TCR avidity assays and uses thereof

Abstract

The present disclosure relates to compositions and methods for characterizing T-cells. In particular, the present disclosure relates to compositions and methods for predicting a cancer patient's treatment response to immune checkpoint blockade and for treating cancers using anti-tumor cells from patients.

Description

Attorney Docket No.: 2025-046-01; 45635.601TCR AVIDITY ASSAYS AND USES THEREOFRELATED APPLICATION INFORMATION

[0001] This application claims priority to U.S. Application No. 63 / 730,020 filed on December 10, 2024, the contents of which is herein incorporated by reference.SEQUENCE LISTING STATEMENT

[0002] The contents of the electronic sequence listing titled UCONN_45635_601_SequenceListing.xml (Size: 3.614 bytes; and Date of Creation: December 9, 2025) are herein incorporated by reference in their entirety.FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to compositions and methods for characterizing T-cells. In particular, the present disclosure relates to compositions and methods for predicting a cancer patient's treatment response to immune checkpoint blockade and for treating cancers using antitumor cells from patients.BACKGROUND

[0004] CD8+T cells recognize antigenic peptides presented by MHC I molecules of cancer cells. On the CD8 T cell side, this recognition is made through T cell receptors (TCRs). Although the affinity of a TCR for a peptide-MHC I complex (pMHCI) is a centerpiece of T cell-tumor cell interaction, other factors, such as the number of TCR molecules on T cells, the number of cognate pMHCI complexes on cancer cells, and the number of other co-receptors on T cells contribute to the strength of the TCR-pMHCI interaction, termed as TCR avidity. Despite such centrality, the role of TCR avidity in endogenous CD8 T cell response to neoepitopes remains entirely un-explored, especially in the areas of developing methods of treatment and predicting the treatment response of a cancer.

[0005] Improved methods for characterizing and utilizing T cells in cancer therapy are needed.Attorney Docket No.: 2025-046-01; 45635.601SUMMARY

[0006] The following brief summary is not intended to include all features and aspects of the present disclosure, nor does it imply that any claim must include all features and aspects discussed in this summary.

[0007] The present disclosure relates to compositions and methods for characterizing T-cells. In particular, the present disclosure relates to compositions and methods for predicting the treatment response of a cancer patient to immune checkpoint blockade and for the treatment of cancers using anti-tumor cells from patients.

[0008] Disclosed herein are methods and compositions to predict the treatment response of a cancer patient to immune checkpoint blockade and for the treatment of cancers including effective anti-tumor cells, wherein the effective anti-tumor cells include anti-tumor T cells.

[0009] In an aspect, disclosed is a method to predict the treatment response of a subject to immune checkpoint blockade as shown and described herein.

[0010] In further aspects, disclosed is a composition for treating cancer in a subject, the composition including a therapeutically effective amount of isolated effective anti-tumor cells from the subject as shown and described herein.

[0011] In an additional aspect, disclosed is a method for the treatment of cancers in a subject, the method including isolating effective anti-tumor cells from the subject, wherein the effective antitumor cells include anti-tumor T cells; expanding the isolated effective anti-tumor cells; and reinfusing them into the subject as shown and described herein.

[0012] Certain aspects provide a method of predicting the likelihood of a subject to respond to immunotherapy, comprising: a) identifying the subject as having low, medium, or high-avidity T-cells; and b) predicting that the subject is likely to respond to immunotherapy when the subject has low-avidity T-cells.

[0013] Further aspects provide a method of predicting the likelihood of a subject to not respond to immunotherapy, comprising: a) identifying the subject as having low, medium, or high-avidity T-cells; and b) predicting that the subject is not likely to respond to immunotherapy when the subject has high-avidity T-cells.

[0014] Also provided is a method of treating cancer in a subject in need thereof, comprising: a) identifying the subject as having low, medium, or high-avidity T-cells; and b) administeringAttorney Docket No.: 2025-046-01; 45635.601 immunotherapy (e.g., checkpoint immunotherapy) to a subject identified as having low-avidity T-cells. In some embodiments, the method comprises repeating the identifying during or after the administering.

[0015] The present disclosure is not limited to particular methods of identifying the T-cells as low, medium, or high-avidity T-cells. In certain exemplary aspects, the method comprises: a) contacting a sample comprising the T-cells with a first peptide tetramer labeled with a first fluorescent dye (Teti), a second peptide tetramer labeled with second fluorescent dye (Tet2), an agent that specifically binds to the tetramer and prevents rebinding of the tetramer to antigenspecific T-cell (e.g., an antibody that specifically binds to the tetramer), and an antibody that specifically binds to the first fluorescent dye; b) measuring the rate of dissociation of the Tet2 from the T-cell; and c) identifying the T-cells as low, medium, or high-avidity based on the dissociation time of the Tet2.

[0016] Also provided is a method of identifying a subject as having low, medium or high-avidity T-cells, comprising: a) contacting a sample comprising the T-cells with a first peptide tetramer labeled with a first fluorescent dye (Teti), a second peptide tetramer labeled with second fluorescent dye (Tet2), an agent that specifically binds to the tetramer and prevents rebinding of the tetramer to antigen-specific T-cell (e.g., an antibody that specifically binds to the tetramer), and an antibody that specifically binds to the first fluorescent dye; b) measuring the rate of dissociation of the Tet2 from the T-cell; and c) identifying the T-cells as low, medium, or high- avidity based on the dissociation time of the Tet2.

[0017] Further aspects provide a method of treating cancer, comprising: administering low- avidity T-cells to a subject in need thereof. In some embodiments, the method further comprises administering checkpoint immunotherapy to the subject. In some cases, the T-cells are isolated from the subject. In certain aspects, the T-cells are obtained using a method, comprising: a) contacting a sample from the subject comprising the T-cells with a first peptide tetramer labeled with a first fluorescent dye (Teti), a second peptide tetramer labeled with second fluorescent dye (Tet2), an antibody that specifically binds to the tetramer, and an antibody that specifically binds to the first fluorescent dye; b) measuring the rate of dissociation of the Tetc from the T-cell; c) identifying the T-cells as low, medium, or high-avidity based on the dissociation time of the Tet2; and d) isolating the low-avidity T-cells.Attorney Docket No.: 2025-046-01; 45635.601

[0018] The present disclosure is not limited to particular fluorescent dyes. In some cases, the first dye is phycoerythrin (PE) and the second dye is brilliant violet 421. In some embodiments, the measuring comprising staining of the tetramers by flow cytometry.

[0019] Any suitable peptide is contemplated for use in the methods described herein. Examples include but are not limited to, a MHC I antigen (e.g., Dd-IGPRALDVL; SEQ ID NO: 1).

[0020] In some embodiments, the method further comprises adding an additional anti-dye antibody.

[0021] In certain aspects, low-avidity T-cells dissociate within about 15 minutes, mediumavidity T-cells dissociate at about 75 minutes, and high-avidity T-cells dissociate after about 105 minutes.In further embodiments, identifying the T-cells as low, medium, or high-avidity comprises identifying altered gene expression associated with T-cell avidity. Exemplary genes include but are not limited to, those described in Tables 2 and / or 3. For example, in some embodiments, the level of expression of one or more of CD38, TOX2, ENTPD1, CTLA4, HAVCR2, TOX, PDCD1, and TIGIT is increased in high-avidity T-cells, and the level of expression of one or more of TCF7, LEF1, IL7R, and CCR7 is increased in low-avidity T-cells. In some embodiments, the method further comprises generating an avidity score based on the altered gene expression.

[0022] In some cases, the T-cells are CD8 T-cells.

[0023] In some embodiments of the disclosure, the T-cells are isolated from tumors or tumordraining lymph nodes. In some embodiments, the T-cells are isolated after the identification.

[0024] Other aspects provide the use of immunotherapy to treat cancer in a subject identified as having low-avidity T-cells.

[0025] In some aspects, the present disclosure provides the use of low-avidity T-cells to treat cancer in a subject in need thereof. Also provided is the use of low-avidity T-cells in combination with checkpoint immunotherapy to treat cancer in a subject in need thereof. In some embodiments, the low-avidity T-cells are ex vivo T-cells.

[0026] These and other aspects of the present invention are described in more detail below.Attorney Docket No.: 2025-046-01; 45635.601BRTEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings are included to provide a further understanding of the methods and compositions of the disclosure, and are incorporated in and constitute a part of this specification. The drawings illustrate one or more embodiment(s) of the disclosure, and together with the description serve to explain the principles and operation of the disclosure.

[0028] FIG. 1 shows that CD8 T cells with low-avidity confer superior anti-tumor immunity in vivo. (A) A method for separating antigen-specific (Ag*) low-, medium-, and high-avidity T cells is shown. (B) A representative decay plot of PDPRMUTspecific CD8 TIL from the Meth A tumor. (C) T cells that were PE+BV421+ or PE+BV421- after 15 minutes were re-stained with BV421 and PE labelled tetramers. The proportions of tetramer-double positive cells at 0 and 15 minutes are shown. (D) Control (tetramer-negative), low, or high-avidity cells were re-stimulated in vitro and stained for ZAP70-pY292. The proportion of positive cells (left) and MFI (right) in CD8+T cells are shown. (E) Control, low, or high-avidity CD8+cells. The proportion of IFNy CD8+T cells is shown. (F) An in vitro killing assay with control, low, or high-avidity CD8+cells was conducted. (G-I) Control, low, or high-avidity CD8+cells of (G, H) PDPRMUTor (I) GTF2bMUTpeptide immunized mice were adoptively transferred (i.v.) into mice bearing 10-day old Meth A tumors. The kinetics of tumor growth in individual mice with the statistical analysis of the tumor growth as calculated per the Tumor Control Index as well as by comparison of summary plots of tumor growth data (G, I, Fig. 9A, B), and the survival of mice (H) are shown. P-values for two-way ANOVA (C, D), one-way ANOVA (E, F), two-tailed t-test (G, I), and Mantel-Cox survival analysis (H) are indicated (* P < 0.05; ** P < 0.01; *** P<0.001;****p<0.0001).

[0029] FIG. 2 shows that CD8+T cells of high-avidity express higher levels of exhaustion markers than those of low-avidity. (A) The proportion of TIM3 positive cells, the MFI of TIM3, the proportion of TOX positive cells, the proportion of cells expressing TIM3 in the TOX+cells, and the MFI of TIM3 in TOX+cells in low and high-avidity PDPRMVTspecific CD8+TIL is shown. (B) The proportion of TfM3+TCFl‘ (left) and TIM3 TCF1+(right) of low and high- avidity PDPRMUTspecific CD8 T cells from lymph nodes of PDPRMLTpeptide-immunized mice. (C) The proportion of TfM3+TCFL (left) and TIM3 TCF1+(right) of low and high-avidity PDPRMUT-specific CD8 T cells from tumor-draining lymph nodes of mice seven days after tumor challenge is shown. (D) Design for the isolation of PDPRMUTspecific CD8+TIL cells forAttorney Docket No.: 2025-046-01; 45635.601 multi-omic sequencing. (E) UMAP of cells obtained as in D, shows unbiased clusters based on their transcriptomes. (F) The cells in the UMAP (E) are labelled with their individual TCR avidities as indicated. (G) Relative mean expression (color intensity) and the proportion of cells expressing (circle size) selected differentially expressed genes in each cluster. (H) Relative expression (color intensity) of each CITE-Seq antibody in each cluster. Asterisks are shown in the matrix plot for significantly upregulated (white) and downregulated (black) expression of surface proteins. (I) Inferred differentiation, based on each cell’s RNA velocity and transcriptome, for all cells obtained from D is shown. (J) The cluster composition of low and high-avidity Ag* CD8+TIL as in E. Each point represents pooled samples from 3-4 mice. P- values for two-tailed paired t-tests are indicated (* P < 0.05; ** P < 0.01; *** P < 0.001).

[0030] FIG. 3 shows avidity-dependent differentiation towards exhaustion within TCR clonotypes of high-avidity. (A) A UMAP of TCRa0+T cells is shown. (B) The proportion of cells in each clonotype that are of low, medium, or high-avidity, as determined experimentally, are shown. (C) The proportion of low, medium, and high-avidity cells in each clonotype are indicated for KM_1 and KM_2. (D) A dot-plot depicting the relative mean expression (color intensity) and the proportion of cells expressing (circle size) differentially expressed genes (Table 1) related to TCR signaling is shown for KM_1 and KM_2. The number of cells in each group is indicated by a horizontal bar graph at the right edge of the dot plot. (E) The proportion of cells in each clonotype that are Stem -Like, Effector-Exhausted or Terminally Exhausted is shown for KM_1 and KM_2. (F, G) The median expression of TIM3 (F) and CX3CR1 (G), as measured by CITE-Seq, for each clonotype in KM_1 and KM_2 are shown. (H, I) PAGA plots depicting the most confidently inferred differentiation pathway, based on each cluster’s RNA velocity and transcriptome, of KM_1(H), and KM_2 (I) are shown. P-values for (G, H) two- tailed paired t-tests and (C, D) two-way ANOVA with corrections for multiple-comparisons for each comparison is shown (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001).

[0031] FIG. 4 shows that TCR avidity of CD8+ TIL associated with differences in TCR signaling and T cell phenotype. (A) A summary image depicting the presence of CD8 TIL with identical TCRs that have substantially different measured TCR avidities. (B,C) Pair-wise comparisons of the mean expression of Cd8a (B, left), Cd8bl (B, middle), Trac (B, right), Nr4al (C, left), and Nfkb2 (C, right) for cells of low or high-avidity within the same clonotype is shown. (D) The proportion of cells low-avidity and high-avidity in the same clonotype that areAttorney Docket No.: 2025-046-01; 45635.601Stem-Like, Effector-Exhausted or Terminally Exhausted is shown. P-values for two-tailed paired t-tests (B, C) and two-way ANOVA with corrections for multiple-comparisons for each comparison (D) are indicated (* P < 0.05; ** P < 0.01; *** P < 0.001).

