Chimeric antigen receptors targeting claudin-3 and methods for treating cancer

Abstract

Described herein is a chimeric antigen receptor (CAR) comprising an antigen binding protein that binds to a discontinuous epitope of human claudin-3 comprising at least N38 and E153 of SEQ ID NO: 13. Also described herein are polynucleotides encoding the antigen binding protein, the CAR, immune effector cells comprising the CAR, pharmaceutical compositions comprising the immune effector cells, and methods of treating cancer with the immune effector cells.

Description

[Technical Field]

[0001] The present invention relates to a chimeric antigen receptor (CAR) comprising an antigen binding protein that binds to at least one epitope of a cell-binding protein, wherein said cell-binding protein is located within a cell-cell junction, and wherein said one or more epitopes of the cell-binding protein are accessible and / or available for binding by said CAR extracellular domain only in cancer cells. [Background technology]

[0002] Adoptive T cell therapy is a groundbreaking, potentially curative treatment for cancer patients. To tailor these powerful autologous cell therapies to individual patients, T cells are obtained from peripheral blood and genetically engineered. These cells are transfected with chimeric antigen receptors (CARs) that enable them to specifically bind to an antigen of choice. These engineered cells are expanded ex vivo and reinfused into the patient for delivery to and subsequent killing of cancer cells expressing the matching antigen (Yeku et al., Am Soc Clin Oncol Educ Book. 2017; 37: 193-204; McBride et al., Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2019; 11(5): e1557).

[0003] CARs are synthetic antigen receptors that redirect T cell specificity, function, and persistence. CARs consist of an antigen-specific region of an antigen-binding protein, such as a single-chain variable fragment (scFv), fused to a T cell activation domain, e.g., the zeta chain of the CD3 complex, and a costimulatory domain, e.g., CD28 or 4-1BB. This configuration promotes antigen-specific activation and enhances proliferation and anti-apoptotic function of human primary T cells.

[0004] CAR-T cells have shown remarkable efficacy against a variety of liquid B-cell malignancies, and early clinical trial results suggest activity in multiple myeloma (Sadelain et al., Nature. 2017; 545(7655): 423-31; June et al., N Engl J Med. 2018; 379(1): 64-73; Brudno et al., Nat Rev Clin Oncol. 2017; 15(1): 31-46). The 2017 FDA approval of CD19 CAR-T for lymphoma and leukemia reinvigorated intensive efforts to develop CAR-T cells for solid tumors. However, CAR-T cell therapy has so far shown limited efficacy in solid tumors.

[0005] To develop safe and effective T cell therapy for solid tumors, T cells must maintain high and effective killing activity during production, be able to migrate to tumors, and overcome the immunosuppressive tumor microenvironment (TME). One of the key success factors is the selection of antigen targets. Typically, antigens selected for targeting are expressed in sufficient amounts on the surface of cancer cells but at low levels in normal tissues, ensuring low cross-reactivity with healthy cells (both off-target and on-target effects). CAR immunotherapy in solid tumors remains challenging, primarily due to the lack of suitable surface antigens whose expression is restricted to malignant tissues. Extratumoral expression of antigen targets can cause on-target toxicity, with varying degrees of severity depending on the affected organ tissue (Watanabe et al., Front Immunol. 2018;9:2486; Park et al., Sci Rep. 2017;7(1):14366). Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, there is a need to identify new targets for CAR immunotherapy, particularly for developing T cell therapy for solid tumors and cancers. [Means for solving the problem]

[0007] According to the first aspect, the following domains: a) an extracellular domain comprising an isolated claudin-3 binding protein that binds to a discontinuous epitope of human claudin-3 comprising at least N38 and E153 of SEQ ID NO: 13; b) a transmembrane domain; and c) one or more intracellular signaling domains A chimeric antigen receptor (CAR) comprising:

[0008] In a further aspect, the following domains: a) an extracellular domain comprising a claudin-3 binding domain comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (CDRH1) sequence of SEQ ID NO: 1; the heavy chain complementarity determining region 2 (CDRH2) sequence of SEQ ID NO: 2; and the heavy chain complementarity determining region 3 (CDRH3) sequence of SEQ ID NO: 3; b) a transmembrane domain; and c) one or more intracellular signaling domains A chimeric antigen receptor (CAR) comprising a polypeptide comprising:

[0009] In a further aspect, an isolated claudin-3 binding protein is provided that binds to a discontinuous epitope of human claudin-3 comprising at least N38 and E153 of SEQ ID NO:13.

[0010] In a further aspect, a claudin-3 binding protein is provided comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (CDRH1) sequence of SEQ ID NO: 1; the heavy chain complementarity determining region 2 (CDRH2) sequence of SEQ ID NO: 2; and the heavy chain complementarity determining region 3 (CDRH3) sequence of SEQ ID NO: 3.

[0011] In a further aspect, there is provided a polypeptide comprising the amino acid sequence of a CAR or claudin-3 binding protein disclosed herein.

[0012] According to other aspects, there are provided polynucleotides comprising sequences encoding the CAR or claudin-3 binding proteins disclosed herein, vectors comprising the polynucleotide sequences disclosed herein, and vector-producing cells comprising the polynucleotide sequences and / or vectors disclosed herein.

[0013] In another aspect, an immune effector cell is provided comprising a CAR, polypeptide, polynucleotide and / or vector disclosed herein.

[0014] In another aspect, there is provided a pharmaceutical composition comprising an immune effector cell or claudin-3 binding protein disclosed herein and a pharmaceutically acceptable excipient.

[0015] In another aspect, a method of producing an immune effector cell comprising a CAR disclosed herein is provided, said method comprising introducing into an immune effector cell a polypeptide, polynucleotide and / or vector disclosed herein.

[0016] In another aspect, there is provided a CAR, claudin-3 binding protein, polypeptide, polynucleotide, vector, immune effector cell, or pharmaceutical composition disclosed herein for use in treating cancer.

[0017] In another aspect, a method of treating cancer in a subject is provided, the method comprising administering to the subject a therapeutically effective amount of a CAR or claudin-3 binding protein, polypeptide, polynucleotide, vector, immune effector cell, or pharmaceutical composition disclosed herein.

[0018] In another aspect, there is provided a method of increasing cytotoxicity against cancer cells in a subject having cancer, said method comprising administering to the subject an effective amount of a CAR or claudin-3 binding protein, polypeptide, polynucleotide, vector, immune effector cell, or pharmaceutical composition disclosed herein.

[0019] In another aspect, a method of reducing the number of cancer cells in a subject having cancer is provided, the method comprising administering to the subject an effective amount of a CAR or claudin-3 binding protein, polypeptide, polynucleotide, vector, immune effector cell, or pharmaceutical composition disclosed herein.

[0020] In another aspect, there is provided a use of a CAR or claudin-3 binding protein, polypeptide, polynucleotide, vector, immune effector cell, or pharmaceutical composition disclosed herein in the manufacture of a medicament for the treatment of cancer.

[0021] In another aspect, there is provided a CAR or claudin-3 binding protein, polypeptide, polynucleotide, vector, immune effector cell, or pharmaceutical composition disclosed herein for use in therapy. [Brief explanation of the drawings]

