progression of the patient harboring CD20-expressing CLL cells, to harboring a population of CD20-low/nearly negative CLL cells, finally to harboring CD20-negative CLL cells and becoming refractory to antibody treatment (Figure 5A-C). Watanabe teaches that the CD20-negative CLL cells isolated from the patient after anti-CD20 monoclonal antibody treatment were considered residual disease that the monoclonal antibody therapy failed to eradicate, indicating the patient as refractory to monoclonal antibody therapy. These residual cells were exposed to the monoclonal antibody therapy and survived. Watanabe teaches the CD20 expression level of these cells was below the effective range of the anti-CD20 monoclonal antibody. Watanabe demonstrated that anti-CD20 CAR T cells efficiently recognized and lysed these cells, and induced production of IFNɣ (p. 918, col. 1, first paragraph). Watanabe teaches “Therefore, the minimum threshold number of surface target molecule that CAR-T recognized and lysed was markedly low, at approximately a few hundred molecules” (p. 915, col. 2). Watanabe teaches “…these results indicated that the threshold of CD20 expression for recognition and lysis by CD20 CAR-T cells, which we termed the “lytic threshold”, was a few hundred molecules” (p. 918, col. 1, last paragraph). Watanabe concludes that CAR T cells can recognize and lyse cells expressing considerably low levels of the target antigen and were activated and expanded upon such stimulation. Watanabe suggests the strategy of administering CAR T cell therapy as a first-line treatment, or in earlier phases with the aim of earlier eradication of target cells to prevent immunological escape by negative conversion of the target antigen. Watanabe suggests CD20 CAR T cell therapy applicable for the treatment of CD20-positive lymphoid malignancies (p. 919, col. 1).
Watanabe provides further motivation and a solution to enhance the lytic threshold (i.e., lyse cancer cells with an even lower level of antigen cell surface expression) by utilizing a CAR-T cell expressing a high affinity receptor. Watanabe notes that endogenous T cells, such as melanoma-specific T cells and virus-specific T cells require 10-100 epitope molecules/cell to trigger specific lysis, and both the lytic threshold and the T cell activating threshold were slightly lower in endogenous T cells compared with CAR-T cells. Watanabe teaches “Obviously, the thresholds are affected by the affinity of the mAb or TCR of the ligand or peptide/HLA complex. In our study, the affinity of the humanized anti-CD20 mAb (OUBM Mab), which was used to construct CD20CAR, was within the same range as that of rituximab…Using a mAb with this range of affinity, both thresholds of CAR-T cells were close to those of endogenous T cells. Furthermore, CD20CAR-T cells recognized and lysed CD20-downregulated target cells that survived after mAb therapy, indicating that manufacturing a CAR with a mAb may reinforce target recognition more than the mAb itself” (p. 918, col. 1-2). Therefore, Watanabe provides further motivation and a solution to lower the lytic threshold of CAR-T cells in order to achieve the targeting levels of endogenous T cells at 10-100 epitope molecules/cell to trigger specific lysis.
Rufener, like Zah, teaches B cell malignancies are successfully treated with CAR T cell therapy targeting CD19 or CD20 antigen (p. 509, col. 1). Rufener, like Zah, recognizes that loss of CD19 expression on tumor cells is an escape mechanism in patients treated by CD19 CAR T cells. Rufener, like Watanabe, also notes that patients previously treated with anti-CD20 monoclonal antibody therapy can suffer CD20 loss. Antigen escape or antigen loss results in relapsed or refractory lymphoma. Rufener, like Zah, suggests a solution to avoiding antigen escape or antigen loss is to simultaneously target multiple antigens with CAR T cell therapy (p. 509, col. 2).
Rufener assessed the effectiveness of an anti-CD20 CAR T cell on hematologic cancer cells expressing low, medium, or high levels of CD20 antigen, and in the presence of various quantities of anti-CD20 antibody rituximab, in order to determine the effects of prior antibody therapy and CD20 antigen density on the efficacy of CAR T cells. Rufener teaches p. 517, col. 1-2 (emphasis added):
Our results also provide insights into CAR T-cell biology, with respect to the effect of high or low target antigen expression on CAR T-cell activation, because varying degrees of blockade of CD20 binding sites at different rituximab concentrations may be thought of as a surrogate for antigen density. The observation that CAR T cells effectively kill target cells in the presence of high concentrations of rituximab, even on target cells with low CD20 expression, suggests that very little antigen is required for CAR T-cell lytic activity, consistent with recently reported data (citing Watanabe above).