[0032] FIG. 5 shows that low-avidity CD8+T cells are the sole responders to PD-1 and CTLA-4 blockade in vivo. (A-E) Low-avidity Ag*, high-avidity Ag*, or control tetramer-negative CD8 T cells were adoptively transferred (i.v.) into mice 10 days after MethA challenge. The kinetics of tumor growth in mice treated with control (A), low-avidity (C), or high-avidity (C) cells are shown. The survival of mice treated with control (B), low-avidity (E), or high-avidity (E) cells is shown. The tumor control index for each low- or high-avidity-treated mouse (D) is shown. Each line and dot in A-E represents data from individual mice. (F) Twenty mice were treated with anti-PD-1 or anti-CTLA-4 antibodies twice weekly, or isotype control antibody, starting 6 days after tumor challenge, for 28 days. The number of low and high-avidity Ag* CD8+TIL that are Stem-like, as determined in Fig. 3F, are shown. Each dot represents data from 6-7 mice. P-values for (B, E) log-rank survival analysis and (D, F) one-way ANOVA analysis with multiple comparison correction is shown (* P < 0.05; ** P < 0.01).

[0033] FIG. 6 shows that low-avidity human CD8+TIL from multiple cancer types have a less exhausted phenotype and predict response to immunotherapy. (A, B) Expression of individual genes comprising the Avidity Score (see Table 2) in the training (A) and test (B) data sets. (C) Distribution of the Avidity Score for mouse CD8+ TIL of low and high-avidity in the test data. P-value for the comparison of Avidity Scores with a Wilcoxon ranked-sums test is shown. (D, E) The distribution of the Avidity Score is shown for the (D) mouse test data and (E) human CD8+ TIL from eight different cancer types. The Avidity Score of low and high-avidity T cells was concordant with measured avidity for low (95% purity) and high-avidity (96% purity) cells. (F) The list of individual genes comprising the Avidity Score was intersected with the transcriptional targets of TCR-related transcription factors as predicted by the Archs4 database. (G) Differentially expressed genes between the low and high-avidity human CD8+ TIL (as determined in F) is shown. (H) The proportion of low and high-avidity human CD8+ TIL for hepatocellular carcinoma, triple-negative breast cancer, and melanoma is shown for patients before treatment with PD-1 or CTLA-4 blockade. P-values for (H) two-way ANOVA analysis are indicated for each comparison (* P < 0.05; ** P < 0.01; **** P < 0.0001).Attorney Docket No.: 2025-046-01; 45635.601

[0034] FIG. 7 that shows surface expression of SEMA4a is predictive of measured TCR avidity regardless of the knowledge of the antigen.

[0035] FIG. 8 shows (A) the proportion of CD8 TILs that are PDPRMUTtetramer from mice bearing MethA(left) or 4T1 (right) 28 days after tumor challenge. (B) PDPRMUTtetramer positive CD8 T cells isolated from lymph nodes of mice seven days after immunization were cocultured with splenocytes pulsed with either control (DMSO only), PDPRWT, PDPRMUTfor 4 hours. The proportion of IFN-y+(left) and the MFI of IFN-y in CD8 T cells detected by flow cytometry is shown. (C) Tetramer decay in the absence of aPE antibody (left) in contrast to (right) lack of such decay (i.e. stabilization of tetramer-binding) in the presence of aPE antibody with the GTF2bMUT-PE (square) or PDPRMUT-PE (circle) tetramers. (D) The proportion of PDPRMUTspecific TILs that are Tetramer-BV421+and Tetramer-PE+after 15 and 105 minutes of decay with anti -PE antibody is shown. (E) The proportion of PDPRMUT-specific cells (from lymph nodes of PDPRMUTpeptide-immunized mice) that are Tetramer-BV421+and Tetramer- PE+through the course of a tetramer decay is shown. (F) The frequency of low (blue) or high (red) avidity cells in PDPRMLT-specific cells from lymph nodes of PDPRMUT-immunized mice or from Meth A TILs is shown. (G) PDPRMUT-specific TILs from 28-day-old Meth A tumors were analyzed for tetramer decay in the presence of buffer alone, or in the presence of anti-H-2Ddblocking antibody, or in the presence of anti-H-2Ddblocking antibody as well as anti-PE antibody.

[0036] FIG 9 shows the tumor control index (A) as well as a summary plot annotated with P values from Tukey’s multiple comparison test (B), corresponding to the tumor growth curves in Fig. 1G. (C) One thousand control (tetramer-negative), one thousand high-avidity, or four thousand high-avidity CD8+ cells from lymph nodes of PDPRMUT (SEQ ID NO:2) peptide- immunized mice were adoptively transferred (i.v.) into mice bearing 10-day-old Meth A tumors. The kinetics of tumor growth in individual mice is shown. (D) The tumor control index corresponding to the tumor growth curves shown in Fig. II is shown. (E) C57BL / 6 mice were immunized with the mutated neoepitope (STFLYFSFF; SEQ ID NO:3) of MC38-FABF colon carcinoma (REF), and low and high-avidity CD8 T cells were sorted as described in Fig. 1A. The kinetics of tumor growth is shown. The survival of recipient mice is shown in F. P values for Tukey’s multiple comparison test (E) or Mantel-Cox test (F) is indicated where statistically significant. (G) A consolidated survival analysis of mice treated with low-avidity (n=48 mice) orAttorney Docket No.: 2025-046-01; 45635.601 high-avidity (n=30 mice) CD8 T cells specific to any of the three neoepitopes or negative control CD8 T cells (n=35 mice), is shown. P values were determined by Mantel-Cox test.

[0037] FIG. 10 shows (A, B) a representative plot for gating strategy for data in Fig. 2A. (A) Live single cells from TIL of 28 Day Meth A-bearing mice are gated for CD8 and TCR-P expression (left), followed by PD-1 expression (right). (B) PD-l+CD8+TCR-+cells from samples after 15 (left) and 105 (right) minutes of decay are gated on PDPRMUTTetramer in PE and BV421. (C) The proportion of TIM3+TCF1' (left) and TIM3‘TCF1+(right) of low and high- avidity PDPRMUT (SEQ ID NO:2) -specific CD8 T cells from vaccine draining lymph nodes, tumor draining lymph nodes, and 21 -day-old TIL. P-values for two-tailed paired t-tests are indicated (* P < 0.05; ** P < 0.01).

[0038] FIG. 11 shows (A) the proportion of TIM3 positive cells, the MFI of TIM3, the proportion TOX positive cells, the MFI of TIM3 in the TOX+cells, and the proportion of cells expressing TIM3 in the TOX cells in low and high-avidity GTF2bMUTspecific CD8+TIL are shown in individual panels as indicated. (B-D) Proportion of exhausted (PD1+ TIM3+) TRIB3MUT, PRPF 19-1MUT’ and STFLYFSFF (SEQ ID NO:3) -specific CD8 T cells of low and high-avidity (left figure of each panel) and the MFI of TIM3 (right figure of each panel) are shown. (E) The proportion of low and high-avidity STFLYFSFF (SEQ ID NO:3) -specific CD8 T cells isolated from 21-day-old MC38-FABF tumors that express stem-like (TCF1+ LY108+) (left) or exhausted (PD1+ TIM3+) (right) markers.

[0039] FIG. 12 shows (A) A flow chart indicating the pre-processing of sequencing data for experiments described in Fig. 2. (B) A UMAP of the filtered cells labeled with clusters from unbiased clustering based on gene expression is shown. (C) UMAPs depicting gene expression of Cd8bl (left), Cd3e (middle), and Trac (right) for all cells after filtration is shown. (D) The nine most enriched Gene Ontology Terms for differentially expressed genes in low (blue) and high (red) avidity Ag* CD8+ TIL obtained as in Fig. 3D are shown. (E, F) Representative flow plots demonstrating the gating strategy for Stem -Like (F), Eff-Exh (E), Term-Exh- 1 (E), and Term-Exh-2 (E) cells for the analysis in Fig. 3 J.

[0040] FIG. 13 shows (A) A dot plot depicting the normalized mean expression (intensity) and proportion (circle size) of cells expressing indicated genes in KM_1 and KM_2. (B, C) Velocity stream plots depicting the inferred differentiation, based on each cell’s RNA velocity and transcriptome, of KM_1 (B), and KM_2 (C) are shown.Attorney Docket No.: 2025-046-01; 45635.601DETAILED DESCRIPTION

[0041] Before the disclosed processes and materials are described, it is to be understood that the aspects described herein are not limited to specific embodiments, or examples, and as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting.

[0042] The present disclosure relates to compositions and methods for characterizing T-cells. In particular, the present disclosure relates to compositions and methods for predicting the treatment response of a cancer patient to immune checkpoint blockade and for the treatment of cancers using anti-tumor cells from patients.

[0043] CD8 T cells which recognize relevant antigens are central to immune control of cancers. T cells, through T cell receptors, recognize cognate antigen (e.g., neoepitope peptide-MHC I) on cancer cells. The strength (or avidity) of the T cell receptor- peptide-MHCI interaction is a critical variable in immune control of cancers in vivo. The present disclosure describes experiments that demonstrate by analyzing neoepitope-specific CD8 T cells of distinct avidities to validated neoepitopes of a mouse tumor, that low-avidity T cells are observed to be the sole mediators of cancer control in vivo. Low-avidity cells are also observed to be solely responsive to checkpoint blockade in mice and in humans. High-avidity T cells are not only ineffective but suppress immune response. While not limited to a particular mechanism, the data supports that the differences in the anti -tumor activity of cells of low and high avidities lies in the higher exhaustion status of high-avidity cells. High-avidity T cells have a distinct transcriptomic profile which creates an Avidity Score, used here to identify in silico, the low and high-avidity T cell populations in mice and humans. Surprisingly, CD8 T cells with identical TCRs exhibit a wide variation in avidities, supporting a novel level of regulation of T cell activity. These observations, and the mechanisms that underlie them, shed new light on the endogenous T cell response to cancer and suggest new avenues in cancer immunotherapy.

[0044] CD8+T cells recognize antigenic peptides presented by MHC I molecules of cancer cells. On the CD8 T cell side, this recognition is made through T cell receptors (TCRs). Although the affinity of a TCR for a peptide-MHC I complex (pMHCI) is a centerpiece of T cell-tumor cell interaction, other factors, such as the number of TCR molecules on T cells, the number ofAttorney Docket No.: 2025-046-01; 45635.601 cognate pMHCT complexes on cancer cells, and the number of other co-receptors on T cells contribute to the strength of the TCR-pMHCI interaction, termed as TCR avidity. TCRs interact with cancer cells in a wide range of avidities. Since T cells play the central role in cancer control in vivo, and since avidity is a substantial measure of T cell-cancer cell interactions, considerable effort has gone into understanding the role of TCR avidity in tumor control. The interaction of TCRs with a wide variety of antigens presented by the cancer cell MHC I has been analyzed. These antigens include un-mutated self-antigens, model foreign antigens, model antigens expressed as tumor antigens, cryptic antigens or oncogene products. Previous studies have typically analyzed monoclonal T cell populations adoptively transferred into mice or humans. Not surprisingly in view of the heterogeneity of the antigens studied and the models used, they have resulted in every possible conclusion: (i) high-avidity responses are more effective, (ii) low as well as high-avidity TCRs can be effective and sometimes, equally so, (iii) high-avidity responses can risk autoimmunity while low-avidity responses are less likely to do so and (iv) low and intermediate avidity responses are more effective at tumor control. Studies in human cancers have tested the influence of T cell avidity to neoepitopes in vivo using adoptive transfers of transgenic TCRs in immune compromised mice rather than in humans.

[0045] Far more than self-antigens, model antigens, oncogene-encoded antigens and foreign antigens, cancer neoepitopes have emerged as potent stimulators and key targets of anti-tumor immune responses in mice and humans. Neoepitopes arise from somatic mutations in cancer cells, and are the only cancer associated alterations that are truly cancer specific. CD8 and CD4 T cell responses to natural neoepitopes are currently the subjects of intense scrutiny. Despite such centrality, the role of TCR avidity in endogenous CD8 T cell response to neoepitopes remain entirely un-explored. Here, that critical lacuna is examined in mice as well as humans. The studies reported here are the first to examine the endogenous polyclonal CD8+T cell response to natural cancer neoepitopes that elicit potent tumor rejection. Analyzing neoepitopespecific CD8 cells of distinct avidities to two validated neoepitopes of a mouse tumor, inventors observed that low-avidity T cells are the sole mediators of cancer control in vivo. Remarkably small numbers of antigen-specific (Ag*) low-avidity T cells are sufficient for tumor rejection in vivo. High-avidity T cells are not only ineffective in controlling tumors in vivo but suppress immune response. Low and high-avidity T cells have distinct transcriptomic profiles, and a unique signature can be used to distinguish one from the other. This avidity-specific signature isAttorney Docket No.: 2025-046-01; 45635.601 shown in TILs of several human cancers as well. Low-avidity CD8 T cells are also the sole responders to PD1 as well as CTLA4 blockade in mice as well as PD1 blockade in humans. Finally, it was observed that endogenous CD8 T cells of an identical clonotype show a wide variation in avidities which is independent of the TCR-pMHC affinity. These observations, and the mechanisms that underlie them improve understanding of endogenous T cell response to cancer in vivo as well as their applications in human cancer characterization and treatment.

[0046] The following terms are used to describe the invention of the present disclosure. In instances where a term is not specifically defined herein, that term is given an art-recognized meaning by those of ordinary skill applying that term in context to its use in describing the present disclosure.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.

[0048] As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” and “protein” have their common meanings as would be understood by a biochemist of ordinary skill in the art. Terms “nucleic acid” or a “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and / or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982)). Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein. The present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogenous or homogenous in composition, and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or doubleAttorney Docket No.: 2025-046-01; 45635.601 stranded form, including homoduplex, heteroduplex, and hybrid states. In some embodiments, a nucleic acid or nucleic acid sequence comprises other kinds of nucleic acid structures such as, for instance, a DNA / RNA helix, peptide nucleic acid (PNA), morpholine nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002)) and U.S. Pat. No. 5,034,506), locked nucleic acid (LNA; see Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 97: 5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, J. Am. Chem. Soc., 122: 8595-8602 (2000)), and / or a ribozyme. Hence, the term “nucleic acid” or “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and / or nonnucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”); further, the term “nucleic acid sequence” as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or double-stranded, and represent the sense or antisense strand. The terms “nucleic acid,” “polynucleotide,” “nucleotide sequence,” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.