[0022] [Figure 1]Figure 1 is a schematic diagram illustrating the accessibility of claudin-3 for binding in cancer cells and healthy, noncancerous cells ("normal cells"). CLDN3 belongs to a large family of integral membrane proteins important for the formation of tight junctions (TJs) between epithelial cells. Disruption of normal tissue architecture is a hallmark of cancer, and altered CLDN3 expression has been linked to the development of various epithelial cancers, including those with significant unmet medical needs, such as colorectal, breast, pancreatic, and ovarian cancers (Singh, Sharma, and Dhawan 2010). As shown, CLDN3 is inappropriately localized outside TJs in tumors, but not in healthy tissues. This mechanism makes CLDN3 a target for CAR-T cells to selectively kill tumor cells while sparing normal cells where CLDN3 is hidden in tight junctions. [Figure 2] Figure 2 shows the frequency of LNGFR+ cells. Figure 2A: Enrichment of LNGFR+ CAR-T batches. CAR-T batches were subjected to EasySep LNGFR positive selection to generate a 100% CAR-T cell population for use in functional assays. Figure 2B: Homogenization of CAR-T batches to achieve the required frequency of LNGFR+ cells. CAR-T batches were homogenized by adding untransduced T cells to achieve a transduction efficiency of 30% across all constructs. [Figure 3A] Figure 3A shows the basal IFNγ cytokine secretion profiles of untransduced and transduced CAR-T cells. Mean and standard deviation of IFNγ secretion concentrations in culture supernatants of CAR-T cells obtained from six different healthy donors (****=p<0.0001 calculated by Bonferroni one-way ANOVA; UT=untransduced). [Figure 3B-G] Figures 3B-G show the basal exhausted / activated phenotype (CD69+, TIM3+, and PD-1+) of untransduced and transduced CAR-T cells. Means and standard deviations are shown (p values ​​ranged from <0.02 to <0.0001, calculated by Bonferroni one-way ANOVA; UT = untransduced). [Figure 3H]Figure 3H shows CAR-specific pCD3ζ levels (assessed by PEGGY-SUE) in transduced CAR-T cells. pCD3ζ staining normalized to the negative control (anti-CD19 CAR) is shown. [Figure 4A] Figure 4A shows the secretion of IFNγ, IL-2, and TNFα in response to claudins. Quantitation of cytokine release (mean + 95% confidence limits, n=3) from anti-claudin-3 or anti-CD19 CAR-T cells cocultured with RKO KO cell lines overexpressing hCLDN3, hCLDN4, hCLDN5, hCLDN6, hCLDN8, hCLDN9, hCLDN17, or mCLDN3. [Figure 4B-C] Figures 4B-C show IFNγ secretion in response to claudin proteins. Cytokine release from anti-claudin-3 CAR T cells, anti-CD19 CAR T cells (control), or untransduced T cells co-cultured with RKO KO overexpressing hCLDN3, hCLDN4, hCLDN6, hCLDN9, or mCLDN3 is shown. Figure 4B: IFNγ concentrations expressed as the mean + 95% confidence limit of six donors. Figure 4C: Fold change in IFNγ secretion from anti-claudin-3 CAR T cells compared to anti-CD19 control T cells or untransduced T cells when cultured with cell lines expressing claudin proteins. [Figure 4D-E] Figures 4D-E show the levels of cytokines secreted in response to claudins. Cytokine concentrations (pg / mL) are overlaid on a heatmap representing the fold change for each condition compared to RKO KO cells cultured with control T cells. Fold changes are calculated and log-transformed within each experiment and donor. Data for IFNγ (top panel) and IL-2 (bottom panel) are shown for the specific donors indicated in the left column. [Figure 4F]Figure 4F shows the levels of cytokines secreted in response to claudins. Cytokine concentrations (pg / mL) are overlaid on a heat map representing the fold change for each condition compared to RKO KO cells cultured with control T cells. Fold changes were calculated and log-transformed within each experiment and donor. TNFα data for specific donors are shown in the left column. [Figure 5A] Figure 5A shows images of anti-claudin-3 CAR-T cells cocultured with RKO KO cells expressing claudin proteins. These images were acquired on day 4 for each cell line expressing hCLDN3, hCLDN4, hCLDN6, hCLDN9, or mCLDN3. Left panel: Images are overlaid with a yellow mask surrounding the cell area used to calculate % confluency. Right panel: Images are overlaid with red fluorescence showing the intensity and localization of the CYTOTOX Red dye. [Figure 5B] Figure 5B shows images of anti-claudin-3 CAR-T cells co-cultured with claudin-expressing RKO KO cells taken every 2 hours for 4 days, and the % confluency for each well at each time point was calculated using INCUCYTE software. Six donors were used in this study, with triplicates. The average of triplicate wells was calculated, and each point in the figure represents the mean ± SEM for six donors. [Figure 6] Figure 6 shows the cytotoxic response of CAR-T cells to claudin family proteins. Anti-claudin-3 CAR ("906-009"), anti-CD19 CAR ("CD19"; control), and untransduced T cells ("UT") were co-cultured with RKO KO cells expressing hCLDN3, hCLDN4, hCLDN6, hCLDN9, or mCLDN3 for 4 days. CYTOTOX Red absorbance and resulting red fluorescence were analyzed using a mask, and this data was used to calculate the % viable cells. Data from one representative donor are presented as the mean ± SEM of triplicate wells. [Figure 7]Figure 7 shows the results of quantification of surface molecules using quantification beads. Figure 7A: Average number of LNGFR molecules per T cell. T cells and Quantum Simply Cellular quantification beads were stained with anti-LNGFR PE and their mean fluorescence intensity (MFI) was measured. A standard curve was generated using the bead MFI and used to interpolate the number of LNGFR molecules per cell. n = 3 for donors 12031 and 92024, and n = 5 for donor D5. Error bars indicate standard deviation. Figure 7B: Quantification of hCLDN3 expression in RKO human colon cancer cells. RKO cells, engineered to express hCLDN3 after knockout of endogenous hCLDN3, were sorted for low or high hCLDN3 expression. RKO cells and Quantum Simply Cellular quantification beads were stained with anti-hCLDN3-PE and their MFI was measured. A standard curve was generated using the bead MFI and used to interpolate the number of hCLDN3 molecules per cell. Measurements were performed in quadruplicate and error bars indicate standard deviation. [Figure 8] Figure 8 shows examples of INCUCYTE and XCELLIGENCE killing assays. The INCUCYTE raw data are shown for LNGFR-enriched anti-CD19 CAR-T cells (Figure 8A) or anti-claudin-3 CAR-T cells (Figure 8B) incubated with RKO-KO CLDN3 L14 for 90 hours. Figure 8C shows an example killing curve based on the raw data from Figures 8A-8B. Triplicate measurements were performed, and error bars indicate standard deviation. Figure 8D shows an example of an XCELLIGENCE killing assay. Normalized data are shown for unsorted anti-claudin-3 CAR-T cells or untransduced T cells cocultured at a 1:1 ratio with the HT-29-LUC target cell line. Data are expressed as the mean ± standard deviation for n=3. [Figure 9] Figure 9 shows an INCUCYTE killing assay using various numbers of RKO-KO hCLDN3-expressing target cells. RKO-KO and RKO-KO hCLDN3 polyclonal cells were mixed at various ratios and co-cultured with anti-CD19 control CAR-T cells or anti-claudin-3 CAR-T cells. The % viable cells were measured over time. Triplicates were measured, and error bars indicate standard deviation. [Figure 10A-B] Figures 10A-B show T cell dose responsiveness with a gradient of hCLDN3 expression. RKO-KO cells were electroporated with a gradient of hCLDN3 mRNA and cocultured with CLDN3 CAR-T cells and anti-CD19 control CAR-T cells generated from three donors. Figure 10A: hCLDN3 expression assessed by flow cytometry versus the nucleofection amount of hCLDN3 mRNA. The data are presented as mean fluorescence intensity or % of the target-positive population. Pearson's R2 was calculated for the correlation between mRNA amount and mean fluorescence intensity. Figure 10B: After coculture, anti-claudin-3 CAR-T cells were stained to determine CD69 expression. % CD69 expression is shown as the mean + 95% CI of three donors. [Figure 10C-D] Figures 10C-D show T cell dose responsiveness according to the gradient of hCLDN3 expression. RKO-KO cells were electroporated with a gradient of hCLDN3 mRNA and cocultured with CLDN3 CAR-T cells and anti-CD19 control CAR-T cells generated from three donors. The coculture supernatant was used to quantify the concentrations of cytokines secreted by T cells. Figure 10C: IFNγ pg / mL is expressed as the mean + 95% confidence interval of three donors. The ratio of IFNγ secretion from anti-claudin-3 CAR-T cells to anti-CD19 control CAR-T cells. Figure 10D: Granzyme B pg / mL is expressed as the mean + 95% confidence interval of three donors. The ratio of granzyme B secretion from anti-claudin-3 CAR-T cells to anti-CD19 control CAR-T cells. [Figure 11A]Figure 11A shows CLDN3 expression and IFNγ secretion by flow cytometry and RT-qPCR in response to indication: colon cancer-derived cell lines. hCLDN3 expression was measured at the protein level by flow cytometry (left) and at the mRNA level by RT-qPCR (center). HT-29-LUC and RKO-KO cell lines were included in all studies as positive and negative controls, respectively. IFNγ secretion in response to indication-derived cell lines was also assessed. T cells were incubated with target cell lines at a 1:1 ratio for 24 hours, and IFNγ secretion was measured by MSD (right). [Figure 11B] Figure 11B shows CLDN3 expression and IFNγ secretion by flow cytometry and RT-qPCR in response to cell lines derived from pancreatic cancer. hCLDN3 expression was measured at the protein level by flow cytometry (left) and at the mRNA level by RT-qPCR (center). HT-29-LUC and RKO-KO cell lines were included in all studies as positive and negative controls, respectively. IFNγ secretion in response to cell lines derived from the indications was also assessed. T cells were incubated with target cell lines at a 1:1 ratio for 24 hours, and IFNγ secretion was measured by MSD (right). [Figure 11C] Figure 11C shows CLDN3 expression and IFNγ secretion by flow cytometry and RT-qPCR in response to breast cancer-derived cell lines. hCLDN3 expression was measured at the protein level by flow cytometry (left) and at the mRNA level by RT-qPCR (center). HT-29-LUC and RKO-KO cell lines were included in all studies as positive and negative controls, respectively. IFNγ secretion in response to the indication-derived cell lines was also assessed. T cells were incubated with the target cell lines at a 1:1 ratio for 24 hours, and IFNγ secretion was measured by MSD (right). [Figure 12]Figure 12 shows examples of killing images and raw data for selected cell lines. Examples of images (top) and raw data (bottom) are shown for two cell lines that showed complete killing (HT-29-LUC and MDA MB468), three cell lines that showed partial killing (HCC1954, HPAC, and BxPC3), and one cell line that showed no killing (COLO-320DM). Raw data is shown as the total CYTOTOX Red per well. Raw data was used because data cannot be normalized across different cell lines and can only be compared on a raw basis. [Figure 13] Figure 13 shows CAR expression determined by protein L-biotin (primary) staining and anti-biotin-PE (secondary) staining. Transduction efficiency was determined by LNGFR-PE staining. Shown here are CAR and LNGFR frequencies for exemplary donor D5 at day 7 after transduction with the designated CAR variants. [Figure 14] Figure 14 shows the results of a killing assay with CAR T cells (donor D5). The frequency of lysed target cells was calculated after measuring luciferase activity in co-cultures of CAR-T cells with luciferase-transduced T-47D cells (each data point represents the mean of technical replicates, n=2). [Figure 15] Figure 15 shows the concentrations of cytokines secreted in response to anti-claudin-3 CAR-T cells bearing various scFv constructs, determined using the MACSPlex Cytokine 12 Kit (human). Supernatants from cocultures of T cells with T-47D cells or T cells alone were collected and analyzed (undiluted). Secreted concentrations of IFNγ (Figure 15A), IL-2 (Figure 15B), and TNF-α (Figure 15C) from T cell donor D5 are shown. [Figure 16A-B]Figures 16A-B show the results of long-term coculture of RKO-KO CLDN3 H1 (labeled as RKO-hCLD3) cells with CAR-T cells (donor G5) (rounds 1 and 2 (Figures 16A-B) for donor G5). CAR-T cells were transferred into fresh RKO-KO CLDN3 H1 cells a total of three times. Growth of GFP-expressing RKO-KO CLDN3 H1 was monitored at % green target confluency using INCUCYTE and normalized to the initial value (4 hours). [Figure 16C-D] Figures 16C-D show the results of long-term coculture of RKO-KO CLDN3 H1 (labeled as RKO-hCLD3) cells with CAR-T cells (donors G5 and H5) (third time for donor G5 (Figure 16C) and first time for donor H5 (Figure 16C). CAR-T cells were transferred into fresh RKO-KO CLDN3 H1 cells a total of three times. Proliferation of GFP-expressing RKO-KO CLDN3 H1 was monitored at % confluency in green using INCUCYTE and normalized to the initial value (4 hours). Conditions without replicates are marked with an asterisk (*). [Figure 16E-F] Figures 16E-F show the results of long-term coculture of RKO-KO CLDN3 H1 (labeled as RKO-hCLD3) cells with CAR-T cells (donor H5) (second and third rounds for donor H5 (Figures 16E-F)). CAR-T cells were transferred into fresh RKO-KO CLDN3 H1 cells a total of three times. Growth of GFP-expressing RKO-KO CLDN3 H1 cells was monitored at % confluency in green using INCUCYTE and normalized to the initial value (4 hours). Conditions without replicates are marked with an asterisk (*). [Figure 17A-B]Figures 17A-B show exhaustion marker expression determined by staining with anti-LAG3 (CD223)-VioBlue, anti-PD-1 (CD279)-PE-Vio770, and anti-TIM3 (CD366)-APC. LNGFR-positive T cells were assessed for double-positive exhaustion marker expression (TIM3, PD-1; indicated by filled circles) and triple-positive exhaustion marker expression (TIM3, PD-1, LAG3; indicated by filled squares). The frequencies of double- and triple-positive CAR T cells are shown for donor H and donor P. Day 0 represents the frequency before target cell addition, and day 1 represents the frequency after the first addition of target cells. Fresh RKO-KO CLDN3 H1 cells were added on days 1, 2, and 3. Results for day 0 (Figures 17A and B) are shown for donor H and donor P, respectively. [Figure 17C-D] Figures 17C-D show exhaustion marker expression determined by staining with anti-LAG3 (CD223)-VioBlue, anti-PD-1 (CD279)-PE-Vio770, and anti-TIM3 (CD366)-APC. LNGFR-positive T cells were assessed for double-positive exhaustion marker expression (TIM3, PD-1; indicated by filled circles) and triple-positive