Therefore, Rufener recognizes CAR T cells are effective against tumor cells expressing very little cell surface antigen.
Godin reviews known super-resolution microscopy methods for imaging molecules on cell surfaces, including STORM, dSTORM, or PALM (p. 1779; Figure 2). Godin teaches photoactivation localization microscopy (PALM) allows for the fluorescence imaging of the density of proteins, takes advantage of the versatility and specificity of fluorescently tagged molecules on cells, and has quickly become the tool of choice for super-resolution live cell imaging (p. 1779, col. 1, second paragraph).
Dietz teaches and demonstrate successfully counting cell surface molecules on single cancer cells utilizing dSTORM (Figure 1; p. 672, section 2.1 in Results; Experimental Section). Dietz teaches (p. 671, col. 2 to p. 672, col. 1):
“Spatial resolution in conventional fluorescence imaging is limited by diffraction to approximately 200 nm. In the last years, several sub-diffraction imaging techniques emerged.[13] One prominent example is single-molecule localization microscopy, including photoactivated-localization microscopy (PALM),[14] fluorescence photoactivation localization microscopy (FPALM),[15] stochastic optical reconstruction microscopy (STORM)[16] and directSTORM (dSTORM).[17] These techniques are based on photoswitchable or photoactivatable fluorescent proteins or organic dyes. By separating nearby fluorophore emission in time, accurate position determination of single fluorophores with nanometer precision is achieved. Super-resolution microscopy is an evolving field with great potential. Recent biological applications include single-molecule counting, cluster and structural analysis and high-density single-particle tracking.[18] Here, we use single-molecule and super-resolution microscopy to determine copy numbers of receptors and ligands as well as binding affinities from single-cell measurements. Typically, receptor densities are too high for conventional microscopy methods so that individual receptors cannot be discerned due to the diffraction limit of light microscopy. In a first step, we use immunofluorescence labelling and single-molecule localization microscopy to determine receptor copy numbers on cells.”
Ghadiali teaches advantages and known methods of using super resolution microscopy to image and quantify cell surface proteins, and suggest imaging CD19 and/or CD20 as well as on B-cells, leukemia, or lymphoma cells:
[0002] The highest resolution of a conventional optical microscope corresponds to about a half of a wavelength, which is referred to as a diffraction limit. As such, conventional microscopes have a limitation in resolving fluorophores which are separated by less than the diffraction limit.
[0003] Circumventing the light diffraction limit, super resolution microscopy produces images with higher spatial resolution measured in nanometers. Signals may be detected from the sequential fluorescence excitation and depletion of fluorophores that have been distributed throughout a sample.
[0004] Among the several super resolution microscopy methods, one approach involves the concept of reversible saturable optically linear fluorescence transitions (RESOLFTs) and encompasses stimulated emission depletion (STED) microscopy and saturated structured-illumination microscopy (SSIM). This approach employs lasers that switch fluorophores between a light emitting “on” state and a dark “off” state. As lasers scan a sample, one laser excites fluorophores meant to aid visualization while another suppresses the fluorescence of fluorophores unnecessary for image resolution.
[0005] Another approach to super resolution microscopy is super-resolved single fluorophore microscopy, a class of techniques including stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM), and fluorescence photoactivation localization microscopy (FPALM). Single fluorophore microscopy techniques account for the diffraction limit by dispersing many photo-switchable fluorescent molecules in a specimen at least 0.2 micrometers apart. After activating just a few fluorescent molecules at a time, a composite image is built by overlaying images taken during each round of excitation. Recent efforts to advance the field of super resolution microscopy include improving upon current features and resolution capabilities, 3D imaging, and multicolor imaging.
[0073] Aspects of the present disclosure include methods for high resolution imaging of a cell. Any of the methods described herein may be applied to the high resolution imaging of a cell of interest. As such, the sample may be a cellular sample that includes a cell of interest. In some instances of the method, the sample is a cell. As used herein, the term “cell” is used in its conventional sense to refer to the basic structural unit of living organisms, both eukaryotic and prokaryotic, having at least a nucleus and a cell membrane. In certain embodiments, cells include prokaryotic cells, such as from bacteria. In other embodiments, cells include eukaryotic cells, such as cells obtained from biological samples from animals, plants or fungi.