[0049] The term “gene” refers to a DNA sequence that comprises control and coding sequences necessary for the production of an RNA having a non-coding function (e.g., a ribosomal or transfer RNA), a polypeptide, or a precursor. The RNA or polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or function is retained. Thus, a “gene” refers to a DNA or RNA, or portion thereof, that encodes a polypeptide or a RNA chain that has functional role to play in an organism. For the purpose of this disclosure it may be considered that genes include regions that regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and / or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions.

[0050] It will be appreciated that expression of a gene is regulated (for example, downregulated) if the expression is reduced by at least about 20% (e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more) as compared to a reference level or control. Expression of a gene is upregulated if the expression is increased by at least about 20%Attorney Docket No.: 2025-046-01; 45635.601(e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more) as compared to a reference level or control.

[0051] The terms “silence” “knock down,” and “silence expression,” as used herein, refer to inhibition of expression of a particular gene. The degree of inhibition may be partially complete (e g., 10% or more, 25% or more, 50% or more, or 75% or more), substantially complete (e.g., 85% or more, 90% or more, or 95% or more), or fully complete (e.g., 98% or more, or 99% or more). Gene silencing may be accomplished using a variety of methods known in the art. In some embodiments, silencing is performed using gene editing. The terms “gene editing,” “genome editing,” or “genome engineering,” may be used interchangeably herein to refer to a type of genetic engineering in which DNA is inserted, deleted, modified, or replaced in the genome of a living organism. For example, gene editing may be used to disrupt or modify an endogenous genomic region of a host cell, inserting an exogenous gene into a host genome, replacing an endogenous nucleotide sequence with an exogenous nucleotide sequence, or any combination thereof. Systems and methods for gene editing are described in detail in, e.g., National Academies of Sciences, Engineering, and Medicine; National Academy of Medicine; National Academy of Sciences; Committee on Human Gene Editing: Scientific, Medical, and Ethical Considerations. Human Genome Editing: Science, Ethics, and Governance. Washington (DC): National Academies Press (US); 2017 Feb. 14. A, The Basic Science of Genome Editing.

[0052] The term “tumor,” as used herein, refers to an abnormal mass of tissue that results when cells divide more than they should or do not die when they should. In the context of the present disclosure, the term tumor may refer to tumor cells and tumor-associated stromal cells or tissue (i.e., the tumor “microenvironment”). Tumors may be benign and non-cancerous if they do not invade nearby tissue or spread to other parts of the organism. In contrast, the terms “cancerous tumor,” “malignant tumor,” “cancer,” and “cancer cells” may be used interchangeably herein to refer to a tumor comprising cells that divide uncontrollably and can invade nearby tissues.Cancer cells also can spread or “metastasize” to other parts of the body through the blood and lymph systems. The cancerous tumor may be a carcinoma (cancer arising from epithelial cells), a sarcoma (cancer arising from bone and soft tissues), a lymphoma (cancer arising from lymphocytes), a blood cancer (e.g., myeloma or leukemia), a melanoma, or brain and spinal cord tumors. The cancerous tumor can be located in the oral cavity (e.g., the tongue and tissues of the mouth) and pharynx, the digestive system, the respiratory system, bones and joints (e.g., bonyAttorney Docket No.: 2025-046-01; 45635.601 metastases), soft tissue, the skin (e.g., melanoma), breast, the genital system, the urinary system, the eye and orbit, the brain and nervous system (e.g., glioma), or the endocrine system (e.g., thyroid) and is not necessarily the primary tumor. More particularly, cancers of the digestive system can affect the esophagus, stomach, small intestine, colon, rectum, anus, liver, gall bladder, and pancreas. Cancers of the respiratory system can affect the larynx, lung, and bronchus and include, for example, non-small cell lung carcinoma. Cancers of the reproductive system can affect the uterine cervix, uterine corpus, ovaries, vulva, vagina, prostate, testis, and penis. Cancers of the urinary system can affect the urinary bladder, kidney, renal pelvis, and ureter. Cancer cells also can be associated with lymphoma (e.g., Hodgkin's disease and NonHodgkin's lymphoma), multiple myeloma, or leukemia (e.g., acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, and the like). In one embodiment, the cancerous tumor is a colorectal cancer or carcinoma (CRC), a breast cancer, or a melanoma.

[0053] Compounds and materials are described using standard nomenclature. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. The following terms are used to describe the invention of the present disclosure. In instances where a term is not specifically defined herein, that term is given an art-recognized meaning by those of ordinary skill applying that term in context to its use in describing the present disclosure.

[0054] A sequence of bases is a succession of bases signified by a series of a set of five different letters that indicate the order of nucleotides forming alleles within a DNA (using GACT) or RNA (GACU) molecule. By convention, sequences are usually presented from the 5' end to the 3' end. For DNA, the sense strand is used. Because nucleic acids are normally linear (unbranched) polymers, specifying the sequence is equivalent to defining the covalent structure of the entire molecule.

[0055] The use of the terms “a” and “an” and “the” and similar referents (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. By way of example, "an element" means one element or more than one element.

[0056] As used herein, the term “substantially” means to a great or significant extent, but not completely.Attorney Docket No.: 2025-046-01; 45635.601

[0057] It should also be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise. Furthermore, the terms first, second, etc., as used herein are not meant to denote any particular ordering, but simply for convenience to denote a plurality of, for example, layers.

[0058] The terms “comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted.

[0059] The terms “about” or “approximately,” as used herein, is inclusive of the stated value and means within an acceptable range of deviation for the particular value as determined by one of ordinary skill in the art, considering the measurement in question and the error associated with measurement of the particular quantity (i.e., the limitations of the measurement system). For example, “about” can mean within one or more standard deviations, or within ± 10% or 5% of the stated value. Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1, 0.2, 0.3, 0.4 . . . 2.0. All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any nonclaimed element as essential to the practice of the invention as used herein.

[0060] The phrase "and / or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to AAttorney Docket No.: 2025-046-01; 45635.601 only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0061] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of."

[0062] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a nonlimiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and / or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0063] The phrase "one or more," as used herein, means at least one, and thus includes individual components as well as mixtures / combinations of the listed components in any combination.

[0064] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients and / or reaction conditions are to be understood as beingAttorney Docket No.: 2025-046-01; 45635.601 modified in all instances by the term "about," meaning within 10% of the indicated number (e.g., "about 10%" means 9%-l 1% and "about 2%" means 1.8%-2.2%).

[0065] All percentages and ratios are calculated by weight unless otherwise indicated. All percentages are calculated based on the total composition unless otherwise indicated. Generally, unless otherwise expressly stated herein, "weight" or "amount" as used herein with respect to the percent amount of an ingredient refers to the amount of the raw material comprising the ingredient, wherein the raw material may be described herein to comprise less than and up to 100% activity of the ingredient. Therefore, weight percent of an active in a composition is represented as the amount of raw material containing the active that is used and may or may not reflect the final percentage of the active, wherein the final percentage of the active is dependent on the weight percent of active in the raw material.

[0066] All ranges and amounts given herein are intended to include subranges and amounts using any disclosed point as an end point. Thus, a range of "1% to 10%, such as 2% to 8%, such as 3% to 5%," is intended to encompass ranges of "1% to 8%," " 1% to 5%," "2% to 10%, " and so on. All numbers, amounts, ranges, etc., are intended to be modified by the term "about," whether or not so expressly stated. Similarly, a range given of "about 1% to 10%" is intended to have the term "about" modifying both the 1% and the 10% endpoints. Further, it is understood that when an amount of a component is given, it is intended to signify the amount of the active material unless otherwise specifically stated.

[0067] As used herein, the term “administering” means the actual physical introduction of a composition into or onto (as appropriate) a subject, a host, or cell. Any and all methods of introducing the composition into the subject, host or cell are contemplated according to the invention; the method is not dependent on any particular means of introduction and is not to be so construed. Means of introduction are well-known to those skilled in the art, and also are exemplified herein. “Providing” means giving, administering, selling, distributing, transferring (for profit or not), manufacturing, compounding, or dispensing.

[0068] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0069] As used herein, the term “pharmaceutically acceptable” refers to compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reactionAttorney Docket No.: 2025-046-01; 45635.601 when administered to a subject, preferably a human or a non-human subject. Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of a federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0070] As used herein, the terms “treat,” “treating,” and “treatment” include inhibiting the pathological condition, disorder, or disease, e.g., arresting or reducing the development of the pathological condition, disorder, or disease or its clinical symptoms; or relieving the pathological condition, disorder, or disease, e.g., causing regression of the pathological condition, disorder, or disease or its clinical symptoms. Treatment means any way the symptoms of a pathological condition, disorder, or disease are ameliorated or otherwise beneficially altered. Preferably, the subject in need of such treatment is a mammal, preferably a human. Treatment also means providing an active compound to a patient in an amount sufficient to measurably reduce any cancer symptom, slow cancer progression or cause cancer regression. These terms also encompass therapy and cure. In certain embodiments treatment of the cancer may be commenced before the patient presents symptoms of the disease.

[0071] As used herein, the term "effective amount" or “therapeutically effective amount” refers to the amount of a therapy, which is sufficient to reduce or ameliorate the severity and / or duration of a disorder or one or more symptoms thereof, inhibit or prevent the advancement of a disorder, cause regression of a disorder, inhibit or prevent the recurrence, development, onset or progression of one or more symptoms associated with a disorder, detect a disorder, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy e.g., prophylactic or therapeutic agent). An effective amount can require more than one dose. As used herein, the term “prophylaxis” refers to preventing or reducing the progression of a disorder, either to a statistically significant degree or to a degree detectable by a person of ordinary skill in the art.

[0072] As used herein, the term “dose” or “dosage” denotes any form of an active ingredient formulation or composition, including cells, that contains an amount sufficient to initiate or produce a therapeutic effect with at least one or more administrations. “Formulation” and “composition” are used interchangeably herein. As used herein, the terms “control,” or “reference” are used herein interchangeably. A “reference” or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. “Control” also refers to control experiments or control cells.Attorney Docket No.: 2025-046-01; 45635.601

[0073] Effective amounts may vary depending upon the biological effect desired in the individual, condition to be treated, and / or the specific characteristics of the composition according to the present invention and the individual. In this respect, any suitable dose of the composition can be administered to the patient (e.g., human), according to the type of disease to be treated. Various general considerations taken into account in determining the “effective amount” are known to those of skill in the art and are described, e.g., in Gilman et al., eds., Goodman And Gilman’s: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990; and Remington’s Pharmaceutical Sciences, 17th Ed., Mack Publishing Co., Easton, Pa., 1990, each of which is herein incorporated by reference.

[0074] The term “subject” or “patient” is used herein to refer to an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, and a whale), a bird (e.g., a duck or a goose), and a shark. In an embodiment, the subject or patient is a human subject or a human patient, such as a human being treated or assessed for a disease, disorder or condition, a human at risk for a disease, disorder or condition, a human having a disease, disorder or condition, and / or human being treated for a disease, disorder or condition as described herein. In one embodiment, the subject is about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years of age. In another embodiment, the subject is about 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100 years of age. Values and ranges intermediate to the above recited ranges are also intended to be part of this invention. In addition, ranges of values using a combination of any of the above-recited values as upper and / or lower limits are intended to be included. As used herein, a subject is “in need of treatment” if such subject would benefit biologically, medically, or in quality of life from such treatment. A subject in need of treatment does not necessarily present symptoms, particular in the case of preventative or prophylaxis treatments. The terms “subject” and “patient” are used interchangeably herein. The term “subject” also refers to an organism, tissue, cell, or collection of cells from a subject.

[0075] All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No languageAttorney Docket No.: 2025-046-01; 45635.601 in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein. Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art of this disclosure.

[0076] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims are introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group.

[0077] All compounds are understood to include all possible isotopes of atoms occurring in the compounds. Isotopes include those atoms having the same atomic number but different mass numbers and encompass heavy isotopes and radioactive isotopes. By way of general example, and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon includenC,13C, and14C. Accordingly, the compounds disclosed herein may include heavy or radioactive isotopes in the structure of the compounds or as substituents attached thereto. Examples of useful heavy or radioactive isotopes include18F,15N,18O,76Br,125I and 131j

[0078] ‘ ‘Pharmaceutical compositions” means compositions comprising at least one active agent, such as a compound or salt of Formula (I), and at least one other substance, such as a carrier. Pharmaceutical compositions meet the U.S. FDA’s GMP (good manufacturing practice) standards for human or non-human drugs.

[0079] “ Carrier” means a diluent, excipient, or vehicle with which an active compound is administered. A “pharmaceutically acceptable carrier” means a substance, e.g., excipient, diluent, or vehicle, that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes a carrier that is acceptable for veterinary use as well as human pharmaceutical use. A “pharmaceutically acceptable carrier” includes both one and more than one such carrier.

[0080] A significant change is any detectable change that is statistically significant in a standard parametric test of statistical significance such as Student’s T-test, where p < 0.05.Attorney Docket No.: 2025-046-01; 45635.601Characterizing T cells

[0081] Embodiments of the disclosure provide compositions and methods for characterizing the avidity of T-cells (e.g., as low, medium, or high-avidity). The present disclosure is not limited to particular methods of identifying the T-cells as low, medium, or high-avidity T-cells. In some embodiments, the methods comprise the use of a tetramer binding assay and / or gene expression analysis.

[0082] T cells detect antigens in the form of peptides bound to self major histocompatibility complex (MHC) molecules at the cell surface. T-cell specificity is determined by the T-cell receptor (TCR), which engages the peptide presentation platform of the peptide-MHC (pMHC) via its highly variable complementarity-determining regions (CDRs). The TCR / pMHC interaction is very weak and classically lasts for no longer than a few seconds at physiological temperatures. Advances over the past decade, however, have produced multimeric forms of soluble pMHC molecules that can be utilized to visualize T cells that bear cognate TCRs.