exhaustion marker expression (TIM3, PD-1, LAG3; indicated by filled squares). The frequencies of double- and triple-positive CAR T cells are shown for donor H and donor P. Day 0 represents the frequency before target cell addition, and day 1 represents the frequency after the first addition of target cells. Fresh RKO-KO CLDN3 H1 cells were added on days 1, 2, and 3. Results are shown for day 1 (Figures 17C and D) for donor H and donor P, respectively. [Figure 17E-F]Figures 17E-F show exhaustion marker expression determined by staining with anti-LAG3 (CD223)-VioBlue, anti-PD-1 (CD279)-PE-Vio770, and anti-TIM3 (CD366)-APC. LNGFR-positive T cells were assessed for double-positive exhaustion marker expression (TIM3, PD-1; indicated by filled circles) and triple-positive exhaustion marker expression (TIM3, PD-1, LAG3; indicated by filled squares). The frequencies of double- and triple-positive CAR T cells are shown for donor H and donor P. Day 0 represents the frequency before target cell addition, and day 1 represents the frequency after the first addition of target cells. Fresh RKO-KO CLDN3 H1 cells were added on days 1, 2, and 3. Results are shown for day 2 (Figures 17E and F) for donor H and donor P, respectively. [Figure 17G-H] Figures 17G-H show exhaustion marker expression determined by staining with anti-LAG3 (CD223)-VioBlue, anti-PD-1 (CD279)-PE-Vio770, and anti-TIM3 (CD366)-APC. LNGFR-positive T cells were assessed for double-positive exhaustion marker expression (TIM3, PD-1; indicated by filled circles) and triple-positive exhaustion marker expression (TIM3, PD-1, LAG3; indicated by filled squares). The frequencies of double- and triple-positive CAR T cells are shown for donor H and donor P. Day 0 represents the frequency before target cell addition, and day 1 represents the frequency after the first addition of target cells. Fresh RKO-KO CLDN3 H1 cells were added on days 1, 2, and 3. Results are shown for day 3 (Figures 17G and H) for donor H and donor P, respectively. [Figure 17I-J]Figures 17I-J show exhaustion marker expression determined by staining with anti-LAG3 (CD223)-VioBlue, anti-PD-1 (CD279)-PE-Vio770, and anti-TIM3 (CD366)-APC. LNGFR-positive T cells were assessed for double-positive exhaustion marker expression (TIM3, PD-1; indicated by filled circles) and triple-positive exhaustion marker expression (TIM3, PD-1, LAG3; indicated by filled squares). The frequencies of double- and triple-positive CAR T cells are shown for donor H and donor P. Day 0 represents the frequency before target cell addition, and day 1 represents the frequency after the first addition of target cells. Fresh RKO-KO CLDN3 H1 cells were added on days 1, 2, and 3. Results are shown for day 6 (Figures 17I and J) for donor H and donor P, respectively. [Figure 18] Figure 18 shows that anti-claudin-3 CAR-T cells (906-009) enhance claudin-3-specific proliferative responses after stimulation with claudin-3-positive target cells compared to anti-claudin-3 CAR-T cells with other spacer or orientation variants (906-002, 906-004, and 906-007). [Figure 19A] Figure 19A shows the growth rate in NSG mice inoculated with the HT-29 human colon adenocarcinoma cell line. Seven days after inoculation, tumors were palpable, and animals were administered PBS (no T cells), anti-CD19 (control CAR), or anti-claudin-3 CAR-T cells at a dose of 1 x 10 cells. The day of administration is designated as D0. Figure 19A: Tumor volume results for each group and each measurement time point are expressed as marginal means with 95% confidence intervals. [Figure 19B]Figure 19B shows the growth rate in NSG mice inoculated with the HT-29 human colon adenocarcinoma cell line. Seven days after inoculation, tumors were palpable, and animals were administered PBS (no T cells), anti-CD19 (control CAR), or anti-claudin-3 CAR-T cells at a dose of 1 x 10 cells. The day of administration is designated as D0. Figure 19B: The difference between the mean tumor volume of each group relative to the negative control anti-CD19 group is shown. A more negative value indicates that the anti-CD19 group had larger tumors than the control group. Stars are used to indicate statistical significance: *p<0.05, **p<0.01, ***p<0.001. [Figure 20] Figure 20 shows the percentage (%) of LNGFR-positive (+) CAR-T cells gated on the human CD3 cell population (CD45+, CD3+ LNGFR+) in the peripheral blood of CAR-T-administered NSG mice analyzed by flow cytometry 28 days after administration. Note that 6 mice in the PBS group (no T cells), 3 mice in the anti-CD19 CAR group, and 8 mice in the anti-claudin-3 CAR-T cell group were still on study at day 28. [Figure 21] Figure 21 shows IFNγ release measured in the serum of HT-29 tumor-bearing NSG mice before and 7 days after administration of PBS (no T cells), anti-CD19 (control CAR)-T cells, or anti-claudin-3 CAR-T cells. N = 6–8 mice per group. Each data point represents the average of a single measurement or two or three technical control replicates per mouse, depending on the blood volume available. Figure 21A: Pre- and post-treatment IFNγ release (pg / mL) per group. HD represents the highest density. Figure 21B: Comparison of pre- and post-treatment IFNγ release between anti-claudin-3 CAR and anti-CD19 CAR controls. The anti-claudin-3 CARs show a 15-fold increase in IFNγ release compared to the anti-CD19 CAR group. The Bayesian posterior probability that this change is greater than a 1-fold change (i.e., the probability that IFNγ increases even slightly with treatment) is 98.2%. This indicates a high probability of increased IFNγ following treatment with anti-claudin-3 CAR-T cells. [Figure 22]Figure 22 shows the results of characterization of colorectal cancer patient-derived xenograft (PDX) models by flow cytometry. The figures summarize the percentage (%) of EpCAM-CLDN3 double-positive (+) cell populations detected after thawing in two separate studies: one using the models CR5052, CR5080, and CR89 (Panel A, left), and the other using the models CR5030 and CR5087 (Panel B, right). Both studies included a CLDN3-positive cell line (HT-29 Luc, designated HT-29) and a negative control (RKO-KO) cell line. The percentage of EpCAM-positive tumor cell populations ranged from 41 to 65% in the CR models. In the first study (Panel A, left), CLDN3-expressing tumor cells ranged from 34.6 to 55.4%. In the second study (Panel B, right), the percentage of CLDN3-expressing tumor cells ranged from 26 to 38%. Furthermore, as expected, high CLDN3 expression was detected in the positive control (HT-29), whereas CLDN3 was not detected in the RKO-KO cells (negative control). OV PDX models were not shown because no populations containing EpCAM-CLDN3 double-positive cells were present in the samples. Each study represents one tumor sample for each model. These studies helped characterize PDX samples with target expression for future in vitro PDX assays, allowing for multiple biological replicates for each model. [Figure 23]Figure 23 shows cytokine release from two co-culture studies, one using models CR5030, CR5087, and OV5287 (Panel A, left; Figures 23A, 23C, and 23E), and the other using models CR5052, CR5080, and CR89 (Panel B, right; Figures 23B, 23D, and 23F). Both studies were performed with the control cell lines HT-29 Luc (HT-29, designated as the positive control) and RKO-KO (the negative control) at 50,000 cells per well in Panel A and 25,000 cells per well in Panel B, and with T cells alone at 50,000 cells per well. CAR-T cells (effector) were added to PDX cells (target) at a 1:1 target to effector ratio. Cytokines were elevated in all cocultures with anti-claudin-3 CAR-T cells, including the low CLDN3-expressing model OV5287, but not in the CLDN3-negative control RKO-KO or T cells alone. Each study used one biological replicate (tumor) per model from one T cell donor, with two or three technical replicates depending on the number of cells available. No statistical analysis was performed on these pilot study data. [Figure 24] Figure 24 shows available CD20 binding sites on anti-claudin-3 CAR-T cells (CD20_906_009) compared to B cells. For each condition, the median fluorescence intensity of CD20+ anti-claudin-3 CAR-T cells (CD20_906_009) and B cells was used to calculate the number of CD20 binding sites. [Figure 25] FIG. 25 shows an outline of the test conditions for complement-dependent cytotoxicity (CDC). [Figure 26] Figure 26 shows a comparison of the proportion of cells lacking CD20 expression under various CDC conditions. A mixed model was fixed to a binomial proportion for the proportion of viable CTV cells after 4 hours. Fixed effects included all combinations of complement and antibody and their interactions on CD20 expression. A random effects split-plot design was then fitted, with donors within random intercepts as random intercepts. [Figure 27A-C]Figures 27A-C show CAR-T deletion by ADCC using CD20+ anti-claudin-3 CAR-T cells (CD20_906_009) and anti-claudin-3 CAR-T cells (906_009) with and without splice site optimization (SO). The ratio of "CTV fraction" between RTX:HI and RTX:RAB under various ADCC conditions is shown. CAR T cells enhanced CAR expression by F(Ab)2. [Figure 27D-E] Figures 27D-E show CAR-T deletion by ADCC using CD20+ anti-claudin-3 CAR-T cells (CD20_906_009) and anti-claudin-3 CAR-T cells (906_009) with and without splice site optimization (SO). The ratio of "CTV fraction" between RTX:HI and RTX:RAB under various ADCC conditions is shown. CAR T cells enhanced CAR expression by F(Ab)2. [Figure 28] Figure 28 shows cell viability of CD20+ anti-claudin-3 CAR-T cells (CD20_906_009) and control anti-claudin-3 CAR-T cells (906_009) surviving at 20 hours in the XCELLIGENCE cytotoxicity assay. A linear mixed-effects model was fit to the data. % survival was modeled as the response, and CAR was modeled as a fixed effect. Because this is a split-plot design, random effects were included for individual assays and donor nested within the assays. Linear contrasts were used to determine the difference in expected % survival between CAR pairs, and these are reported along with p-values ​​and 95% confidence intervals. [Figure 29] Figure 29 shows XCELLIGENCE KT50 values ​​for anti-claudin-3 CAR-T cells (906_009) and CD20+ anti-claudin-3 CAR-T cells (CD20_906_009). A linear model was fitted to KT50, with a fixed effect of CAR (vector) and nested random effects for individual assays and donors. A log10 transformation was used for KT50. [Figure 30]Figure 30 shows the effect of splice site optimization on cell survival at 20 hours in the XCELLIGENCE cytotoxicity assay. A linear mixed-effects model was fit to the data. % survival was modeled as the response, and CARs were modeled as fixed effects. Because this is a split-plot design, random effects were included for individual assays and donors nested within assays. Linear contrasts were used to determine the difference in expected % survival between CAR pairs, and these are reported along with p-values ​​and 95% confidence intervals. [Figure 31] Figure 31 shows the effect of splice site optimization on XCELLIGENCE KT50 values. KT50 was fitted with a linear model, with a fixed effect of CAR (vector) and nested random effects for individual assays and donors. KT50 was log10 transformed. [Figure 32] Figure 32 shows the effect of CD20 on calcium flux in CAR-T cells. Calcium flux is shown for untransduced T cells, anti-claudin-3 CAR-T cells (906_009), and CD20+ anti-claudin-3 CAR-T cells (CD20_906_009) stimulated with ionomycin after pretreatment with thapsigargin (Figure 32A) or DMSO (Figure 32B). [Figure 33] Figure 33 shows the results of a plasma membrane protein array. Pre-screen studies using untransduced cells, BCMA-CAR T cells, and anti-claudin-3 CAR-T cells (906-009) from donor 90928 are shown. ZsGreen Keyspotting patterns of protein expression in HEK293 cells (Figure 33A), untransduced T cells (Figure 33B), BCMA CAR-T cells (Figure 33C), and anti-claudin-3 CAR-T cells (906-009; Figure 33D). [Figure 34] Figure 34 shows the results of the plasma membrane protein array validation screen. Key to spotting patterns (Figure 34A) in untransduced cells (Figure 34B), BCMA CAR-T cells (Figure 34C), and anti-claudin-3 CAR-T cells (906-009; Figure 34D). [Figure 35A-B] Figures 35A-B show the time course of IFNγ secretion in NSG tumor-bearing mice after administration of 902_007-LNGFR, SO-CD20-906_009, or CD20-CD19. IFNγ secretion levels were measured in the serum of HT-29Luc tumor-bearing NSG mice (Figures 35A-35B) before T cell administration (baseline) or 3, 4, 5, 7, and 14 days after T cell administration with 902_007-LNGFR, SO-CD20-906_009, or CD20-CD19. Secretion levels are shown in pg / mL (y-axis). Each dot represents the cytokine concentration at a given time point for a given mouse. The graph (Figure 35A) shows the data as the mean and 95% confidence intervals for all time points. The graph (Figure 35B) shows the marginal means and 95% confidence intervals from a linear mixed model, overlaid on the raw data for all time points except baseline. [Fig. 35C-D] Figures 35C-D show the time course of IL-2 secretion in NSG tumor-bearing mice after administration of 902_007-LNGFR, SO-CD20-906_009, or CD20-CD19. IL-2 secretion levels were measured in the serum of HT-29Luc tumor-bearing NSG mice (Figures 35C-35D) before T cell administration (baseline) or 3, 4, 5, 7, and 14 days after T cell administration with 902_007-LNGFR, SO-CD20-906_009, or CD20-CD19. Secretion levels are shown in pg / mL (y-axis). Each dot represents the cytokine concentration at a given time point for a given mouse. Graph (35C) shows the data as the mean and 95% confidence intervals for all time points. Graph (35D) shows the marginal means and 95% confidence intervals from a linear mixed model, overlaid on the raw data for all time points except baseline. [Figure 35E-F]Figures 35E-F show the time course of TNFα secretion in NSG tumor-bearing mice after administration of 902_007-LNGFR, SO-CD20-906_009, or CD20-CD19. Secreted TNFα levels were measured in the serum of HT-29Luc tumor-bearing NSG mice (Figures 35E-35F) before T cell administration (baseline) or 3, 4, 5, 7, and 14 days after T cell administration with 902_007-LNGFR, SO-CD20-906_009, or CD20-CD19. Secreted levels are shown in pg / mL (y-axis). Each dot represents the cytokine concentration at a given time point for a given mouse. The graph (Figure 35E) shows the data as the mean and 95% confidence intervals for all time points. The graph (Figure 35F) shows the marginal means and 95% confidence intervals from a linear mixed model, overlaid on the raw data for all time points except baseline. [Figure 36] Figure 36 shows the time course of