[0074] A variety of cells may be targeted for imaging using the subject methods. Target cells of interest include, but are not limited to, stem cells, e.g., pluripotent stem cells, hematopoietic stem cells, T cells, T regulator cells, dendritic cells, B Cells, e.g., memory B cells, antigen specific B cells, granulocytes, leukemia cells, lymphoma cells, virus cells (e.g., HIV cells) NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and erythroid cells. Target cells of interest include cells that have a convenient cell surface marker or antigen. In some embodiments, the target cell is selected from HIV containing cell, a Treg cell, an antigen-specific T -cell populations, tumor cells or hematopoetic progenitor cells (CD34+) from whole blood, bone marrow or cord blood. Any convenient cell surface proteins, cell markers or intracellular targets may be targeted for specific binding to polymeric dyes (e.g., polymeric dye conjugates) in the subject methods. In some embodiments, the target cell includes a cell surface marker selected from a cell receptor and a cell surface antigen. For example, the target cell may include a cell surface antigen such as CD11b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25, CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61, CD7, CD8, CD34, CD1c, CD23, CD304, CD235a, T cell receptor alpha/beta, T cell receptor gamma/delta, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-Terminal), SSEA-3, TRA-1-60 Antigen, Disialoganglioside GD2 and CD71.
Ghadiali suggests utilizing a binding member to CD20 for detection in the super resolution microscopy methods:
[0143] In some cases, a specific binding member can specifically bind a molecule expressed on the surface of a target cell of interest. In certain instances, a specific binding member is a ligand to a target cell receptor. Thus, it can have specific binding affinity for molecules such as T cell surface molecules (e.g., CD3 polypeptides, CD4, CD8, CD2, CD7, cytokine or growth factor receptors, or TCR), B cell surface molecules (e.g., CD19, CD20, CD22, cytokine or growth factor receptors, or Ig molecules), molecules expressed on tumor cells, and molecules expressed on the surface of infected target cells (e.g., viral proteins and glycoproteins). I some cases, the specific binding member may be an antibody, or a binding fragment thereof, against a tumor associated antigen, e.g. the carcinoembryonic antigen. For example, carcinoembryonic antigen (CEA) is a tumor marker which can be present on the membrane cells surface of various cancer such as pancreatic, gastric, colonic, ovarian and breast carcinoma. Other tumor associated antigens of interest are, among others, oncofetal antigens, MART-1, Mage-1, Mage-3, gp 100, tyrosinase, CEA, her2/neu, PSA, CA-125, erb-2, Muc-1, Muc-2, point mutated ras oncogenes, point mutated p53 oncogenes, and TAG-72.
[0147] Specific binding members which specifically bind any convenient target molecules may be selected. Any convenient specific binding members may be utilized. In some embodiments, the specific binding member specifically binds a target selected from the group consisting of, CD11b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25, CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61, CD7, CD8, CD34, CD1c, CD23, CD304, CD235a, T cell receptor alpha/beta, T cell receptor gamma/delta, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-Terminal), SSEA-3, TRA-1-60 Antigen, Disialoganglioside GD2 and CD71.
[0148] In some embodiments, the specific binding member specifically binds a target cell of interest. In certain embodiments of the method, the target analyte is a cellular target analyte, such as a subcellular target analyte. Specific binding members of interest include, but are not limited to, those affinity agents that specifically bind cell surface proteins of a variety of cell types, including but not limited to, stem cells, T cells, dendritic cells, B Cells, granulocytes, leukemia cells, lymphoma cells, NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells and erythroid cells. In certain instances, the specific binding member may be targeted to a subcellular component of the cell, including, but not limited to, extracellular space, cytoplasm, nucleus, mitochondria, Golgi apparatus, endoplasmic reticulum (ER), peroxisome, vacuoles, cytoskeleton, nucleoplasm, nucleolus, nuclear matrix and ribosomes.
Hartley teaches known super-resolution imaging techniques PALM, STORM and dSTORM (p. 1725, col. 2), and demonstrates successfully applying dSTORM to image and quantify CD20 molecules on hematologic tumor cells (Figure 3).