[0083] Aspects of the present disclosure provide improved tetramer binding assays that allow for the characterization and optionally isolation of T-cells of different avidities. In certain exemplary aspects, the method comprises: a) contacting a sample comprising the T-cells with a first peptide tetramer labeled with a first fluorescent dye (Teti), a second peptide tetramer labeled with second fluorescent dye (Tet2), an agent (e.g., antibody) that specifically binds to the tetramer and prevents rebinding of the tetramer to antigen specific T-cells, and an antibody that specifically binds to the first fluorescent dye; b) measuring the rate of dissociation of the Tet2 from the T- cell; and c) identifying the T-cells as low, medium, or high-avidity based on the dissociation time of the Tet2.

[0084] The present disclosure is not limited to particular fluorescent dyes. In some cases the first dye is phycoerythrin (PE) and the second dye is brilliant violet 421. In some embodiments, the measuring comprises staining of the tetramers by flow cytometry.

[0085] Any suitable peptide is contemplated for use in the methods described herein. Examples include but are not limited to, a MHC I antigen (e.g., Dd-IGPRALDVL; SEQ ID NO: 1).

[0086] In some embodiments, the method further comprises adding additional anti-dye antibody.

[0087] In certain aspects, low-avidity T-cells dissociated within about 15 minutes, mediumavidity T-cell dissociate at about 75 minutes, and high-avidity T-cells dissociate after about 105 minutes.Attorney Docket No.: 2025-046-01; 45635.601

[0088] In further embodiments, identifying the T-cells as low, medium or high-avidity comprises identifying altered gene expression associated with T-cell avidity (e.g., by generating an Avidity score based on the gene expression levels). For example, in some embodiments, the level of expression of one or more of CD38, TOX2, ENTPD1, CTLA4, HAVCR2, TOX, PDCD1 and TIGIT is increased in high-avidity T-cells and the level of expression of one or more of TCF7, LEF1, IL7R, and CCR7 is increased in low-avidity T-cells.

[0089] Table 2 shows an exemplary list of genes for use in generating an Avidity score. Example 1 and the below discussion provide additional information on generating an Avidity score.Table 2: Differential gene expression analysis between cells of low and high measured avidities in the training data set was used to identify 375 genesAttorney Docket No.: 2025-046-01; 45635.601

[0090] In some cases, the T-cells are CD8 T-cells.

[0091] In some aspects, T-cells are separated by avidity. In embodiments of the disclosure, the T-cells are isolated from tumors or tumor draining-lymph nodes. In some embodiments, the T- cells are further isolated after the identifying and separating (e.g., low-avidity T-cells are separately isolated in a different fraction than medium-avidity and high-avidity T-cells, etc.). Methods for isolating T-cells are described in more detail below and in Example 1.

[0092] Isolated T-cells find use in a variety of applications (e.g., those discussed below).Attorney Docket No.: 2025-046-01; 45635.601Methods of treating cancer

[0093] Embodiments of the present disclosure provide compositions and methods for treating cancer. For example, in some embodiments, the present disclosure provides methods for identifying subjects likely to respond to a cancer therapy (e.g., checkpoint therapy) and optionally administering the therapy to the subject.

[0094] In an aspect, disclosed is a method to predict a treatment response of a subject to an immune checkpoint blockade using small quantities of biological samples (for example, blood or tumor samples). In an embodiment, the method relies on phenotypic analysis using either genomic or protein information about T cells (a crucial cell type in anti-tumor immunity) in subject samples to identify a key characteristic (TCR Avidity) that is predictive of cancer treatment response in subjects like mice and humans. In an embodiment, the method predicts the TCR avidity with -95% accuracy in mice and has a strong mechanistic basis for its prognostic activity. In an embodiment, the method can be utilized for predicting patient outcomes to immune checkpoint blockade. In an embodiment, the subject is a mammal. In an embodiment, the subject is a human. In an embodiment, the subject is a cancer patient.

[0095] In an embodiment, the method includes: (1) collecting a biological sample from a subject; (2) isolating CD8 T cell from the biological sample; (3) conducting a genomic analysis by either whole exome gene sequencing or targeted sequencing to collect a genomic data; (4) analyzing the collected genomic data by a computational method, wherein the computational method includes gene scoring, random forest model, or neural network model; and (5) predicting the subject’s response to immune checkpoint blockade.

[0096] In further embodiments, the method of predicting the likelihood of a subject to respond to immunotherapy, comprises: a) identifying the subject as having low, medium, or high-avidity T- cells; and b) predicting that the subject is likely to respond to immunotherapy when the subject has low-avidity T-cells or that the subject is unlikely to respond to immunotherapy when the subject has high-avidity T-cells.

[0097] In further embodiments, the method includes: a) contacting a sample comprising the T- cells with a first peptide tetramer labeled with a first fluorescent dye (Teti), a second peptide tetramer labeled with second fluorescent dye (Tet ), an agent that specifically binds to the tetramer and prevents rebinding of the tetramer to antigen-specific T-cell (e.g., an antibody that specifically binds to the tetramer), and an antibody that specifically binds to the first fluorescentAttorney Docket No.: 2025-046-01; 45635.601 dye; b) measuring the rate of dissociation of the Tet2 from the T-cell; and c) identifying the T- cells as low, medium, or high-avidity based on the dissociation time of the Tet2.

[0098] In an embodiment, the method utilizes blood or tumor samples from patients that would be subjected to CD8 T cell isolation (either by FACS sorting or magnetic isolation), followed by genomic analysis by either whole exome gene sequencing or targeted sequencing. These genomic data is then analyzed by any suitable method (e.g., those disclosed herein). In some aspects, the method is a computational method that includes gene scoring, random forest model, or neural network model) to yield a prediction of the patient’s response to immune checkpoint blockade, which can be used to guide clinical care.

[0099] The compositions and methods of the disclosure solve a crucial problem in isolation of these cells by eliminating the need to determine what antigen the T cell is responding to, therefore making it viable to isolate these cells from patients in the clinical setting. Through the use of a single or a small number of phenotypic marker (protein expression of selected markers) T cells that can mediate potent anti-tumor activity with >80% accuracy can be identified and isolated. These T cells can then be used for adoptive cellular therapy to the patient from which these cells were derived.

[0100] Accordingly, in some embodiments, methods for treating cancer comprise a) identifying the subject as having low, medium, or high-avidity T-cells; and b) administering immunotherapy (e g., checkpoint immunotherapy) to a subject identified as having low-avidity T-cells and not administering immunotherapy to a subject identified as having high-avidity T-cells (e.g., administering an alternate therapy). In some embodiments, the method comprises repeating the identifying during or after the administering.

[0101] Additional embodiments provide compositions and methods of treating cancer utilizing low-avidity T-cells.

[0102] For example, also provided are low-avidity T-cell compositions for use in cancer therapy. In an embodiment, the composition includes anti -tumor T cells subjected to flow cytometry, magnetic separation or other suitable methods to isolate T cells of low and high-avidity based on any suitable method (e.g., expression of selected surface proteins or selected internal proteins for autologous T cell transfer). In an embodiment, the composition includes the anti-tumor T cells subjected to flow cytometry. In an embodiment, the composition includes the anti-tumor T cells subjected to magnetic separation. In an embodiment, the composition includes the anti-tumor TAttorney Docket No.: 2025-046-01; 45635.601 cells sorted using at least one of the genes from Table 2. Tn an embodiment, the composition includes the anti-tumor T cells sorted using an variant including a splice variant of at least one of the genes from Table 2. In an embodiment, the composition includes the anti-tumor T cells sorted using a splice variant of at least one of the genes from Table 2. In an embodiment, the composition includes the anti-tumor T cells sorted using genes including SEMA4a (Figure 7). In an embodiment, the composition includes the anti-tumor T cells that can be expanded in vitro or modified genetically before reinfusion into the subject to improve the anti -tumor activity of these cells.

[0103] In an aspect, disclosed is a composition for treating cancer, the composition including effective anti-tumor cells from a subject, wherein the effective anti-tumor cells include antitumor T cells. In an embodiment, the composition includes the anti-tumor T cells that bind weakly to tumors (that is, the T cells having low TCR avidity) having improved potent antitumor activity. In an embodiment, the composition includes the anti-tumor T cells isolated from blood or tumors from subjects such as humans or other mammals.

[0104] Accordingly, in an aspect, disclosed is a method for treating cancer, the method including: (1) isolating effective anti-tumor cells from a subject, wherein the effective anti-tumor cells include anti-tumor T cells; (2) expanding the anti-tumor cells; and (3) infusing / reinfusing the expanded anti -tumor cells in the subject.

[0105] In an embodiment, the method includes the anti-tumor T cells that bind weakly to tumors (that is, the T cells having low TCR avidity) having improved potent anti-tumor activity. In an embodiment, the method includes the anti-tumor T cells isolated from blood or tumors from subjects such as humans or other mammals. In an embodiment, the method includes the antitumor T cells subjected to flow cytometry to isolate T cells of low and high-avidity based on expression of selected surface proteins for autologous T cell transfer. In an embodiment, the method includes the anti -tumor T cells subjected to flow cytometry. In an embodiment, the method includes the anti -tumor T cells subjected to magnetic separation. In an embodiment, the method includes the anti-tumor T cells sorted using at least one of the genes from Table 2. In an embodiment, the method includes the anti-tumor T cells that can be expanded in vitro or modified genetically before reinfusion into the subject to improve the anti -turn or activity of these cells.Attorney Docket No.: 2025-046-01; 45635.601

[0106] In an embodiment, the method includes the composition including isolated T cells from subject’s tumors or blood and reinfusing them back into the subject to help control their cancer.

[0107] The compositions of the present disclosure can be formulated into any of many possible forms of application, such as, but not limited to, encapsulation in a hydrogel, encapsulated in a biocompatible foam, encapsulated in a biocompatible construct, injected in a polymerizable construct, in a 3D formulated construct to fill cavity created by surgical removal of tumor, in the form of modified fibroblasts adhered to silicon, in the form of direct injection of cells suspended in aqueous solution, in the form of cell suspension in decell ularized matrix, in the form of fibroblast suspension on a biomimetic construct, in the form of fibroblast suspension with other cells such as, but not limited to, macrophages, NK cells, vascular cells, mesenchymal stromal cells, adipocyte derived stromal cells.

[0108] The compositions of the present disclosure can be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, emulsions or microemulsions, and enemas. The compositions of the present disclosure can also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions can further contain substances which increase the viscosity of the suspension including, for example, sodium carboxy methyl cellulose, sorbitol or dextran. The suspension can also contain stabilizers. The pharmaceutical composition of the present disclosure may also include a pharmaceutical carrier or excipient. A pharmaceutical carrier or excipient is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more cargoes such as T-cells to a subject.

[0109] Ideally, the methods described above result in the containment of cancerous tumor to prevent its metastasis. As used herein, the terms “treatment” and “treating” can include reversing, alleviating, inhibiting the progression of, preventing or reducing the likelihood of cancer malignancy, or one or more symptoms or manifestations of a cancer metastatic spread. Accordingly, the disclosed compositions can be administered prophylactically to prevent or reduce the incidence or recurrence of a cancer. In some embodiments, the disclosed agents, compositions, and methods promote inhibition of tumor cell dissemination, increase the sensitivity of chemotherapy, prevention of cancer cells to spread into stroma, or to metastasize to proximal or distal lymph nodes, or vasculature, such that chance of recurrent metastasis is reduced in mammals (e.g. a human, horse, or dog, or cat). “Treatment of cancer” meansAttorney Docket No.: 2025-046-01; 45635.601 alleviation of cancer in whole or in part. In an embodiment, the disclosed methods and compositions reduce the size of a cancerous tumor by at least about 20% (e.g., at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%). In an embodiment, the disclosed methods and compositions contained the spread of cancer to other organs more than 90% (e.g. at least about 95%, 100%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%).

[0110] Generally, the methods and compositions described herein can be administered as monotherapy or as a part of combination therapy. In an embodiment, the methods described herein are administered as monotherapy. In an embodiment, the methods described herein are administered as a part of combination therapy. In an embodiment, the composition described herein is administered as monotherapy. In an embodiment, the composition described herein is administered as a part of combination therapy.[OHl] In some embodiments, subjects identified as having low-avidity T cells are administered checkpoint immunotherapy. In some embodiments checkpoint therapy is administered in combination low-avidity T-cells isolated from the subject (e.g., autologous or heterologous T- cells). In some embodiments, checkpoint therapy is administered in combination with chemotherapy, surgery, radiation, or other cancer therapies.

[0112] The present disclosure is not limited to particular checkpoint immunotherapy agents. Examples include but are not limited to, pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, ipilimumab, tremelimumab, relatlimab, or any combinations thereof.

[0113] Any suitable cancer can be treated using the compositions and methods of the disclosure. Examples include but are not limited to, breast cancer, bladder cancer, cervical cancer, colon cancer, head and neck cancer, Hodgkin lymphoma, liver cancer, lung cancer, renal cell cancer, skin cancer, including melanoma, stomach cancer, or colorectal cancer.

[0114] EXAMPLES

[0115] Experimental system

[0116] Previous experiments identified several naturally occurring neoepitopes of a murine sarcoma Meth A by mass spectrometry (MS). Of these, GTF2bMUTand PDPRMurwere shown to elicit CD8+T cell responses and mediate tumor control in vivo. Ag* CD8+tumor infiltrating lymphocytes (TIL) are identified as cells that stain equivalently for the same tetramer labeled with two distinct fluorophores (Fig. 8A, left). These same tetramers do not stain any TIL fromAttorney Docket No.: 2025-046-01; 45635.601 the murine breast carcinoma 4T1 , which does not express PDPRMUT(Fig. 8A, right) thus establishing their specificity of binding. Tetramer-sorted CD8 T cells produced interferon gamma (IFNy) only upon re-stimulation with their cognate mutant but not the corresponding wild type peptides (Fig. 8B). Here, this system is used to understand the heterogeneity of T cell phenotypes among the endogenous CD8+T cells specific to natural cancer neoepitopes which mediate tumor rejection in vivo. The isolated, purified sub-populations of Ag* CD8 T cells were examined for their individual roles in tumor rejection in vivo and the mechanisms behind such roles.