IFNγ, IL-10, IL-12p70, IL-13, IL-1β, IL-2, IL-4, IL-6, IL-8, and TNF-α secretion in NSG tumor-bearing mice after administration of 902_007-LNGFR, SO-CD20-906_009, or CD20-CD19. This heat map shows the changes in IFNγ, IL-10, IL-12p70, IL-13, IL-1β, IL-2, IL-4, IL-6, IL-8, and TNF-α secretion at each time point (3, 4, 5, 7, and 14 days) after T cell administration compared to baseline (before T cell administration) in HT-29Luc tumor-bearing NSG mice administered 902_007-LNGFR, SO-CD20-906_009, or CD20-CD19. Linear contrast was used to calculate the change in secretion at various time points after T cell administration relative to baseline levels and is expressed here as fold change. [Figure 37]Figure 37 shows tumor growth rates in NSG tumor-bearing mice administered 902_007-LNGFR, SO-CD20-906_009, or CD20-CD19 from the endpoint "day 14." Tumor growth rates in NSG tumor-bearing mice administered 902_007-LNGFR, SO-CD20-906_009, or CD20-CD19 from the endpoint "day 14." Mice were inoculated with HT-29Luc cells on SD0 and administered CAR T cells on SD23, when tumors reached approximately 320 mm3. Mice from the endpoint "day 14" were sacrificed on SD37, 14 days after T cell administration. The y-axis indicates tumor volume (mm3), and the x-axis indicates the test date for all caliper measurements. Two-way ANOVA followed by Bonferroni multiple comparisons was performed to compare all CAR T groups at all time points. Error bars indicate standard error of the mean. ns>0.05, **p<0.01, ****p<0.0001. [Figure 38] Figure 38 shows tumor growth in NSG tumor-bearing mice administered with 902_007-LNGFR, SO-CD20-906_009, or CD20-CD19 from endpoints "day 4" and "day 7." Tumor growth is shown in NSG tumor-bearing mice administered with 902_007-LNGFR, SO-CD20-906_009, or CD20-CD19 from (Figure 38A) endpoint "day 4" and (Figure 38B) "day 7." Mice were inoculated with HT-29Luc cells on SD0 and administered CAR T cells on SD23, when tumors reached approximately 320 mm3. Mice from endpoint "day 4" were sacrificed on SD27, 4 days after T-cell administration. Mice from endpoint "day 7" were sacrificed on SD30, 7 days after T-cell administration. The y-axis indicates tumor volume (mm3), and the x-axis indicates CAR T group. One-way ANOVA followed by Tukey's multiple comparison test was performed to compare CAR T groups at the indicated endpoints. Error bars represent the standard error of the mean. ns>0.05. All comparisons were not significant. [Figure 39]Figure 39 is a schematic diagram showing the study design. Briefly, female NSG mice were inoculated with HT-29Luc on study day (SD) 0. On SD23 (when tumors reached approximately 320 mm3), mice were administered CAR T cells. Blood samples were collected on SD5, SD26, SD27, SD28, SD30, and SD37. Tissues and tumors were collected on SD26, SD27, SD30, and SD37. [Figure 40] Figure 40 shows the mouse model. NSG-SGM3 mice harbor mouse macrophages and transgenically express human cytokines. Human PBMCs and human target cells (SO-CD20-906_009 T cells) are co-injected. These cells were generated from the same healthy donor. Anti-CD20 mAb rituximab is injected to induce the killing of SO-CD20-906_009 T cells in peripheral blood and tissues. Control mice receive isotype or vehicle in the presence of SO-CD20-906_009 T cells, or rituximab in the absence of SO-CD20-906_009 T cells. [Figure 41] Figure 41 shows the study timeline. On day -1 (D-1), cells were inoculated (1x107 hPBMCs, 1x107 T cells with a transduction efficiency of 38% for SO-CD20-906_009). On day 0 (D0), blood was collected from each mouse (pre-RTX blood as baseline), followed by RTX, isotype, or vehicle injection via the ip route. Blood was collected from each mouse again 24 and 72 hours after mAb administration. On days 7 and 8 after mAb administration, mice were humanely sacrificed, and terminal blood and tissue collections were performed in an alternating fashion to ensure sample quality and feasibility. [Figure 42A] Figure 42A shows the characterization of the inoculum on the day of injection. Gating strategy for identifying SO-CD20-906_009 and PBMC subpopulations is shown. F(ab')2+ CD20+ = SO-CD20-906_009. F(ab')2 = CAR. [Fig. 42B-C]Figures 42B-C show the characterization of the inoculum on the day of injection. Figure 42B shows the PBMC composition in the inoculum. Figure 42C shows the characterization of CAR and CD20 expression on T cells in the inoculum. The graph shows the mean (n=3 replicates) percentage of CAR-expressing T cells and T cells co-expressing CAR and CD20. Error bars indicate standard deviation. F(ab')2+ CD20+ = SO-CD20-906_009. F(ab')2 = CAR [Figure 43A-B] Figures 43A-B show the characterization and enumeration (flow cytometry) of SO-CD20-906_009 and hPBMCs in terminal mouse blood. (A) Comparison of CD3+ (T cell) and f(ab')2 counts in terminal blood 7 and 8 days after mAb / isotype treatment. (B) Total f(ab')2 counts in terminal mouse blood. Each dot in Figure 43B represents one mouse with marginal mean and 95% confidence interval. F(ab')2 = CAR, CD3+ = T cells, mAb = monoclonal antibody (rituximab), ISO = anti-RSV antibody. * = p-value ≤ 0.05, ** = p-value ≤ 0.01, *** = p-value ≤ 0.001. [Fig. 43C-D] Figures 43C-D show the characterization and enumeration (flow cytometry) of SO-CD20-906_009 and hPBMCs in terminal mouse blood. (C) Total CD3+ counts in terminal mouse blood. (D) Percentage of SO-CD20-906_009 relative to CD3+ counts in terminal mouse blood. Each dot represents one mouse with marginal mean and 95% confidence interval. F(ab')2 = CAR, CD3+ = T cells, mAb = monoclonal antibody (rituximab), ISO = anti-RSV antibody. * = p-value ≤ 0.05, ** = p-value ≤ 0.01, *** = p-value ≤ 0.001. [Figure 44] Figure 44 shows a comparison of CAR and CD20 expression on SO-CD20-906_009 before and after inoculation. Histograms show CD20 and CAR (F(ab')2) co-expression on SO-CD20-906_009 T cells in the inoculum on the day of injection (day of inoculation) and from mouse blood on the day of sacrifice (day 7 or 8 after mAb administration). F(ab')2 = CAR expression. [Figure 45]Figure 45 shows that SO-CD20-906_009 in the blood decreases in mAb-treated mice by 24 hours after mAb administration. Blood samples were collected from mice before mAb administration and 24 hours, 72 hours, and 7 / 8 days (final day) after mAb treatment. HIV DNA copies were measured using ddPCR as a marker for the presence of SO-CD20-906_009 in mouse blood. (A) Mice treated with pre-mAb, SO-CD20-906_009 with and without mAb controls, SO-CD20-906_009 and isotype mAb control, and SO-CD20-906_009 and mAb showed comparable SO-CD20-906_009 expression in the blood. Twenty-four hours after mAb administration, the mAb-treated group had a significant reduction in SO-CD20-906_009 compared to the SO-CD20-906_009 and isotype mAb groups, which persisted until the end of the study. Graphs show mean % HIV copy change and 95% confidence intervals. [Clear triangles] represent mice treated with SO-CD20-906_009 and a no-mAb control; [Clear squares] represent mice treated with SO-CD20-906_009 and an isotype mAb control; and [Diagrams] represent mice treated with SO-CD20-906_009 and a mAb. Graphs show the geometric mean and 95% confidence interval for each mouse. (B) The percent change in HIV copies between SO-CD20-906_009 and mAb versus SO-CD20-906_009 and isotype mAb was calculated, showing an 85.11% reduction in HIV copies at 24 hours in the mAb treatment group compared to the isotype mAb treatment group. At 72 hours after mAb treatment and at the end of the study, there was a 70.44% and 61.56% reduction in HIV copies for SO-CD20-906_009 and mAb compared to SO-CD20-906_009 and isotype mAb control. The graph shows the mean percent change in HIV copies and 95% confidence intervals. [Figure 46A]Figure 46A shows that SO-CD20-906_009 is reduced in the bone marrow, liver, lungs, and spleen of mAb-treated mice. At the end of the study (day 7 / 8 after mAb administration), bone marrow, liver, lungs, and spleen were harvested and HIV DNA copies were measured as a marker of the presence of SO-CD20-906_009 in mouse tissues. Significant reductions in HIV copies were observed in the SO-CD20-906_009 and mAb groups compared with the SO-CD20-906_009 and isotype mAb groups in bone marrow, liver, lungs, and spleen (p<0.0001 for all tissues). Significantly lower HIV copy numbers were also observed in the SO-CD20-906_009 and no mAb control group compared with the SO-CD20-906_009 and isotype mAb group. Graphs show the geometric mean and 95% confidence intervals for each mouse. Closed triangles indicate mice treated with SO-CD20-906_009 and no mAb control; closed squares indicate mice treated with SO-CD20-906_009 and isotype mAb control; and open squares indicate mice treated with SO-CD20-906_009 and mAb. (B) The percent change in HIV copies between SO-CD20-906_009 and mAb and SO-CD20-906_009 and isotype mAb control was calculated. HIV copy reductions of 95.75% in bone marrow, 88.05% in liver, 95.75% in lung, and 98.66% in spleen were observed. Graphs show the mean percent change in HIV copies and 95% confidence intervals. [Figure 46B] Figure 46B shows that SO-CD20-906_009 is reduced in the bone marrow, liver, lungs, and spleen of mAb-treated mice. At the end of the study (day 7 / 8 after mAb administration), bone marrow, liver, lungs, and spleen were harvested and HIV DNA copies were measured as a marker for the presence of SO-CD20-906_009 in mouse tissues. The percent change in HIV copies between SO-CD20-906_009 and mAb and SO-CD20-906_009 and isotype mAb control was calculated. HIV copy reductions of 95.75% in bone marrow, 88.05% in liver, 95.75% in lungs, and 98.66% in spleen were observed. The graph shows the mean percent change in HIV copies and 95% confidence intervals. [Figure 47] Figure 47 shows the expression of CLDN3 by a panel of NSCLC cell lines. (A) CLDN3 expression was measured by PCR and expressed as 2-ΔCT. (B) CLDN3 expression was measured by flow cytometry and the % CLDN3-positive population is shown. HT-29 and RKO KO were used as positive and negative controls, respectively. Cells were cultured for 6 weeks, and three separate experiments were performed. The data are shown as the mean + standard error. Orange cell lines were used for functional studies. [Figure 48] Figure 48 shows CLDN3 expression by a panel of NSCLC and CRC cell lines for use in functional assays. (A) Relative CLDN3 expression was measured by qPCR and expressed as 2-ΔCT. The data are mean + / - standard error (n=2 technical replicates). (B) hCLDN3 expression was measured by flow cytometry and expressed as MFI of hCLDN3 normalized to isotype control. Testing was performed on the day cells were seeded for functional testing. Data in both graphs are organized from lowest to highest relative CLDN3. [Figure 49] Figure 49 shows the quantification of activators from co-cultures of NSCLC cell lines with 906-009_LNGFR, CD19-LNGFR, and UT. Five CRC cell lines with different CLDN3 expression levels were used as controls. This represents activator levels after 24 hours of co-culture at a 1:1 CAR:target ratio. Data are organized by relative CLDN3 (2-ΔCT) expression on the day of target cell seeding and are presented as mean + / - standard error (n=3 donors). (A) IFNγ pg / mL. (B) Granzyme B pg / mL. [Figure 50] Figure 50 shows a modeled relationship between T cell activation and relative CLDN3 mRNA expression (quantified by dCT aka 2-ΔCT). (A) IFNγ. (B) Granzyme B. Points on the graph represent secretion of activating factors in co-cultures of cell lines (varying in CLDN3 expression) with 906-009_LNGFR (3 donors): 906-009_LNGFR (black), CD19_LNGFR (dark gray), UT (light gray). [Figure 51]Figure 51 shows images of target cell killing of colon cancer cell lines. Images of co-cultures of colon cancer cell lines with 906-009_LNGFR or CD19_LNGFR CAR-T cells. Images are shown for three donors. Images show Annexin V staining in blue, with purple outlines indicating masks. Images from the assay endpoint are shown to demonstrate target cell killing of HT-29 and DLD1 cell lines in co-culture with 906-009_LNGFR. RKO-KO did not show target cell killing with 906-009_LNGFR. [Figure 52] Figure 52 shows images of target cell killing in NSCLC cell lines. Images of co-cultures of 906-009_LNGFR CAR-T cells or CD19_LNGFR CAR-T cells with various NSCLC cell lines. Images are shown for three donors. Images from the assay endpoint are shown to demonstrate target cell killing. [Figure 53] Figure 53 shows images of co-cultures of 906-009_LNGFR or CD19_LNGFR CAR-T cells with low CLDN3 expression. Images are shown for three donors. Images from the assay endpoint are shown, demonstrating partial cytotoxicity at this time point. Colo320DM showed partial target cell killing only with donor PR19K133900 using 906-009_LNGFR, which was not seen with donors PR19C133904 and PR19W133916. NCI-H1650 showed partial killing with co-cultures of donors PR19K133900 and PR19C133904 with 906-009_LNGFR. [Fig. 54A-F] Figures 54A-F show cell lines: RKO KO (CLDN3 protein KO), NCI-H1650, and NCI-H2023, ranked by CLDN3 mRNA expression. (A, C, E) Example plots of isotype stained cell lines. (B, D, F) Example plots of CLDN3 antibody stained plots. Gates were set based on the isotype staining control. [Figure 54G-J]Figures 54A-F show cell lines: NCI-H1650 and NCI-H2023, ranked by CLDN3 mRNA expression. (G, I) Example plots of isotype-stained cell lines. (H, J) Example plots of CLDN3 antibody staining. Gates were set based on the isotype-stained control. [Figure 55A] Figure 55A shows the effect of CLDN3 mutations on 906-mAb binding. The gating strategy used to determine the GFP-FITC and 906-mAb-PE positive populations is shown. [Fig. 55B-C] Figures 55B-C show the effect of CLDN3 mutations on 906-mAb binding. Figure 55B shows an overlay of representative histograms of GFP expression in RKO KO target cells. Figure 55C is a graph showing the fold change in 906-mAb binding to mutant cell lines compared to WT, represented by median fluorescence intensity (MFI). (C n=2, 95% confidence intervals shown) [Figure 55D] Figure 55D shows the effect of CLDN3 mutations on 906-mAb binding. A graph showing the change in the % population of 906-mAb positive cells (n=2, 95% confidence intervals shown). [Figure 56] Figure 56 shows the effect of CLDN3 mutations on the activation of 906-009_LNGFR after co-culture with RKO KO target cells. IFNγ release is shown after 24 hours of co-culture of 906-009_LNGFR with RKO KO target cells at a 1:1 target:transduced CAR T ratio. Data are expressed as % change in IFNγ compared to WT and normalized to non-transduced T cells (n=3). Data are the average of three T cell donors (n=3). 95% confidence intervals are shown. DETAILED DESCRIPTION OF THE INVENTION