Mattila demonstrates successfully using super-resolution imaging technique dSTORM to quantify cell surface CD19 expression on primary B cells (p. 467, col. 2; Supplementary Figures S3K and S3L).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to utilize super resolution microscopy, such as dSTORM, to quantify CD20 or CD19 cell surface tumor antigen in the method of Zah, and to identity and treat subjects having between 4-200 cell surface CD19 or CD20 antigens on their tumor cells, wherein the tumor cells were identified as CD20 or CD19 negative by flow cytometry. One would have been motivated to and have a reasonable expectation of success to because: (1) Zah, Weijtens, Watanabe, and Rufener teach that B cell malignancies comprise tumor cells that are negative by flow cytometry or are expressing very low levels of CD19 or CD20 tumor antigens, and these tumor cells escape immune recognition by adoptively transferred T lymphocytes or monoclonal antibody therapy leading to relapsed/refractory cancer; (2) Zah, Weijtens, Watanabe, and Rufener recognize there is a need to identify and effectively treat patients having these tumor cells, and solutions for treatment include administering bispecific CAR T cells simultaneously targeting CD19 and CD20, or producing CAR T cells that express high levels of high affinity CARs; (3) Zah, Weijtens, Watanabe, and Rufener teach or demonstrate CAR T cells targeting CD20 or both CD19/CD20 successfully target cancer cells, lyse cancer cells, or treat cancers comprising cancer cells having negative antigen cell surface expression or very low cell surface expression comprising a few hundred cell surface antigen molecule; wherein Zah demonstrates bispecific CD19/CD20 CAR T cells successfully lysed tumor cells having negative CD19 or CD20 antigen expression and successfully treated subjects having CD19 negative tumor cells identified by flow cytometry; (4) Weijtens demonstrates that flow cytometry failed to detect low antigen expression levels on tumor cells, flow cytometry falsely identified tumor cells as negative that were actually positive for low antigen expression, and Weijtens exemplified using alternative detection assays to verify positive antigen expression in the negative cells; and (5) Godin, Dietz, Ghadiali, Hartley, and Mattila teach super resolution microscopy, such as dSTORM, is a known alternative for quantifying ultra-low levels or individual cell surface antigens, including CD19 and CD20; Hartley exemplifies successfully conducting dSTORM to quantify CD20 cell surface expression; and Matilla exemplifies successfully conducting dSTORM to quantify CD19 cell surface expression.
Thus, the prior art teaches the known need to identify and treat B cell malignancies expressing negative or low levels of CD19 and/or CD20 in order to avoid antigen escape and refractory/relapsed cancer; and teach the known successful solution to treating such cancers by administering bispecific CAR T cells simultaneously targeting CD19 and CD20, or CAR T cells that express high levels of high affinity CARs. The prior art also recognizes that flow cytometry, frequently used to identify antigen expression on tumor cells, fails to detect near negative or low antigen expression levels on tumor cells, wherein flow cytometry falsely identifies tumor cells as negative that are actually positive for low antigen expression. The prior art suggests alternative detection assays to more accurately quantify and verify positive antigen expression in the negative cell, and the prior art teaches and successfully demonstrates known alternative detection assays encompassing super resolution microscopy, such as dSTORM, accurately quantify CD19 and CD20 cell surface expression in cells.
Given the identified needs, problems, and successful solutions taught and demonstrated by the prior art, the prior art provides both motivation and reasonable expectation of success to treat a patient having a B cell malignancy expressing nearly negative or very low cell surface CD19 or CD20 antigen levels encompassing the range of 4-200 cell surface antigens/cell, by administering to the patient an improved CAR T cell therapy that simultaneously targets CD19 and CD20, and/or expresses high levels of high affinity CARs, in order to reduce antigen escape and the emergence of relapsed/refractory cancer.
The cited prior art also identified the need, problem, and successful solutions to improve identification of cancer cells that are negative or have low CD19 or CD20 antigen expression by applying super resolution microscopy, such as dSTORM, that can quantify cell surface antigens in the very low to near negative range of 4-200 surface antigens/cell superior to flow cytometry, in order to more accurately identify the negative to low antigen expression of CD19 or CD20 on the tumor cells that contribute to antigen escape and to identify patients having negative to very low CD19 or CD20 antigen expression in need of the improved CAR T cell therapy.
4. All other rejections recited in the Office Action mailed September 23, 2025 are hereby withdrawn in view of amendments. The claim amendments overcome the previous rejection under 35 USC 103(a) because the claims limit the cell surface antigen to CD19 or “CD19 and CD20”. The previous rejection under 35 USC 103(a) was based on primary reference Watanabe et al (Journal of Immunology, 2015, 194:911-920) that focused on CD20 CAR T cell therapy, therefore the claim amendment requiring CD19 or CD19 and CD20 excludes the previous prior art rejection focused solely on CD20 and the rejection is withdrawn.
5. Conclusion: No claim is allowed.
Conclusion
6. 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.
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/Laura B Goddard/Primary Examiner, Art Unit 1642
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