[0117] A tetramer decay assay for preparative isolation of low, medium, and high-avidity T cells

[0118] Savage et al. developed a tetramer decay assay to measure the dissociation of pMHC tetramers from T cells, thus providing a measure of TCR avidity [P. A. Savage, et al., A Kinetic Basis For T Cell Receptor Repertoire Selection during an Immune Response. Immunity. 10, 485-492 (1999)]. They incubated tetramer-stained T cells for various time points to assess dissociation of tetramers as a reflection of TCR avidity. The longer a tetramer stays bound to a T cell, the higher the TCR avidity of the T cell for that antigen. Cells with persistent binding of tetramers at the end of the experiment are deemed high-avidity. If one aims to isolate T cell populations of low and medium avidities, they will always be contaminated with high-avidity T cells, because they retain the tetramers throughout the experiment. This problem has hindered the isolation of pure populations of T cells of low or medium-avidity.

[0119] In order to enable preparative isolation and functional characterization of low, medium, and high-avidity sub-populations of Ag* T cells from a heterogenous population of T cells, the assay of Savage et al. was modified. TIL were isolated from Meth A tumors 28 days after implantation and stained with a mixture of Dd-IGPRALDVL (SEQ ID NO: 1) tetramers labeled with Phycoerythrin (PE) (TetpE) or Brillian Violet 421 (TetBV42i), and the tetramers were allowed to dissociate from CD8+T cells for varying periods of time in medium containing anti-MHC I antibody to prevent re-association of dissociated tetramers. T cells which dissociate early are of low-avidity, those that dissociate later are of medium-avidity and those that remain associated even after a prolonged time, are of high-avidity (Fig. 1A, B). Kinetic separation of T cells by tetramer decay has been shown to be superior to tetramer intensity as a surrogate for TCRAttorney Docket No.: 2025-046-01; 45635.601 avidity. The modified tetramer decay assay disclosed herein, described in detail in Methods, is a further improvement over the original assay.

[0120] Characterization of sorted Ag* CD8 T cells of low and high-avidity. The studies in Fig. IB are carried out with TILs. Functional studies of PDPRMUT-specific CD8 T cells in vitro were pursued with the LN-derived CD8 T cells rather than TILs because of the relative paucity of Ag* CD8 TILs. CD8 T cells from LNs of mice immunized with PDPRIUTwere tested for tetramer decay as in Fig. IB. Indeed, CD8 T cells of low and high-avidity were observed in equal proportions between the LNs and TILs (Fig. 8E, F). To test if the low and high-avidity T cells isolated as in Fig. 8E are truly of distinct avidities, they were re-stained and re-decayed to determine if they “sort true” as described under Tetramer Re-decay in Methods. Almost all the cells that were TetpE+TetBV42i+at tis remained TetpE+TetBV42 after re-decay for 15 minutes, while the majority of cells that were TetpE+TetBV42f at tis became TetpE+TetBV42f upon re-decay for 15 minutes (Fig. 1C). This was not attempted for TetpE+TetBV42r and TetpE+TetBV42f at 105 minutes because TCR-signaling mediated TCR downregulation would be a confounding variable due to the very long durations of tetramer staining.

[0121] The purified low and high-avidity populations were characterized with respect to classical markers of phosphorylation and cytokine secretion. Phosphorylation ofZAP70 at tyrosine 292 (ZAP70-pY292) is a TCR-pMHC affinity dependent event. CD45.1 congenic PDPRMUT-pulsed splenocytes were co-cultured (as APCs) with low and high-avidity PDPRMUTspecific T cells sorted from vaccine-draining lymph nodes (LNs) of PDPRMUTimmunized mice and stained for ZAP70-pY292. LN-derived CD8+T cells were used rather than TIL because TIL express high levels of co-inhibitory receptors that can dephosphorylate ZAP70, thus confounding the interpretation of the result. High-avidity PDPRMUTspecific T cells had a significantly greater proportion of cells that were ZAP70-pY292+and had significantly greater ZAP-pY292 MFI than low-avidity PDPRMUTspecific T cells and tetramer-negative T cells from the same LNs (Fig. ID).

[0122] Production of IFNg by low and high-avidity PDPRMUTspecific T cells was tested in absence or presence of PDPRMUTpeptide. No IFNg was detected in un-stimulated cells, while both low and high-avidity T cells made significant IFNg upon stimulation. Control (tetramernegative) T cells did not make any IFNg without or with stimulation. A higher proportion of high-avidity T cells made IFNg than low-avidity T cells (Fig. IE). Killing of PDPRMUTpeptide-Attorney Docket No.: 2025-046-01; 45635.601 pulsed target cells by low and high-avidity T cells, as well as tetramer-negative cells, was tested; low and high-avidity cells were equally cytotoxic while tetramer-negative cells showed negligible killing (Fig. IF), consistent with previous reports.

[0123] Anti-tumor activity of low and high-avidity T cells in vivo. The Ag* specific low and high-avidity populations were tested individually for their ability to control tumors in vivo. One thousand (1,000) control (tetramer-negative), low or high-avidity cells (all obtained from LNs of immunized mice and isolated as in Fig. 8E) were adoptively transferred (i.v.) into mice bearing progressively growing, 10-day old palpable Meth A tumors. The kinetics of tumor growth in individual mice was monitored (Fig. 1G). The tumors in mice that had received high-avidity T cells grew faster than those in control -treated mice, while tumors in mice that received low- avidity PDPRMUT-specific T cells grew significantly slower, or regressed (Fig. 1G, Fig. 9A, B). These results are also reflected in a significantly greater survival of mice that received low- avidity T cells over those that received tetramer-negative T cells or high-avidity T cells (Fig. 1H). Adoptive transfer of as many as 4,000 high-avidity PDPRMUT-specific T cells did not mediate tumor control (Fig. 9C). Anti -tumor activity of low-avidity over high-avidity T cells was also observed in case of Ag* T cells recognizing the neoepitope GTF2bMUT(Fig. II, Fig.9D). The generality of this phenomenon was further tested in an additional mutated neoepitope (STFLYFSFF; SEQ ID NO: 3) of the MC38-FABF colon carcinoma of C57BL / 6 mice. Once again, low-avidity cells were observed to be the sole drivers of significant tumor control and significantly improved survival of recipient mice (Fig.9E, F).

[0124] An additional analysis of consolidated data of survival of mice that had received low- avidity, high-avidity, or control CD8 T cells for all neoepitopes was performed. This analysis confirms the exclusive power of low-avidity T cells to mediate tumor control with enormous statistical significance (P<0.0001 between control and low-avidity T cells, as well as between low-avidity and high-avidity T cells) (Fig. 9G).

[0125] Greater expression of exhaustion-related markers in high-avidity CD8 T cells from any source. The absence of anti-tumor activity of high-avidity CD8 T cells in vivo (Fig. 1G-I) sharply contrasts with the equivalent cytotoxic activity of the low and high-avidity T cells in vitro (Fig. IF). The hypothesis that this discrepancy results from differential exhaustion of CD8 T cells in vivo was considered; hence, low and high-avidity T cells were examined for markers of exhaustion. Twenty-eight-day TIL-derived low and high-avidity T cells specific forAttorney Docket No.: 2025-046-01; 45635.601PDPRMUT were characterized for exhaustion-related markers TOX and TIM3 (Fig. 2A). High- avidity TIL showed a significantly higher proportion of TIMS cells as well as a higher MFI of TIM3. As compared to low-avidity TIL, high-avidity TIL also showed a higher proportion of TOX+cells, which also have a higher proportion of TIM3+cells as well as a higher MFI of TIM3. Regardless of the source of sampling (TILs or vaccine draining LNs or tumor draining LNs), high-avidity T cells are observed to be more exhausted than low-avidity T cells by every parameter tested (Fig. IOC).

[0126] The association of avidity with exhaustion is not restricted to T cells against PDPRMUTsince similar observations were made with respect to the low and high-avidity CD8 TILs specific for the neoepitope GTF2bMUT(Fig. 11 A). CD8 T cell responses against two additional neoepitopes of Meth A, TRIB3MUT(Derestricted) and PRPF19-1MUT(Kd-restricted) reproduced the association between TCR avidity and the exhaustion phenotype seen with PDPRMUTand GTF2bMUT(Fig. 11 B ,C). The findings were further reproduced in the CD8 T cells specific for the Kb-restricted STFLYFSFF (SEQ ID NO: 3) neoepitope of MC38-FABF (Fig. 11 D, E), a tumor of a different haplotype and distinct histological origin from the Meth A.

[0127] Expression of exhaustion-related markers in low and high-avidity T cells was examined in vaccine draining as well as tumor draining LNs. The high-avidity T cell population has a significantly greater proportion TIM3+ TCFL cells (exhausted population) than the low-avidity T cell population in vaccine draining LNs (Fig. 2B left) as well as tumor-draining LNs (Fig. 2C left). Conversely, the high-avidity T cell population has a significantly lower proportion TIM3- TCF1+ (stem like population) cells than the low-avidity T cell population in both vaccine draining and tumor draining LNs (Fig. 2B right, 2C right).

[0128] Multi-omic sequencing of PDPRMUT-specific TIL of varying avidities. TILs were isolated from tumor bearing mice (n=l 1) 28 days after tumor challenge and stained with surface CITE-Seq antibodies against PD-1, CX3CR1, TIM3, and LY108. These markers identify well- known sub-sets of exhausted T cells in cancers and chronic infections. These cells were sorted into low, medium, and high-avidity as in Fig. 1A, B and subjected to single cell RNA, TCR, and CITE-Seq sequencing (Fig. 2D). Unbiased clustering of the filtered CD8+TIL based on their transcriptomes revealed five clusters: Stem cell Like (Stem-Like), Natural Killer Like (NK- Like), Effector Exhausted (Eff-Exh), Terminally Exhausted 1 (Term-Exh-1), and Terminally Exhausted 2 (Term-Exh-2) (Fig. 2E, G, H). See Methods for description of clusters. A UMAPAttorney Docket No.: 2025-046-01; 45635.601 showing the TCR avidities of each T cell illustrates that T cells of all avidities are represented in every cluster (Fig. 2F).

[0129] The distinctions between low and high-avidity T cells are not restricted to exhaustion- related pathways. Other pathways with significant differences include those related to ribosomal content, oxidative phosphorylation, cytoskeletal rearrangement, responsiveness of IFNg and regulation of apoptosis (Fig. 12D).

[0130] Trajectory inference, based on transcriptional dynamics and RNA velocity, was used to understand the differentiation of PDPRMUT-specific CD8+TIL. When inferring trajectories from TIL of all avidities, Stem-Like cells are predicted to give rise to NK-Like and Eff-Exh cells; Eff- Exh cells then differentiate into either Term-Exh-1 or Term-Exh-2 cells (Fig. 21).

[0131] Flow cytometry of PDPRMUT-specific CD8+TIL from 28-day Meth A-bearing mice (n=20) was used to identify the phenotypes observed in Fig. 2E and to determine if they are differentially distributed among cells of low and high-avidity. Validation of the transcriptionally derived signatures (Fig. 2G) by CITE-Seq (Fig. 2H) allows this testing by flow cytometry. The proportion of low or high-avidity PDPRMUT-specific CD8+TIL that are Stem-Like (PD-1+ TIM3" LY108+TCFl+), Eff-Exh (PD-1+CX3CR1+TOX+), Term-Exh- 1 (PD-1+CX3CRL TIM3+), or Term-Exh-2 (PD-llllghCX3CRr TIM3 ) were compared (gating strategy in Fig. 10A, B, 5E, F). Stem-like and Eff-Exh cells formed a significantly greater proportion of low-avidity than high- avidity PDPRMUT-specific CD8+TIL. Additionally, a significantly smaller proportion of Term- Exh-1 and Term-Exh-2 cells were found in PDPRMUT-specific TIL of low-avidity than high- avidity (Fig. 2J). Broadly, all four phenotypes were observed, albeit in varying proportions, in low as well as high-avidity CD8+TIL.

[0132] Avidity-dependent exhaustion. PDPRMUTspecific CD8+TIL, which had a single pair of TCRaP sequences and belonged to clonotypes of >40 cells were selected from the multi-omic sequencing for further analysis (Fig. 3A). The threshold of 40 cells per clonotype was chosen to overcome extreme skewness and outliers. Cells within a clonotype had identical nucleotide sequences for CDR1, 2, and 3 for both TCRa and TCR[3. Each clonotype was assigned a unique numerical identifier as well as a color (Fig. 3A). Unbiased K-means clustering on the proportion of low, medium and high-avidity T cells within each clonotype (shown in Fig. 3B), identified two clusters, KM_1 (containing clonotypes 2, 3, 5, 6, 11, 13, 27, 29, 43, and 50) and KM_2 (containing clonotypes 8, 9, 24, 32, and 44). The KM_1 group had a significantly greaterAttorney Docket No.: 2025-046-01; 45635.601 proportion of cells with measured low or medium-avidity than KM_2. The KM_2 group had a significantly greater proportion of cells with measured high-avidity than KM_1 (Fig. 3C). Cells in the KM 2 group expressed significantly higher levels of genes (see Table 1 for a full list) related to TCR signaling or TCR-pMHC affinity than those in KM_1 (Fig. 3D). In an embodiment, the composition includes the anti-tumor T cells sorted using a variant including a splice variant of at least one of the genes from Table 1. In an embodiment, the composition includes the anti-tumor T cells sorted using a splice variant of at least one of the genes from Table 1Table 1: Differentially expressed pathways in Low and High-avidity CD8 TIL*Attorney Docket No.: 2025-046-01; 45635.601Attorney Docket No.: 2025-046-01; 45635.601low and high-avidity CD8 TIL. All significantly differentially expressed pathways are shown for low and high-avidity CD8 TIL

[0133] Since chronic and excessive TCR signaling has been described to contribute to T cell exhaustion, the role of TCR avidity on CD8+TIL differentiation towards the exhausted phenotype was examined. The proportion of cells within each clonotype that was Stem-Like, Eff-Exh, and Term-Exh (the sum of Term-Exh- 1 and Term-Exh-2) was compared between the two KM groups. The KM 2 group had a significantly greater proportion of terminally exhausted cells and a significantly smaller proportion of Eff-Exh cells than KM_1 (Fig. 3E). There was no difference in the proportion of Stem-Like cells between the two KM groups (Fig. 3E). Median surface expression of CX3CR1 (Fig. 3G) was significantly higher, while that of TIM3 (Fig. 3F)Attorney Docket No.: 2025-046-01; 45635.601 was significantly lower, in the KM_1 group. Cells in KM_1 express higher levels of cytotoxicity-associated genes Gzmk and Nkg7, while those in KM_2 express higher levels of suppression-associated genes such as Tgfbl, Cd274 n Entpdl (Fig. 13A).