[0023] Detailed Description of the Invention As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "peptide chain" refers to one or more peptide chains and includes equivalents thereof known to those skilled in the art.

[0024] As used in this specification and claims, the term "comprising" includes "comprises" or "consisting of," e.g., a composition "comprising" X may consist exclusively of X or may include additional elements, e.g., X+Y.

[0025] The term "consisting essentially of" limits the scope of the feature to specific materials or steps and those that do not materially affect the basic characteristics of the claimed feature. The term "consisting of" excludes the presence of any additional components.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any compositions and methods similar or equivalent to those described herein can be used to practice or test the methods of the present disclosure, exemplary compositions and methods are described herein. Any aspect and embodiment of the disclosure described herein can be combined. For example, the subject matter of any dependent or independent claim disclosed herein can be combined in multiple ways (e.g., one or more statements from each dependent claim can be combined into a single claim based on the independent claim to which it depends).

[0027] The ranges set forth herein include all values ​​within the specified ranges described, as well as values ​​at the endpoints of the specified ranges. Additionally, the figures and tables of this disclosure set forth ranges and discrete values ​​that may comprise any element of the methods disclosed herein.

[0028] The concentrations described herein are determined at ambient temperature and pressure. This can be, for example, room temperature or the temperature and pressure within a particular portion of the process stream. Preferably, the concentrations are determined at standard conditions of 25°C and 1 bar pressure.

[0029] The term "about" means a value within two standard deviations of the mean for a particular measurement.

[0030] overview Recognition of neoepitopes or neoantigens (cancer-specific mutations or proteins expressed exclusively by cancer cells) has been crucial for the development of anticancer therapies. These neoepitopes distinguish cancer cells from healthy, non-cancerous cells, allowing anticancer drugs and the patient's own immune system to specifically target them while leaving healthy, non-cancerous cells unaffected. A similar but less specific approach involves targeting tumor-associated autoantigens (proteins or other cellular components that are upregulated or overexpressed in cancer cells compared to healthy, non-cancerous cells). However, drawbacks of these approaches include the fact that truly cancer-specific neoepitopes are rare and not easily predictable, and that targeting tumor-associated autoantigens can result in off-target effects due to their expression in healthy, non-cancerous cells.

[0031] To address these shortcomings, provided herein is a chimeric antigen receptor (CAR) that binds to at least one epitope of a cell-binding protein, where the cell-binding protein is located within a cell-cell junction, and the at least one epitope of the cell-binding protein is accessible for binding by the extracellular domain of the CAR only in cancer cells. Such one or more epitopes are present or expressed in both cancer cells and healthy, non-cancerous cells and cells in organized tissue, but off-target effects are reduced in cells in organized tissue due to the inaccessibility and / or unavailability of the one or more epitopes for binding.

[0032] Chimeric antigen receptor (CAR) In various embodiments, genetically engineered receptors are provided that redirect immune effector cells to cancer cells expressing the epitopes described herein. These genetically engineered receptors, referred to herein as chimeric antigen receptors (CARs), are molecules that combine antibody-based specificity for a desired antigen / epitope with a T cell receptor activation intracellular domain to generate chimeric proteins that exhibit specific cellular immune activity. The term "chimeric" refers to a molecule that is composed of different protein or DNA segments derived from different sources.

[0033] In certain embodiments, the following domains: a) an extracellular domain comprising a claudin-3 binding domain comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (CDRH1) sequence of SEQ ID NO: 1; the heavy chain complementarity determining region 2 (CDRH2) sequence of SEQ ID NO: 2; and the heavy chain complementarity determining region 3 (CDRH3) sequence of SEQ ID NO: 3; b) a transmembrane domain; and c) one or more intracellular signaling domains A chimeric antigen receptor (CAR) comprising:

[0034] When the antigen-binding domain of the CAR engages with the target antigen on the surface of the target cell, the CAR clusters and provides an activation stimulus to the CAR-bearing cell. A key feature of CARs is their ability to utilize the cell-specific targeting capabilities of monoclonal antibodies, soluble ligands, or cell-specific coreceptors to redirect the specificity of immune effector cells in a major histocompatibility (MHC)-independent manner, thereby inducing the production of molecules that can mediate proliferation, cytokine production, phagocytosis, or cell death of target antigen-expressing cells.

[0035] Extracellular domain In various embodiments, the CAR comprises an extracellular binding domain comprising an antigen binding domain (e.g., a claudin-3 specific binding domain); a transmembrane domain; one or more costimulatory signaling domains; and one or more intracellular signaling domains.

[0036] The term "chimeric antigen receptor" ("CAR"), as used herein, refers to an engineered receptor comprising an extracellular antigen-binding domain (usually derived from a monoclonal antibody or a fragment thereof, e.g., the VH domain in the form of a single-domain antibody (sdAb) or the VH and VL domains in the form of an scFv), an optional spacer region, a transmembrane region, and one or more intracellular effector domains. In certain embodiments, a CAR further comprises a hinge region between the antigen-binding domain and the intracellular signaling domain. A CAR may also comprise a hinge or spacer domain between any of the extracellular binding domain, the transmembrane domain, the costimulatory domain, and / or the intracellular signaling domain. CARs are also called chimeric T cell receptors or chimeric immune receptors (CIRs). CARs are genetically introduced into hematopoietic cells, such as T cells, to redirect T cell specificity to a desired cell surface antigen, resulting in CAR-T therapeutics. The term "spacer domain," as used herein, refers to an oligopeptide or polypeptide that serves to link the transmembrane domain and the extracellular antigen / target-binding domain. This region may also be referred to as the "hinge domain" or "stalk domain." The size of the spacer may vary depending on the location of the target epitope to ensure optimal function of CAR:target / antigen binding. In some cases, without being bound by theory, optimal function may be achieved by maintaining a set distance (e.g., 14 nm) for CAR:target / antigen binding.

[0037] In certain embodiments, a CAR comprises an extracellular binding domain comprising an antigen-binding protein that specifically binds to an epitope, where the epitope is present on multiple cells but is accessible and / or available for binding only on target cells, e.g., cancer cells. As used herein, the terms "binding domain," "antigen-binding domain," "extracellular domain," "extracellular binding domain," "antigen-specific binding domain," and "extracellular antigen-specific binding domain" are used interchangeably and provide a CAR with the ability to specifically bind to a target antigen / epitope of interest. The binding domain may be derived from natural, synthetic, semi-synthetic, or recombinant sources.

[0038] The term "antigen-binding protein" as used herein refers to proteins, antibodies, antibody fragments (e.g., Fab, scFv, etc.), and other antibody-derived protein constructs, such as antibody-derived protein constructs comprising domain antibodies (dAbs) and sdAbs, capable of binding to a target antigen. The terms "antigen-binding protein" and "epitope-binding protein" are used interchangeably herein. This does not include natural cognate ligands or receptors. In some embodiments, the antigen-binding protein can bind to claudin-3 (also known as RVP1, HRVP1, C7orf1, CPE-R2, CPETR2) and can be referred to as a "claudin-3-binding protein" or a "claudin-3-specific binding protein." A "claudin-3-binding protein" refers to proteins, antibodies, antibody fragments (e.g., Fab, scFv, etc.), and other antibody-derived protein constructs, such as antibody-derived protein constructs comprising domains (e.g., dAbs, sdAbs, etc.) capable of binding to claudin-3, preferably human claudin-3.

[0039] The term "antigen," as used herein, refers to a macromolecular structure selectively recognized by an antigen-binding protein. Antigens include, but are not limited to, proteins (with or without polysaccharides) or protein compositions comprising one or more T-cell epitopes.

[0040] The term "epitope," as used herein, refers to the portion of an antigen that contacts a specific binding domain of an antigen-binding protein, also known as a paratope. Epitopes can be linear or conformational / discontinuous. Conformational or discontinuous epitopes comprise amino acid residues that are separated by other sequences, i.e., that are not contiguous in the primary sequence of the antigen, but that can be assembled by the three-dimensional folding of the polypeptide chain. These residues may originate from different regions of the polypeptide chain, but are adjacent in the three-dimensional structure of the antigen. In the case of multimeric antigens, conformational or discontinuous epitopes may include residues from different peptide chains. The specific residues contained within an epitope can be determined by three-dimensional structures obtained by computer modeling programs or methods known in the art, such as X-ray crystallography. Epitope mapping can be performed using a variety of techniques known to those skilled in the art, including, but not limited to, those described in publications such as "Methods in Molecular Biology 'Epitope Mapping Protocols'" by Mike Schutkowski and Ulrich Reineke (volume 524, 2009) and Johan Rockberg and Johan Nilvebrant (volume 1785, 2018). Non-limiting examples of methods include peptide-based approaches, such as pep scans, which screen a series of overlapping peptides for binding using techniques such as ELISA, or in vitro display of large libraries of peptide or protein variants, e.g., by phage. Detailed epitope information can be determined by structural techniques, including, but not limited to, X-ray crystallography, liquid-phase nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM). Mutagenesis, such as alanine scanning, is another effective approach to epitope mapping that uses loss-of-binding analysis. Another method combines hydrogen / deuterium exchange (HDX) with proteolysis and liquid chromatography-mass spectrometry (LC-MS) to characterize discontinuous or conformational epitopes.

[0041] In certain embodiments, the extracellular binding domain of the CAR comprises an antibody or its antigen-binding domain.

[0042] The term "antibody" is used herein in the broadest sense to refer to molecules having immunoglobulin-like domains (e.g., IgG, IgM, IgA, IgD, or IgE) and includes monoclonal antibodies, recombinant antibodies, polyclonal antibodies, chimeric antibodies, human antibodies, humanized antibodies, multispecific antibodies (including bispecific antibodies), and heteroconjugate antibodies; dAbs, sdAbs, antigen-binding antibody fragments, Fab, F(ab'), Fv, disulfide-linked Fv, scFv, disulfide-linked scFv, diabodies, TANDABs, etc., as well as modified forms of any of the above (for a review of select "antibody" formats, see Holliger and Hudson 2005 Nature Biotechnology 23(9):1126-1136).

[0043] The terms "full antibody," "whole antibody," or "intact antibody," used interchangeably herein, refer to a heterotetrameric glycoprotein with an approximate molecular weight of 150,000 daltons. An intact antibody is composed of two identical heavy chains (HC) and two identical light chains (LC) linked by covalent disulfide bonds. This H2L2 structure folds to form three functional domains, including two antigen-binding fragments (known as "Fab" fragments) and an "Fc" crystallizable fragment. The Fab fragment consists of an amino-terminal variable domain (heavy chain variable (VH) or light chain variable (VL))) and a carboxyl-terminal constant domain (CH1 (heavy chain) and CL (light chain)). The Fc fragment consists of two domains formed by the dimerization of paired CH2 and CH3 regions, respectively. The Fc can elicit effector functions by binding to receptors on immune cells or by binding to C1q, the first component of the classical complement pathway. The five classes of antibodies, IgM, IgA, IgG, IgE, and IgD, are defined by different heavy chain amino acid sequences, called μ, α, γ, ε, and δ, respectively, and each heavy chain can pair with either a κ or λ light chain. The majority of antibodies in serum belong to the IgG class, and there are four isotypes of human IgG (IgG1, IgG2, IgG3, and IgG4), whose sequences differ primarily in their hinge regions.

[0044] Fully human antibodies can be obtained using a variety of methods, for example, using yeast-based libraries or transgenic animals (e.g., mice) capable of producing a repertoire of human antibodies. Yeast displaying human antibodies that bind to the antigen of interest on their surface can be selected using FACS (fluorescence-activated cell sorting)-based methods or by bead capture using labeled antigen. Transgenic animals modified to express human immunoglobulin genes can be immunized with the antigen of interest, and then B cell sorting techniques can be used to obtain isolated antigen-specific human antibodies. Human antibodies generated using these techniques can then be characterized for desirable properties, such as affinity, developability, and selectivity.

[0045] Alternative antibody formats also include other scaffolds in which one or more CDRs of an antigen binding protein are arranged on a suitable non-immunoglobulin protein scaffold or framework, such as affibodies, SpA scaffolds, LDL receptor class A domains, avimers (see, e.g., U.S. Patent Application Publication Nos. 2005 / 0053973, 2005 / 0089932, and 2005 / 0164301), or EGF domains.