[0134] In order to identify avidity-dependent differences in TIL differentiation, trajectory inference based on transcriptomes and RNA velocity was carried out at two levels, i.e. clusters as well as individual cells, and identical results were obtained. The most confidently inferred trajectory at the cluster level is shown for KM_1 (Fig. 3H) and KM_2 (Fig. 31). The analysis at single cell level is shown in Fig. 13B, C. At both levels of analyses, differentiation of Stem-Like cells towards Eff-Exh cells and NK-Like cells does not differ between the two KM groups. However, Eff-Exh cells in the KM_1 group are restricted to differentiation towards Term-Exh-1, while Eff-Exh cells in the KM_2 group differentiate predominantly towards Term-Exh-2.

[0135] Substantial variation in measured TCR avidity within populations of cells harboring identical TCRs. Fig. 4A, B shows that each clonotype consists of cells with low, medium as well as high avidities in spite of their identical TCR sequences for both the a and the b chains. Ten clonotypes (KM_1) are of predominantly low-avidity and five (KM_2) are of predominantly high-avidity (Fig. 4B). Binding of the CD8 molecule to MHC I has been shown to greatly increase the strength of the TCR-pMHCI interaction. To further understand the differences in the measured avidity within cells of the same clonotype, the mean expression of Cd8a and Cd8bl were compared between cells of a low or high measured avidity. Within a given clonotype, cells with a high measured avidity had significantly higher mean expression of Cd8a, but not Cd8bl, than cells with a low measured avidity (Fig. 4B). Variation in the number of TCR molecules on the T cell surface can also contribute to differences in TCR avidity. Indeed, cells of a high measured avidity had significantly higher mean expression of the constant region of TCRa chain (Trac) than cells of a low measured avidity in the same clonotype (Fig. 4B).

[0136] Changes in TCR avidity are known to be associated with changes in TCR signaling. Cells of low and high-avidity within clonotypes were analyzed for corresponding changes in TCR signaling-related transcription factors Nr4al and Nfkb2. Indeed, when compared in a pair-wise fashion, mean expression of Nr4al and N kb2 is significantly higher in high-avidity T cells than low-avidity T cells of the same clonotype (Fig. 4C). Low and high-avidity cells of the same clonotype were also compared for avidity -dependent differences in T cell phenotype. High- avidity populations were observed to consist of significantly more Term-Exh and fewer Eff-ExhAttorney Docket No.: 2025-046-01; 45635.601 cells than the low-avidity populations within the same clonotype (Fig.4D), as previously observed in the total TIL population agnostic of the clonotypic composition (Fig. 2J ,3E).

[0137] Differential response of PD1 and CTLA4 blockade on low and high-avidity T cells in vivo

[0138] It was tested if TCR avidity determines response to immune checkpoint blockade in vivo. As controls, 1,000 tetramer-negative CD8 T cells were adoptively transferred (i.v.) into mice bearing 10-day old MethA tumors, which were also treated with anti-PD-1, CTLA-4, or isotype control antibody. Neither treatment had any effect on tumor growth as measured by growth kinetics or by survival (Fig. 5A, B).

[0139] In another arm, 1,000 low, or high-avidity CD8+T cells (isolated as in Fig. 1A, B) were adoptively transferred into mice bearing 10-day old MethA tumors, followed by treatment with anti-PD-1, CTLA-4, or isotype control antibody. Two distinct observations are made. First, as shown in Fig. 1G-I, adoptively transferred low-avidity T cells mediated tumor control, while high-avidity T cells did not. Second, PD-1 or CTLA-4 blockade enhanced the anti -tumor activity of low-avidity PDPRMUT-specific T cells but had no effect on the activity of high-avidity T cells (Fig. 5 C, D). This improvement in tumor control was also reflected in the improved survival of mice receiving low-avidity PDPRMUT-specific T cells with either PD-1 or CTLA-4 blockade in vivo. No such improvement in survival was seen in mice receiving high-avidity PDPRMUT- specific T cells (Fig. 5E).

[0140] The effect of checkpoint blockade on the endogenous response to PDPRMUTwas examined. Six-day old tumor-bearing mice (n=20 per group) were treated with anti-PD-1, anti- CTLA-4, or isotype control antibody. The total number of low and high-avidity PDPRMUT- specific TILs in mice treated with anti-PDl, anti-CTLA4 or isotype control antibodies was compared. Several studies have demonstrated that Stem-Like cells are the key responders and mediators of immune checkpoint blockade. The number of Stem-Like TILs of low and high- avidity was compared in mice treated with anti-PDl, anti-CTLA4 or isotype control antibodies. It was observed the number of low-avidity Stem-Like TILs increased in mice treated with antibodies to PD1 or CTLA4; no such change was noted in high-avidity Stem -Like TILs (Fig. 5F).

[0141] High-avidity CD8 TILs in several human cancers are more exhausted. A method for the determination in silico of TCR avidity using only single cell gene expression data wasAttorney Docket No.: 2025-046-01; 45635.601 developed. All the Ag* CD8 TILs were divided evenly into a training and a test data set. Differential gene expression analysis between cells of low and high measured avidities in the training data set was used to identify 375 genes (Table 2). Expression of these genes in the low, medium and high-avidity cells of the training and test sets is shown in Fig. 6A, B. The test and training data sets show nearly identical patterns of gene expression (Pearson r2=0.998). Using these differentially expressed genes, a composite Avidity Score was derived using the method described herein. This score was applied to the low and high-avidity TILs of the test data set; the Avidity Score distinguishes the low and high-avidity populations with an enormous statistical significance (P<10‘275) (Fig. 6C). Populations of 95% purity (for low-avidity) and 96% purity (for high-avidity) can be obtained by taking cells one SD below and above the mean Avidity Score respectively (Fig. 6D).

[0142] TILs in the human Pan Cancer TIL Atlas were analyzed by Avidity Score. It is known that the proximal TCR-signaling network has a high degree of qualitative and quantitative conservation across human and mouse T cells and that this network should behave similarly when applied to human and mouse T cells. This TIL Atlas comprises single cell RNASeq data from CD8+ TILs of 36 cancer patients across eight cancer types. Unlike the mouse data (Fig. 6D), the distribution of Avidity Score in human TILs is bimodal and not normal (Fig. 6E). This lack of a normal distribution warranted refinement of the mouse Avidity Score for human TILs. In order to eliminate noise, inventors looked at the genes that are common between the 375 DE genes (that constitute the basis of mouse Avidity Score) and the 2,703 gene targets of TCR signaling related transcription factors (Fig. 6F) as predicted by the Archs4 database. The resulting 97-gene list (which constitutes the basis of the human Avidity Score) was used to characterize the human TIL population (Table 3) as in Fig. 6D and resulted in a positively skewed normal distribution (Fig. 6F). Differential gene expression analysis of the pseudo-bulk transcriptomes of human TILs of low and high-avidity based on the human Avidity Score showed significantly higher expression of exhaustion related genes (CD38, TOX2, ENTPD1, CTLA4, HAVCR2, TOX, PDCD1 and TIGIT) in high-avidity TILs, while low-avidity TILs expressed significantly higher levels of Stem-ness related genes (TCF7, LERI, I 7R. and CCR7) (Fig. 6G). In an embodiment, the composition includes the anti-tumor T cells sorted using an variant including a splice variant of at least one of the genes from Table 3. In an embodiment, theAttorney Docket No.: 2025-046-01; 45635.601 composition includes the anti-tumor T cells sorted using a splice variant of at least one of the genes from Table 3.Table 3: Genes used to characterize the human TIL population

[0143] Low-avidity human CD8 TILs predict response to immune checkpoint blockade.Pre-treatment scRNASeq of human CD8 TILs of 37 patients with hepatocellular carcinoma, melanoma, and triple negative breast cancer and was used for estimation of proportion of low and high -avidity T cells in each patient by the human Avidity Score. Responders (by RECIST or mRECIST criteria) to PD1 or CTLA4 blockade had significantly higher proportions of low- avidity CD8 TILs (P<0.05) and lower proportions of high-avidity CD8 TILs than non-responders (P<0.001) (Fig. 6H).

[0144] Materials and Methods

[0145] Materials

[0146] Mice

[0147] Female BALB / c mice (strain no. 000651) and CD45.1 -B ALB / c (strain no. 006584) were obtained from Jackson Laboratory. Mice were housed in the specific pathogen-free animal facilities at University of Connecticut Health Center. All experiments conducted here were part of animal protocols approved by the Animal Care and Use Committee of University of Connecticut School of Medicine: AP-2000633-0325.

[0148] Functional AssaysAttorney Docket No.: 2025-046-01; 45635.601

[0149] Tumor Growth Assay

[0150] Meth A cells were grown in ascites of mice for four days before injection (95,000 live cells z.tZ) into the right flank of mice. MC38-FABF cells were grown in culture as described [Brennick CA, et al., An unbiased approach to defining bona fide cancer neoepitopes that elicit immune-mediated cancer rejection. J Clin Invest. 13 l(3):el42823. (2021)]. Tumors on mice were measured twice weekly with calipers for about six weeks. Mice with tumors exceeding 15 mm diameter were euthanized. Tumor volumes were calculated by the equation 0.4 * I * w2(1, length; w, width of tumor). Tumor Control index was calculated, as described in [Corwin, W. L. et al. Tumor Control Index as a new tool to assess tumor growth in experimental animals. Journal of Immunological Methods 445, 71-76 (2017)], using average tumor diameter measurements.

[0151] Immune Checkpoint Blockade In Vivo

[0152] Isotype-treated mice were injected (i.p.) with 65 pg of Mouse IgG2b (clone MPC-11; twice weekly) and 30 pg of Rat IgG2a (clone 2A3; weekly) antibody. Anti-PD-1 -treated mice were injected ( / . / ?.) with 65 pg of Mouse IgG2b (twice weekly) and 30 pg of aPD-1 (clone RPMI-14; weekly) antibody. Anti-CTLA-4-treated mice were injected (i.p.) with 65 pg of aCTLA-4 (clone 9D9; twice weekly) and 30 pg of Rat IgG2a (weekly) antibody.

[0153] Isolation of CD8 T cells from tumors and from LNs

[0154] Tumors were excised from mice, using scalpels, 28 days after tumor challenge and digested using the Tumor Dissociation Kit, mouse on a gentleMACS Octo Dissociator (Miltenyi Biotec). The digested tumor solution was then washed twice with cold PBS and subjected to density -based separation of lymphocytes using Lympholite M (Cedarlane Laboratories). LNs were excised and subject to Easy Sep Mouse CD8+ T cell Isolation Kit (STEMCELL Technologies).

[0155] Modified Tetramer Decay Assay

[0156] CD8 T cells, extracted as in Isolation of CD8 T cells from tumors and from LNs, were then subjected to Fc blocking with Mouse IgG Isotype antibody (Invitrogen; 1 :50) for 20 minutes on ice. The cells were washed and resuspended in tetramer staining solution containing 1% v / v of tetramers (MBL Technologies) loaded with same antigen labelled with either BV421 or PE at concentration of 10 million cells per mL for 30 minutes on ice. The tetramer staining solutionAttorney Docket No.: 2025-046-01; 45635.601 was then washed off and cells were stained with a surface antibody staining solution for 20 minutes on ice.

[0157] It was observed empirically that inclusion of an anti-PE antibody during tetramer decay prevents the dissociation of TetpE over 120 minutes; the tetramer is thus stabilized (Fig. 8C, right, Fig. 8G). In contrast, the non-stabilized Tetnv42i dissociates normally (Fig. 8D). The stabilized binding of the TetpE serves as the reference point against which decay of the TetBV42i is compared. The addition of the stabilizing antibody is a critical innovation in this assay: in its absence, low and medium-avidity cells are indistinguishable from antigen non-specific T cells because they will also be tetramer negative.

[0158] Therefore, cells are split into three samples and subjected to tetramer decay at room temperature in anti-H2-Dd(clone 34-5-8S; 20 pg / mL) antibody for either 15 (low-avidity), 75 (medium-avidity), or 105 (high-avidity) minutes. Anti-PE (clone PE0001; 1 : 100) was added at either 0 (low and high-avidity) or 45 (medium-avidity) minutes to stabilize binding of the PE- labelled tetramer.

[0159] Staining was then analyzed by flow cytometry (BD Sciences LSR-II & Cytek Aurora) or isolated by FACS sorting (BD Sciences Aria-II). If cells are to be isolated, the samples are immediately placed in ice and subject to sorting at 4C. If the cells are to be used for flow cytometry, the cells are mixed into a 1 mL fix and permeabilization solution, made using the Foxp3 / Transcription Factor Staining Buffer (Invitrogen), for 30 minutes on ice before intracellular staining with indicated flow cytometry antibodies for 60 minutes on ice.

[0160] Operationally, T cells which shed the tetramers during the first 15 minutes are defined as of low-avidity (Fig. IB) and are sorted as TetpE+TetBV42f. Tetramer-associated T cells after 105 minutes of dissociation are defined as of high-avidity and are sorted as TetpE+TetBV42i+. For the isolation of T cells of medium-avidity, in a separate reaction, TetpE and TetBV42i are allowed to dissociate from T cells for 45 min to allow for the exclusion of low-avidity T cells. Anti-PE stabilizing antibody is added to the assay at t45 to prevent further dissociation of TetpE while still allowing dissociation of TetBV42i for 30 minutes. Thus, T cells of medium-avidity are sorted as TetpE+TetBV42i' at t?5 (Fig. IB).

[0161] This assay has been repeated more than 100 times with reproducible results for all antigens tested.