[0046] Throughout this specification, amino acid residues in variable domain sequences and in variable domain regions within full-length antigen-binding sequences, e.g., in antibody heavy chain sequences or antibody light chain sequences, are numbered according to the Kabat numbering convention. Similarly, the terms "CDR," "CDRL1," "CDRL2," "CDRL3," "CDRH1," "CDRH2," and "CDRH3" used in the examples also follow the Kabat numbering convention. Further information can be found in Kabat et al., Sequences of Proteins of Immunological Interest, 4 th Ed., US Department of Health and Human Services, National Institutes of Health (1987).

[0047] Those skilled in the art will understand that there are other numbering rules for the amino acid residues of variable domain sequences and full-length antibody sequences.For example, there are other numbering rules for CDR sequences, such as the numbering rules shown in Chothia et al., 1989 Nature 342(6252):877-83.The structure and protein folding of antigen-binding proteins may mean that other residues are also considered as part of CDR sequences, and those skilled in the art will understand this.

[0048] Other numbering methods for CDR sequences available to those skilled in the art include the "AbM" method (University of Bath) and the "contact" method (University College London). The minimum overlapping region using at least two of the Kabat, Chothia, AbM, and contact methods can be determined to obtain a "minimum binding unit." The minimum binding unit can be a portion of a CDR.

[0049] Table 1 below provides one definition using each numbering convention for each CDR or binding unit. In Table 1 below, the Kabat numbering scheme is used to number the variable domain amino acid sequences. Note that the definition of a CDR may vary depending on the individual publication used.

[0050] [Table 1]

[0051] Exemplary claudin-3 binding proteins include any one or combination of the following CDRs: CDRH1 of SEQ ID NO:1; CDRH2 of SEQ ID NO:2; CDRH3 of SEQ ID NO:3; CDRL1 of SEQ ID NO:4; CDRL2 of SEQ ID NO: 5; and / or CDRL3 of SEQ ID NO: 6 or CDRH1, CDRH2, CDRH3 in SEQ ID NO: 7; and / or CDRL1, CDRL2, CDRL3 in SEQ ID NO: 8 or CDRH1, CDRH2, CDRH3 in SEQ ID NO: 7; and CDRL1, CDRL2, CDRL3 in SEQ ID NO: 8 The compound comprises:

[0052] The CDRs may be modified by at least one amino acid substitution, deletion or addition, wherein the variant antigen binding protein substantially retains the biological characteristics of the unmodified protein.

[0053] It is understood that each of CDR H1, H2, H3, L1, L2, L3 can be modified alone or in any order or combination with any other CDR.In one embodiment, CDR is modified by substitution, deletion or addition of up to 3 amino acids, for example, 1 or 2 amino acids, for example, 1 amino acid.Generally, modification is substitution, particularly conservative substitution, as shown in Table 2 below.

[0054] [Table 2]

[0055] For example, in a variant CDR, the flanking residues that comprise the CDR as part of an alternative definition, such as Kabat or Chothia, may be substituted with conservative amino acid residues.

[0056] Such antigen binding proteins comprising variant CDRs as described above may be referred to herein as "functional CDR variants."

[0057] In one embodiment, the claudin-3 binding protein comprises a heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (CDRH1) sequence of SEQ ID NO: 1; the heavy chain complementarity determining region 2 (CDRH2) sequence of SEQ ID NO: 2; and the heavy chain complementarity determining region 3 (CDRH3) sequence of SEQ ID NO: 3. In one embodiment, the claudin-3 binding protein further comprises a light chain variable region (VL) comprising the light chain complementarity determining region 1 (CDRL1) sequence of SEQ ID NO: 4; the light chain complementarity determining region 2 (CDRL2) sequence of SEQ ID NO: 5; and the light chain complementarity determining region 3 (CDRL3) sequence of SEQ ID NO: 6. In certain embodiments, the extracellular domain of a CAR provided herein comprises a claudin-3 binding protein disclosed herein.

[0058] In one embodiment, the claudin-3 binding proteins of the present disclosure exhibit cross-reactivity between human claudin-3 and claudin-3 from another species, such as mouse claudin-3 and / or cynomolgus monkey claudin-3. In one embodiment, the claudin-3 binding proteins described herein specifically bind to human, cynomolgus monkey, and mouse claudin-3. This is particularly useful because drug development typically requires testing lead drug candidates in mouse systems before testing the drug in humans. By providing a drug that can bind to both human and mouse species, results can be tested in these systems and data can be compared side-by-side using the same agent. This avoids the complexity of having to find a drug that acts on mouse claudin-3 and another that acts on human claudin-3, and also avoids the need to compare human and mouse results using non-identical drugs. Cross-reactivity is also anticipated between other species used in disease models, such as dogs or monkeys, e.g., cynomolgus monkeys.

[0059] An "antigen-binding domain" refers to a domain of an antigen-binding protein that can specifically bind to an antigen, and may be a single variable domain or a VH / VL domain pair such as found in standard antibodies. An sdAb or scFv domain may also provide an antigen-binding site. In one embodiment, the antigen-binding protein is a claudin-3 binding protein. In one embodiment, the claudin-3 binding protein is an sdAb and comprises a heavy chain variable region (VH). In one embodiment, the claudin-3 binding protein is an scFv and comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the VL is located at the N-terminus of the VH, or the VH is located at the N-terminus of the VL. In some embodiments, the VL and VH are directly fused to each other via a peptide bond or linked to each other via a peptide linker.

[0060] The sequences of framework regions of different light or heavy chains are relatively conserved within a species, such as humans. The framework region of an antibody, which is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space. The CDRs are primarily responsible for binding to an epitope of an antigen.

[0061] A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or by a cell transfected with the light and heavy chain genes of a single antibody. Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by creating hybrid antibody-forming cells from the fusion of myeloma cells and immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0062] A "chimeric antibody" is a type of engineered Ab that combines naturally occurring variable regions (light and heavy chains) from a donor Ab with light and heavy chain constant regions from an acceptor Ab. Thus, in certain embodiments, a CAR contemplated herein comprises an antigen-binding domain that is a chimeric antibody or antigen-binding domain thereof.

[0063] In some embodiments, the antibody is a human antibody (such as a human monoclonal antibody) or a fragment thereof that specifically binds to human claudin-3 protein.

[0064] In one embodiment, the CAR comprises a "humanized" antibody or antibody-binding fragment. A "humanized antibody" refers to a type of engineered antibody that has CDRs derived from a non-human donor immunoglobulin, with the remaining immunoglobulin-derived portions of the molecule derived from one or more human immunoglobulins. Additionally, framework support residues may be altered to preserve binding affinity (see, e.g., Queen, et al., Proc. Natl Acad Sci USA. 1989; 86(24): 10029-10032 and Hodgson, et al., Biotechnology, 1991; 9(5): 421-5). A suitable human acceptor antibody may be selected from conventional databases, such as the KABAT database, the Los Alamos database, and the Swiss Protein database, based on homology with the nucleotide and amino acid sequences of the donor antibody. Human antibodies characterized by homology (amino acid-based) with the framework regions of the donor antibody may be suitable for providing heavy chain constant and / or heavy chain variable framework regions for insertion of the donor CDRs. Suitable acceptor antibodies that can provide light chain constant or variable framework regions can be selected in a similar manner. Note that the heavy and light chains of the acceptor antibody do not have to originate from the same acceptor antibody. Non-limiting examples of methods for producing such humanized antibodies are detailed in EP-A-0239400 and EP-A-054951.

[0065] "Percent identity" between a query nucleic acid sequence and a subject nucleic acid sequence is the "identity" value, expressed as a percentage, calculated over the entire length of the query sequence after performing a pairwise global sequence alignment using a suitable algorithm or software, such as BLASTN, FASTA, DNASTAR Lasergene, GeneDoc, Bioedit, EMBOSS needle, or EMBOSS infoalign. Importantly, the query nucleic acid sequence may be described herein by a nucleic acid sequence identified in one or more claims.

[0066] The "percent identity" between a query amino acid sequence and a subject amino acid sequence is the "identity" value, expressed as a percentage, calculated over the entire length of the query sequence after performing a pairwise global sequence alignment using a suitable algorithm / software, such as BLASTP, FASTA, DNASTAR Lasergene, GeneDoc, Bioedit, EMBOSS needle, or EMBOSS infoalign. Importantly, the query amino acid sequence may be described by an amino acid sequence specified in one or more claims herein.

[0067] The query sequence may be 100% identical to the subject sequence, or may contain up to a certain integer number of amino acid or nucleotide changes relative to the subject sequence, resulting in a percent identity of less than 100%. For example, the query sequence may be at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the subject sequence. Such changes include at least one amino acid deletion, substitution (including conservative and non-conservative substitutions), or insertion, which may occur at the amino- or carboxy-terminal position of the query sequence, or anywhere between those terminal positions, interspersed individually among the amino acids or nucleotides in the query sequence, or anywhere interspersed in one or more contiguous groups within the query sequence.

[0068] The percent identity can be determined over the entire length of the query sequence, including the CDRs. Alternatively, the percent identity can exclude one or more or all of the CDRs, e.g., all CDRs are 100% identical to the subject sequence, with the percent identity varying in the remainder of the query sequence, e.g., the framework sequences, so that the CDR sequences are fixed and intact.

[0069] Those skilled in the art will recognize that the VH and / or VL domains disclosed herein can be incorporated into CAR-T therapeutics, for example, in the form of an sdAb or scFv.

[0070] Disorganization of tissue architecture is a hallmark of cancer, and as a result, disruption of cell-cell junctions can make epitopes that remain "hidden" in orderly or healthy tissues accessible / available in cancer tissues and cells [Corsini et al. (2018) Oncotarget]. Alterations in cell-cell contacts also lead to a loss of cell polarity and exposure of several extracellular signals, such as signals from growth factors. In the absence of apical-basal polarity, epithelial cells in the apical region, as well as those receiving growth signals, tend to proliferate by out-of-plane division, driven by misdirection of the division axis.

[0071] In some embodiments, the CAR comprises an extracellular domain comprising an antigen-binding protein that binds to at least one epitope of a cell junction protein (e.g., claudin-3) located within an intercellular junction. The at least one epitope of the cell junction protein (e.g., claudin-3) is accessible for binding by the extracellular domain of the CAR only when the cell junction protein is inappropriately localized outside the intercellular junction and thereby exposed on the cell surface. A cell junction protein inappropriately localized outside the intercellular junction refers to abnormal localization of the cell junction protein, such that the cell junction protein is not confined to the intercellular junction, e.g., tight junction. As an illustrative and non-limiting example, a cell junction protein (e.g., claudin-3) is inappropriately localized outside the intercellular junction in cancer and is exposed on the cell surface, where the CAR or antibody disclosed herein can recognize and bind. In contrast, in healthy or non-cancerous tissue, cell junction proteins located within tight junctions (e.g., claudin-3) are confined to the tight junctions, thereby preventing access by CARs or antibodies that target the cell junction proteins.