[0162] Staining for Flow Cytometry and CITE-SeqAttorney Docket No.: 2025-046-01; 45635.601

[0163] Before surface staining, cells were incubated with Mouse IgG Tsotype Antibody (Invitrogen;l:50) for 20 minutes on ice. Cells were then stained with labelled antibodies against CD8a (clone KT15; 1 :200), CD45.1 (cl one A-20; 1 : 100), PD-1 (clone RMPI-30; 1 :200), TCR-p (clone H57-597; 1 :200), TIM3 (clone 5D12; 1 :200), TOX (clone TXRX10; 1 :50), LY108 (clone 13G3; 1:200), CX3CR1 (clone SA011F11; 1: 100), pZAP70-Y292 (clone Al 6048B;1 :20), or TCF1 (clone C63D9; 1 :50) when indicated.

[0164] Immunization and isolation of CD8+T cells

[0165] Bone marrows isolated from the tibia and femurs from syngeneic mice were cultured in complete Roswell Park Memorial Institute 1640 (cRPMI) with 20ng / mL of murine GM-CSF (PeproTech) for one week to generate BMDCs. Mice were immunized i.d.) with six to nine million BMDCs pulsed with 100 pM of the indicated peptide in cRPMI for 2 hours at 37°C. Five to seven days after immunization vaccine-draining lymph nodes were excised and subject to EasySep Mouse CD8+ T cell Isolation Kit (STEMCELL Technologies) followed by sorting of control (tetramer-negative), low-avidity, or high-avidity as described in Modified Tetramer Decay Assay.

[0166] Tetramer Re-decay

[0167] Cells obtained by the method described in Immunization and isolation ofCD8+T cells were sorted for TetpE+TetBV42i' and TetpE+TetBV42i+at tis after tetramer staining and tetramer decay. These cells were then re-stained with tetramers in both colors, so that all cells are TetpE+TetBV42i+again and then they were subjected to another tetramer decay for 15 minutes before analysis by flow cytometry.

[0168] Ex-vivo Restimulation

[0169] Ten thousand control, low, or high-avidity PDPRMUTspecific T cells were isolated as described in Immunization and isolation of CD8+T cells and co-cultured with 1,000,000 congenic splenocytes pulsed with 10 pM of PDPRMUT. For determination of phosphorylation of ZAP70, cells were co-cultured in cRPMI for 5 minutes at 37°C. For determination of IFN-y production, cells were co-cultured with 1,000,000 BMDCs pulsed with 10 pM of PDPRMUTand Brefeldin A (1 : 1000) in cRPMI for 4 hours at 37°C. Cells were first stained for expression of surface molecules for 20 minutes on ice then fixed and permeabilized using the Foxp3 / Transcription Factor Staining Buffer (Invitrogen) for 30 minutes on ice before intracellular staining with indicated flow cytometry antibodies for 60 minutes on ice. StainingAttorney Docket No.: 2025-046-01; 45635.601 was then detected by flow cytometry (Cytek Aurora) followed by analysis using the FlowJo Software.

[0170] In vitro Killing Assay

[0171] Five thousand congenic splenocytes were pulsed with 10 pM of PDPRMUTin cRPMI for 2 hours at 37°C and used as target cells for the killing assay. Five thousand CTV-labelled congenic splenocytes pulsed in 37C with DMSO for two hours and used as antigen-negative cells for the killing assay. Ten thousand control, low, or high-avidity PDPRMUT-specific T cells were isolated as described in Immunization and Isolation of CD8+ T cells and co-cultured with the target cells and the antigen-negative cells for 4 hours at 37 °C. Killing of the target and bystander cells were assessed by flow cytometry (Cytek Aurora) followed by analysis using the FlowJo Software. Specific killing, defined by the difference in the proportion of dead cells in the target population and the bystander population, is shown.

[0172] Adoptive Cellular Transfer

[0173] One thousand cells, isolated as in Immunization and Isolation of CD8 ' T cells, were injected retro-orbitally into mice 10 days after tumor challenge. In experiments testing the efficacy of immune checkpoint blockade, mice receiving control, low, or high-avidity cells were treated with Isotype, aPD-1, or aCTLA-4, as indicated in Immune Checkpoint Blockade In Vivo, starting 13 days after tumor challenge.

[0174] Surface expression of SEMA4a is predictive of measured TCR avidity regardless of the knowledge of the antigen

[0175] BALB / c mice were immunized with the PDPRMl Tneoepitope. Vaccine draining lymph nodes were dissociated into single cells and were stained with tetramers specific for PDPRMUTneoepitope as well as antibody to SEMA4a, a cell surface marker which appears among the list of markers for the Avidity Scores for mice as well as humans. Fig. 7 shows that surface expression of SEMA4a is predictive of TCR avidity in a statistically significant manner (P<0.01).

[0176] Single cell and multi-omic sequencing

[0177] Preparation of Samples

[0178] Low, medium, and high-avidity PDPRMUT-specific CD8 TIL were stained with TotalSeq-C antibodies against PD-1, TIM3, LY108, and CX3CR1 (Biolegend Inc.) and sorted from 11 tumor bearing mice as in Modified Tetramer Decay Assay. About 6,000 cells for eachAttorney Docket No.: 2025-046-01; 45635.601 condition were captured, using the Next GEM Single Cell 5’ GEM Kit v2 (10X Genomics). Three libraries were generated for each condition using the Library Construction Kit, 5’ Feature Barcode Kit, and Single Cell Mouse TCR Amplification Kit (10X Genomics) using the Chromium Next GEM Single Cell 5’ Reagent Kit v2 (Dual Index) CG000330 Rev D protocol before Next Generation Sequencing with NovaSeq (Illumina Inc.).

[0179] Pre-processing of scRN A Seq Data

[0180] Sequencing data was pre-processed as described in Fig. 12A. Briefly, separate FastQ files for gene expression, TCR sequences, and CITE-Seq were generated using CellRanger (65). Loom files containing RNA splicing information for each sample were then generated from FastQ files for gene expression using Velocyto. Outputs from these analyses were integrated using the scanpy, scirpy, muon, and scVelo package on Python. Normalization of CITE-Seq antibody counts was conducted using DSB, as implemented in the muon package of python. Low quality cells (with less than 500 genes, greater than 5% mitochondrial gene content) were filtered out based on gene expression. Remaining cells were subject to unbiased clustering based on their transcriptomes (Fig. 12B). Only clusters 0, 1, 3, 4, and 8, with significantly upregulated expression of CD8+ T cell marker genes (Cd8bl, Trac, and Cd3e), were used for subsequent analysis (Fig. 12C).

[0181] scRNA Seq Processing

[0182] The remaining cells were then subject to principal component analysis, neighbor graph analysis, UMAP graph analysis before being clustered by the Leiden algorithm at a resolution of 0.6, using scanpy. The intersection of differentially expressed genes between clusters by three different statistical methods (Wilcoxon Rank-Sums, t-test with overestimated variance, and logistic regression) were then used to characterize the clusters. All plots for subsequent analyses were made using the scanpy and scVelo package on Python.

[0183] Cluster Annotation

[0184] The description of the clusters is based solely on differentially expressed genes (Fig. 2G). Stem-Like cells express significantly higher memory-associated markers (Tcf7,Xcll, and Bcl6) and significantly lower exhaustion associated markers (Pdcdl, Havcr2, Lag3, Tigit, Entpdl, and Cd38) (29,76-78). Eff-Exh cells express significantly higher levels of transcription factors associated with effector function (Tbx21, Ikzf2) than Stem-like and Term- Exh cells. They also express significantly higher levels of exhaustion-associated markers thanAttorney Docket No.: 2025-046-01; 45635.601Stem-Like cells, but significantly less than Term-Exh-1 cells. Term-Exh-2 is characterized by significantly high expression of certain markers of exhaustion (Tox, Entpdl, and Cd38), but low expression of others such as Havcr2. Finally, NK-Like cells are characterized by significantly high expression of natural killer cell receptors such as Klrcl, Klrc2, Klrdl, Klrkl, and Klrel (Fig. 2G).

[0185] Validation of these transcriptional signatures was sought by CITE-Seq using antibodies to PD-1, LY108, TIM3 and CX3CR1 (Fig. 2H). This is particularly important for genes that are poorly detected by RNA sequencing (Cx3crl and Slamf6). Stem-Like cells expressed significantly higher levels of LY108 and significantly lower levels of PD-1 and TIM3. Eff-Exh cells expressed significantly higher levels of the effector marker CX3CR1 and significantly lower levels of TIM3. Term-Exh-1 cells expressed significantly higher levels of TIM3 and significantly lower levels of CX3CR1 and LY108. Term-Exh-2 cells expressed significantly higher levels of PD-1 and significantly lower levels of TIM3 and CX3CR1. Finally, NK-Like cells expressed significantly higher levels of TIM3 and significantly lower levels of PD-1.Altogether surface expression of these four proteins, corroborated the phenotypic descriptions of the clusters based on their transcriptomes (Fig. 2G, H).

[0186] TCR Sequence and Trajectory Inference Analysis

[0187] Cells with single chain TCRaP were grouped into clonotypes based on identical CDR1, 2, and 3 amino acid sequences on both chains using the scirpy. Only clonotypes with greater than 40 cells were used for subsequent analysis. Clonotypes were then clustered on the proportion of cells in each clonotype measured to have low, medium, or high-avidity via the K-means algorithm using the scikit-learn package on Python. The resulting two clusters were deemed low and high-avidity clonotypes based on the significant difference of cells in each clonotype with low or high-avidity, as measured by Fig. IB. Trajectory inference of low and high-avidity clonotypes was then conducted using scvelo.

[0188] Gene Score Development

[0189] Sequenced cells were randomly assigned to either the test or training dataset using the sampling function of pandas with frac= 0.5. Low and high-avidity T cells in the training dataset were then subjected to differential gene expression analysis with DESeq2 (avidity and cluster were used as design variables) using pydeseq2. Genes with a False Discovery Rate < 0.1, a log2 fold change > 0.75, and a base mean > 1 were used as a gene score for the determination ofAttorney Docket No.: 2025-046-01; 45635.601 low and high-avidity in silica. This list was then intersected with predicted targets of TCR related transcription factors, as annotated in the Archs4 database, to develop a second gene list.

[0190] Human Samples

[0191] Pan Cancer TIL Atlas

[0192] Preprocessed transcriptomic data from the Pan Cancer TIL atlas was obtained from the Gene Expression Omnibus Database (GSE156728). Gene scoring on all CD8+ T cells obtained was implemented on the scanpy package of Python through the scanpy.tl.score genes function with the genes described in Gene Score Development as inputs for the gene_list parameter. Cells one SD below and above the mean of the gene score were deemed low and high-avidity respectively. Differential Gene Expression analysis by DESeq with avidity as the only design variable was used to compare the low and high-avidity T cells. Differentially expressed (as defined in Gene Score Development) genes were plotted using the volcano function of pydeseq2.

[0193] Immune Checkpoint Blockade Response Prediction

[0194] Transcriptomic data of pre-treatment TIL was obtained from the GEO database for Hepatocellular Carcinoma, Melanoma, and Triple Negative Breast Cancer patients. CD8 T cell clusters, as defined by the Leiden algorithm, were then extracted for determination of avidity in silico by the methods described in Pan Cancer TIL Atlas. The proportion of CD8+ T cells of low and high-avidity was then compared between responders and non-responders using a two-way ANOVA analysis.

[0195] Statistical Methods

[0196] Statistical tests, indicated when used, were conducted using GraphPad Prism. Statistical tests include paired t-tests, unpaired t-tests, one-way ANOVA, two-way ANOVA, and Mantel- Cox Survival Analysis. Paired t-tests were used in place of an unpaired t-test when comparing matched low and high-avidity T cells derived from the same samples. When present, error bars denote standard deviation of samples.Conclusions

[0197] Results disclosed herein place CD8+ T cells of low-avidity at the center of the natural immune response to several neoepitopes of two mouse cancers of different haplotypes and histologies. Retrospectively analyzed data from a small series of human cancers are consistent with the data from murine models. The relative resistance of low-avidity cells to exhaustion is, inAttorney Docket No.: 2025-046-01; 45635.601 significant part, the mechanistic basis for their superior anti-tumor activity. At every level of resolution tested, by flow cytometric analysis of antigen-specific CD8+T cells against five cancer neoepitopes, by scRNA Seq of the antigen-specific CD8+T cell population examined or by each cluster within the population, at the level of TCR clonotypes, and at the level of individual cells within each clonotype, the connection between avidity and exhaustion is persistent. Low and high-avidity CD8 T cells within the same clonotype (Fig. 3B) show the same association of avidity and exhaustion as observed between the total low and high-avidity populations. Identical contribution of TCR avidity on T cell phenotype are seen in interclonotypic (Fig 3D, E) as well as intraclonotypic comparisons (Fig 4C, D). Analysis of CD8+ TILs of 36 cancer patients across eight cancer types, agnostic of their antigenic specificity arrives at the same linkage between high-avidity and higher exhaustion.

[0198] These results also show that the low-avidity neoepitope-specific CD8 T cells are the sole responders to checkpoint blockade of PD1 and CTLA4 in two mouse models. The preferential expansion of low-avidity CD8 T cells upon PD1 blockade was also shown recently in a different mouse model by Sugiyarto et al [Sugiyarto G. et al. Reactivation of low-avidity tumor-specific CD8+ T cells associates with immunotherapeutic efficacy of anti-PD-1. J Immunother Cancer 1 l(8):e007114 (2023)]. Among cancer patients treated with PD1 blockade as well, responders to PD1 blockade had significantly higher proportions of low-avidity CD8 TILs and lower proportions of high-avidity CD8 TILs than non-responders. The correlation between response of cancer patients to PD1 blockade and the activity is described.

[0199] Experiments described herein further demonstrated that not only are the high-avidity CD8 T cells incapable of mediating tumor control, but also that the mice that received the high- avidity T cells did worse than control mice in terms of tumor growth as well as survival. The recent work of Vignali et al. [Vignali PDA et al. Hypoxia drives CD39-dependent suppressor function in exhausted T cells to limit antitumor immunity. Nat Immunol. 2023 Feb;24(2):267- 279. doi: 10.1038 / s41590-022-01379-9] observed that exhausted CD8+ TILs (which constitute a large proportion of high-avidity T cells) possess transcriptional features of CD4+ regulatory T cells and suppress T cell proliferation ex vivo, sheds some light on this observation. The high- avidity T cells differentially express higher levels of PDL1 and TGFb, which are also among the transcriptional features of T regulatory cells (Fig. 13A). Vignali et al. also showed the requirement of CD39 on the exhausted T cells for their immunosuppressive property. High-Attorney Docket No.: 2025-046-01; 45635.601 avidity T cells also differentially express higher levels of CD39 (Entpdl) as well as Hifla, a marker of hypoxia (Fig. 13A).

[0200] Studies disclosed herein also reveal an unexpected diversity of avidities among individual cells of a single clonotype in vivo. This observation is not limited to one or few clonotypes but is seen in all clonotypes. This variation derives, in significant part, from a natural variation of CD8a expression among individual daughter cells of the same clonotype. Kroger et al. have previously reported how CD8+T cells dynamically modulate CD8 expression in vitro, possibly fine-tuning the sensitivity of the T cell to the antigen [C. J. Kroger, et al., Dose-dependent modulation of CD8 and functional avidity as a result of peptide encounter. Immunology. 122, 167-178 (2007)]. The results disclosed herein show for the first time that this variation occurs in vivo in an endogenous response and is the rule rather than the exception. It is quite conceivable, even likely, that the studies of monoclonal CD8 T cell populations that do not recognize the variable avidities within the monoclonal populations, underestimate the influence of TCR avidity.

[0201] Accordingly, the results disclosed herein find use in cancer immunotherapy. Avidityenhancement of cancer-reactive T cells continues to be actively sought, and while that approach is intuitively attractive, adoptive transfer of high-avidity T cells into cancer patients has culminated in acute, immunologically-dependent, severe toxicity or death in the majority of patients. These results support that the opposite direction, i.e. avidity-reduction of cancer- reactive T cells, by modifying the TCR or CD8a or other methods, is more appropriate. An indirect approach to avidity -reduction in vivo may lie in enhancement of the CD4 response to neoepitopes; CD4 help promotes priming of low-avidity CD8 T cells and also helps prevent exhaustion. Finally, the Avidity Score derived from the quantitative expression of the genes described herein and the separation method that is specifically associated with high-avidity mouse T cells can also distinguish human high-avidity T cells from human low-avidity T cells. Thus, in some embodiments, the avidity Score of pre-treatment TILs finds use in identifying responders to T-cell therapy and other immunotherapies.

[0202] For reasons of completeness, various aspects of the disclosure are set out in the following numbered clauses:

[0203] Clause 1. A method of predicting the likelihood of a subject to respond to immunotherapy, comprising:Attorney Docket No.: 2025-046-01; 45635.601 a) identifying the subject as having low, medium, or high-avidity T-cells; and b) predicting that the subject is likely to respond to immunotherapy when said subject has low-avidity T-cell.

[0204] Clause 2. A method of treating cancer in a subject in need thereof, comprising: a) identifying the subject as having low, medium, or high-avidity T-cells; and b) administering immunotherapy to a subject identified as having low-avidity T-cells.

[0205] Clause 3. The method of clause 1 or 2, wherein the immunotherapy is checkpoint immunotherapy.

[0206] Clause 4. The method of clause 2 or 3, further comprising repeating the identifying during or after the administering.

[0207] Clause 5. The method of any one of clauses 1 to 4, wherein the identifying the T-cells as low, medium, or high-avidity T-cells comprises: a) contacting a sample comprising the T-cells with a first peptide tetramer labeled with a first fluorescent dye (Teti), a second peptide tetramer labeled with second fluorescent dye (Tet ), an antibody that specifically binds to the tetramer, and an antibody that specifically binds to the first fluorescent dye; b) measuring the rate of dissociation of the Tet2 from the T-cell; and c) identifying the T-cells as low, medium, or high-avidity based on the dissociation time of the Tet2.

[0208] Clause 6. A method of identifying a subject as having low, medium or high-avidity T- cells, comprising: a) contacting a sample comprising the T-cells with a first peptide tetramer labeled with a first fluorescent dye (Teti), a second peptide tetramer labeled with a second fluorescent dye (Tet2), an agent that specifically binds to the tetramer and prevents rebinding of the tetramer to antigen-specific T-cells, and an antibody that specifically binds to the first fluorescent dye; b) measuring the rate of dissociation of the Tet2 from the T-cell; and c) identifying the T-cells as low, medium, or high-avidity based on the dissociation time of the Tet2.

[0209] Clause 7. A method of treating cancer, comprising: administering low-avidity T-cells to a subject in need thereof.Attorney Docket No.: 2025-046-01; 45635.601

[0210] Clause 8. The method of clause 7, further comprising administering checkpoint immunotherapy to the subject.

[0211] Clause 9. The method of clause 7 or 8, wherein the T-cells are isolated from the subject.

[0212] Clause 10. The method of any one of clauses 7 to 9, wherein the T-cells are obtained using a method, comprising: a) contacting a sample from the subject comprising the T-cells with a first peptide tetramer labeled with a first fluorescent dye (Teti), a second peptide tetramer labeled with second fluorescent dye (Tet2), an agent that specifically binds to the tetramer and prevents rebinding of the tetramer to antigen-specific T-cells, and an antibody that specifically binds to the first fluorescent dye; b) measuring the rate of dissociation of the Tet2 from the T-cell; c) identifying the T-cells as low, medium, or high-avidity based on the dissociation time of the Tet2; and d) isolating the low-avidity T-cells.

[0213] Clause 11. The method of any one of clauses 5, 6 or 10, wherein the first dye is phycoerythrin (PE).

[0214] Clause 12. The method of any one of clauses 5 to 7 or 10, wherein the second dye is brilliant violet 421.

[0215] Clause 13. The method of any one of clauses 5 to 7 and 10 to 12, wherein the measuring comprising staining of the tetramers by flow cytometry.

[0216] Clause 14. The method of any one of clauses 5 to 7 and 10 to 13, wherein the peptide is an MHC I antigen.

[0217] Clause 15. The method of clause 14, wherein the antigen is Dd-IGPRALDVL (SEQ ID NO: 1).

[0218] Clause 16. The method of any one of clauses 5 to 7 and 10 to 15, further comprising adding additional anti-dye antibody.

[0219] Clause 17. The method of any one of clauses 5 to 7 and 10 to 16, wherein low-avidity T- cells dissociated within about 15 minutes, medium-avidity T-cell dissociate at about 75 minutes, and high-avidity T-cells dissociate after about 105 minutes.Attorney Docket No.: 2025-046-01; 45635.601

[0220] Clause 18. The method of any one of clauses 5 to 17, wherein the agent that specifically binds to the tetramer and prevents rebinding of the tetramer to antigen-specific T-cells is an antibody that specifically binds to the tetramer.

[0221] Clause 19. The method of any one of the preceding clauses, wherein the identifying the T-cells as low, medium or high-avidity comprises identifying altered gene expression associated with T-cell avidity.

[0222] Clause 20. The method of clause 19, wherein the level of expression of one or more of CD38, TOX2, ENTPD1, CTLA4, HAVCR2, TOX, PDCD1 and TIGIT is increased in high- avidity T-cells and the level of expression of one or more of TCF7, LEF1, IL7R, and CCR7 is increased in low-avidity T-cells.

[0223] Clause 21. The method of clause 19 or 20, further comprising generating an avidity score based on the altered gene expression.

[0224] Clause 22. The method of any one of the preceding clauses, wherein the T-cells are CD8 T-cells.

[0225] Clause 23. The method of any one of the preceding clauses, wherein the T-cells are isolated from tumors or tumor draining-lymph nodes.

[0226] Clause 24. The method of any one of the preceding clauses, wherein the T-cells are isolated after the identifying.

[0227] Clause 25. The use of immunotherapy to treat cancer in a subject identified as having low-avidity T-cells.

[0228] Clause 26. The use of low-avidity T-cells to treat cancer in a subject in need thereof.

[0229] Clause 27. The use of low-avidity T-cells in combination with checkpoint immunotherapy to treat cancer in a subject in need thereof.

[0230] Clause 28. The use of clause 25, 26, or 27, wherein the low-avidity T-cells are ex vivo T- cells.

[0231] Clause 29. A method of predicting the likelihood of a subject to not respond to immunotherapy, comprising: a) identifying the subject as having low, medium, or high-avidity T-cells; and b) predicting that the subject is not likely to respond to immunotherapy when the subject has high-avidity T-cells.Attorney Docket No.: 2025-046-01; 45635.601Incorporation by Reference

[0232] All U.S. and PCT patent publications and U.S. patents mentioned herein are hereby incorporated by reference in their entirety as if each individual patent publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.Other Embodiments

[0233] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

Claims

Attorney Docket No.: 2025-046-01; 45635.601CLAIMSWhat is claimed is:

1. A method of predicting the likelihood of a subject to respond to immunotherapy, comprising: a) identifying the subject as having low, medium, or high-avidity T-cells; and b) predicting that the subject is likely to respond to immunotherapy when said subject has low-avidity T-cell.

2. A method of predicting the likelihood of a subject to not respond to immunotherapy, comprising: a) identifying the subject as having low, medium, or high-avidity T-cells; and b) predicting that the subject is not likely to respond to immunotherapy when the subject has high- avidity T-cells.

3. A method of treating cancer in a subject in need thereof, comprising: a) identifying the subject as having low, medium, or high-avidity T-cells; and b) administering immunotherapy to a subject identified as having low-avidity T-cells.

4. The method of any one of claims 1 to 3, wherein the immunotherapy is checkpoint immunotherapy.

5. The method of claim 3 or 4, further comprising repeating the identifying during or after the administering.

6. The method of any one of claims 1 to 5, wherein the identifying the T-cells as low, medium, or high-avidity T-cells comprises: a) contacting a sample comprising the T-cells with a first peptide tetramer labeled with a first fluorescent dye (Teti ), a second peptide tetramer labeled with second fluorescent dye (Tet2), an agent that specifically binds to the tetramer and prevents rebinding of the tetramer to antigenspecific T-cell, and an antibody that specifically binds to the first fluorescent dye;Attorney Docket No.: 2025-046-01; 45635.601 b) measuring the rate of dissociation of the Tet2 from the T-cell; and c) identifying the T-cells as low, medium, or high-avidity based on the dissociation time of the Tet2.

7. A method of identifying a subject as having low, medium, or high-avidity T-cells, comprising : a) contacting a sample comprising the T-cells with a first peptide tetramer labeled with a first fluorescent dye (Teti), a second peptide tetramer labeled with a second fluorescent dye (Tet2), an agent that specifically binds to the tetramer and prevents rebinding of the tetramer to antigen-specific T-cell, and an antibody that specifically binds to the first fluorescent dye; b) measuring the rate of dissociation of the Tet2 from the T-cell; and c) identifying the T-cells as low, medium, or high-avidity based on the dissociation time of the Tet2.

8. The method of claim 6 or 7, wherein the agent that specifically binds to the tetramer and prevents rebinding of the tetramer to antigen-specific T-cell is an antibody that specifically binds to the tetramer.

9. A method of treating cancer, comprising: administering low-avidity T-cells to a subject in need thereof.

10. The method of claim 9, further comprising administering checkpoint immunotherapy to the subject.

11. The method of claim 9 or 10, wherein the T-cells are isolated from the subject.

12. The method of any one of claims 9 to 11, wherein the T-cells are obtained using a method, comprising: a) contacting a sample from the subject comprising the T-cells with a first peptide tetramer labeled with a first fluorescent dye (Teti), a second peptide tetramer labeled with second fluorescent dye (Tet2), an agent that specifically binds to the tetramer and prevents rebinding ofAttorney Docket No.: 2025-046-01; 45635.601 the tetramer to antigen-specific T-cell, and an antibody that specifically binds to the first fluorescent dye; b) measuring the rate of dissociation of the Tet2 from the T-cell; c) identifying the T-cells as low, medium, or high-avidity based on the dissociation time of the Tet2; and d) isolating the low-avidity T-cells.

13. The method of any one of claims 6, 7 or 12, wherein the first dye is phycoerythrin (PE).

14. The method of any one of claims 6, 7 or 12, wherein the second dye is brilliant violet 421.

15. The method of any one of claims 6, 7 and 12 to 14, wherein the measuring comprises staining of the tetramers by flow cytometry.

16. The method of any one of claims 6, 7 and 12 to 15, wherein the peptide is an MHC I antigen.

17. The method of claim 16, wherein the antigen is Dd-IGPRALDVL (SEQ ID NO: 1).

18. The method of any one of claims 6, 7 and 12 to 17, further comprising adding additional anti-dye antibody.

19. The method of any one of claims 6, 7 and 12 to 18, wherein low-avidity T-cells dissociated within about 15 minutes, medium-avidity T-cell dissociate at about 75 minutes, and high-avidity T-cells dissociate after about 105 minutes.

20. The method of any one of the preceding claims, wherein the identifying the T-cells as low, medium or high-avidity comprises identifying altered gene expression associated with T- cell avidity.Attorney Docket No.: 2025-046-01; 45635.60121 . The method of claim 20, wherein the level of expression of one or more of CD38, TOX2, ENTPD1, CTLA4, HAVCR2, TOX, PDCD1 and TIGIT is increased in high-avidity T-cells and the level of expression of one or more of TCF7, LEF1, IL7R, and CCR7 is increased in low- avidity T-cells.

22. The method of claim 20 or 21, further comprising generating an avidity score based on the altered gene expression.

23. The method of any one of the preceding claims, wherein the T-cells are CD8 T-cells.

24. The method of any one of the preceding claims, wherein the T-cells are isolated from tumors or tumor draining-lymph nodes.

25. The method of any one of the preceding claims, wherein the T-cells are isolated after the identifying.

26. The use of immunotherapy to treat cancer in a subject identified as having low-avidity T- cells.

27. The use of low-avidity T-cells to treat cancer in a subject in need thereof.

28. The use of low-avidity T-cells in combination with checkpoint immunotherapy to treat cancer in a subject in need thereof.

29. The use of any one of claims 26 to 28, wherein the low-avidity T-cells are ex vivo T- cells.

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