[0072] Thus, the epitopes targeted by the CARs described herein are found in cell-binding proteins expressed in both healthy (non-cancerous) cells and cancer cells. However, the epitopes of the cell-binding proteins bound by the antigen-binding proteins of the CAR extracellular domains are available and / or accessible for binding when the epitopes are inappropriately localized or displayed outside the cell-cell junctions, exposing the cell-binding proteins on the cell surface (e.g., cancer cells or cells in disordered tissues). Alternatively, the epitopes bound by the antigen-binding proteins of the CAR extracellular domains are available and / or accessible for binding in cancer cells when the cell-cell junctions are impaired (e.g., leaky) or destroyed. For example, in healthy non-cancerous cells or cells in disordered tissues, one or more of these epitopes are hidden because they are located within the cell-cell junctions, and therefore the CAR extracellular domain, antibody, or antigen-binding fragment is blocked from binding to the epitope. Thus, at healthy, non-cancerous cells or at cell-cell junctions between healthy, non-cancerous cells or at cell-cell junctions located between cells in an ordered tissue, these one or more epitopes are inaccessible / unavailable for binding of the CAR extracellular domain.

[0073] Thus, without wishing to be bound by any particular theory, it is hypothesized that the CAR extracellular domain described herein binds to one or more epitopes present in both healthy, non-cancerous cells and cancer cells, but that the epitopes are accessible and / or available for binding only in cancer cells or between cells in disordered tissue (see Figure 1). Such accessibility and availability for binding by the CAR extracellular binding domain may be due to a conformational change within the cell-binding protein that results in the formation or exposure of one or more epitopes to which the CAR extracellular domain binds, for example, by unfolding a loop buried within the cell-binding protein or by bringing amino acids that are not in close proximity in cancer cells that are not in close proximity in healthy, non-cancerous cells or in ordered tissue. Alternatively, the accessibility and / or accessibility may be due to the cell-binding protein not forming a complex with or associating with a binding partner in cancer cells compared to healthy, non-cancerous cells or in ordered tissue. It is recognized in this regard that cell junction proteins located within cell junctions may comprise particularly attractive epitopes for targeting by CARs, and that intact or intact (e.g., undisrupted) cell junctions in ordered tissues will prevent access of the CAR, rendering the epitope inaccessible and / or unavailable for binding.

[0074] Thus, in certain embodiments, one or more epitopes are present on healthy, non-cancerous cells and are inaccessible for binding by the CAR ectodomain, and / or one or more epitopes are located within an intercellular junction and are inaccessible for binding by the CAR ectodomain when the intercellular junction is present between healthy, non-cancerous cells. In further embodiments, the CAR ectodomain is sterically hindered from binding to one or more epitopes in healthy, non-cancerous cells and / or is sterically hindered from binding to one or more epitopes located within an intercellular junction between healthy, non-cancerous cells.

[0075] Thus, in one embodiment, cell-cell junctions are disrupted, e.g., disrupted between cells in disordered tissue or between cancer cells, compared to cell-cell junctions present between cells in ordered tissue, e.g., between healthy, non-cancerous cells. In a further embodiment, cell-cell junctions are impaired, e.g., impaired when cell-cell junctions exist between cells in disordered tissue, between cancer cells, or between cancer cells and healthy, non-cancerous cells. Thus, it will be appreciated that the terms "impaired" and "disrupted" can be used interchangeably herein and include cases where cell-cell junctions are physically disrupted, e.g., their structure is altered, and / or where cell-cell junctions are functionally impaired, e.g., leaky. In yet a further embodiment, cell-cell junctions are impaired and / or disrupted when proteins contained within the junctions are improperly localized. In another embodiment, proteins contained within the cell-cell junctions are improperly localized when the junctions are impaired and / or disrupted.

[0076] Without wishing to be bound by any particular theory, it is hypothesized that structurally disrupted cell-cell junctions can make (e.g., expose) epitopes that are normally "hidden" at non-disrupted cell-cell junctions accessible / available for binding (e.g., sterically accessible / available for binding), and / or functionally impaired (e.g., more "leaky") cell-cell junctions can increase lymphocyte infiltration / migration through cell-cell junctions, e.g., resulting in the accessibility / availability of certain epitopes for binding by antigen binding proteins (including antigen binding proteins incorporated into CARs).

[0077] In one embodiment, the one or more epitopes are inaccessible / unavailable for binding by the CAR extracellular domain when cell-cell junctions are not disrupted, e.g., when cell-cell junctions exist between healthy, non-cancerous cells or between cells in an ordered tissue (see Figure 1, left panel). In another embodiment, the one or more epitopes are accessible / available for binding by the CAR extracellular domain when cell-cell junctions are disrupted, e.g., when cell-cell junctions exist between cancer or tumor cells, or when cell-cell junctions exist between healthy, non-cancerous cells and cancer cells, or when cell-cell junctions exist between cells in a disordered tissue (see Figure 1, right panel). In a further embodiment, the one or more epitopes are accessible / available for binding by the CAR extracellular domain only when cell-cell junctions are disrupted, e.g., when cell-cell junctions exist between cancer or tumor cells, or when cell-cell junctions exist between healthy, non-cancerous cells and cancer or tumor cells, or when cell-cell junctions exist between cells in a disordered tissue.

[0078] In a further embodiment, one or more epitopes are inaccessible / unavailable for binding by the CAR extracellular domain when cell-cell junctions are intact, e.g., when cell-cell junctions exist between healthy, non-cancerous cells or between cells in an ordered tissue. In another embodiment, one or more epitopes are accessible / available for binding by the CAR extracellular domain when cell-cell junctions are impaired, e.g., when cell-cell junctions exist between cancer or tumor cells, when cell-cell junctions exist between healthy, non-cancerous cells and cancer cells, or when cell-cell junctions exist between cells in a disordered tissue. In a further embodiment, one or more epitopes are accessible / available for binding by the CAR extracellular domain only when cell-cell junctions are impaired, e.g., when cell-cell junctions exist between cancer or tumor cells, when cell-cell junctions exist between healthy, non-cancerous cells and cancer cells, or when cell-cell junctions exist between cells in a disordered tissue.

[0079] Further examples of epitopes that are accessible and / or available for binding only in cancer include those present in cellular components (e.g., cell-binding proteins) that are inappropriately localized in cancer cells compared to healthy, non-cancerous cells. Such inappropriate localization can be the result of overexpression or upregulation of the cellular component, mutation, changes in post-translational modification of proteins, changes in cell polarization, and / or tissue disorganization. Thus, in some embodiments, one or more epitopes are inaccessible / unavailable for binding by the CAR extracellular domain when the cell-cell junction comprises a cell-binding protein with a target epitope that is not inappropriately localized, for example, when the cell-cell junction exists between healthy, non-cancerous cells or between cells in an organized tissue. In further embodiments, the one or more epitopes are accessible / available for binding by the CAR extracellular domain when the cell-cell junction comprises a cell-binding protein with an inappropriately localized target epitope, e.g., when the cell-cell junction exists between cancer or tumor cells, when the cell-cell junction exists between healthy non-cancerous cells and cancer or tumor cells, or when the cell-cell junction exists between cells in disordered tissue. In other embodiments, the one or more epitopes are accessible / available for binding by the CAR extracellular domain only when the cell-cell junction comprises a cell-binding protein with an inappropriately localized target epitope, e.g., when the cell-cell junction exists between cancer or tumor cells, when the cell-cell junction exists between healthy non-cancerous cells and cancer cells, or when the cell-cell junction exists between cells in disordered tissue. In still further embodiments, the one or more epitopes are accessible / available for binding by the CAR extracellular domain only when the cell-cell junction comprises a cell-binding protein with the target epitope inappropriately localized outside the cell-cell junction, e.g., when the cell-cell junction exists between cancer cells, when the cell-cell junction exists between healthy and cancer cells, or when the cell-cell junction is otherwise impaired or disrupted (e.g., between cells in disorganized tissue).

[0080] Thus, as will be appreciated, the terms "accessible" and "available" are used interchangeably herein and can refer to the spatial and / or steric accessibility / availability of an epitope, or alternatively, the expression of an epitope in cancer cells relative to healthy, non-cancerous cells. Similarly, the terms "inaccessible" and "unavailable" are also used interchangeably herein.

[0081] In certain embodiments, the one or more epitopes are present in a cell junction protein located within a tight junction.

[0082] Tight junctions (also known as occluding junctions or zonula occludens) are multiprotein complexes between epithelial cells whose general function is to prevent the leakage of transported solutes and water across the epithelial barrier and seal the paracellular pathway. Tight junctions can also provide leaky pathways by forming selective channels for small molecules such as cations, anions, or water. Whether an epithelial barrier is classified as "tight" or "leaky" depends on the ability of the tight junctions between cells to prevent the movement of solutes and water. A non-limiting example of a "tight" epithelial barrier is the blood-brain barrier, and a non-limiting example of a "leaky" epithelial barrier is the kidney proximal tubule. Thus, tight junctions not only function to hold cells together to form an epithelial barrier, but also to prevent / control the passage of molecules and ions through the space between the membranes of adjacent cells and maintain cell polarity by preventing lateral diffusion of cell membrane components between the apical and basal / lateral surfaces.

[0083] Tight junctions consist of a branching network of sealing strands, each of which functions independently of the others. Each strand is formed from an array of transmembrane proteins embedded in the plasma membrane of epithelial cells, with their extracellular domains directly connected to each other. At least 40 different proteins are present in tight junctions, including both transmembrane and cytoplasmic proteins. The three major transmembrane proteins found in tight junctions are occludin, claudins, and junction adhesion molecule (JAM) proteins. These associate with different peripheral membrane proteins, such as ZO-1, located on the intracellular side of the plasma membrane, and anchor the strands to the actin component of the cytoskeleton. In this way, tight junctions connect the cytoskeletons of adjacent cells.

[0084] Occludin is an NADH oxidase that influences certain aspects of cellular metabolism, such as glucose uptake, ATP production, and gene expression. Occludin is also important for tight junction function and has been shown to interact with tight junction protein 1, tight junction protein 2, and YES1. While not required for tight junction assembly, it plays a role in maintaining barrier properties. Mutations or absence of occludin increase epithelial leakiness, and loss or aberrant expression of occludin has been shown to cause increased invasion, decreased adhesion, and significantly impaired tight junction function in breast cancer tissue.

[0085] Claudins are small (20–27 kDa) transmembrane proteins found in many organisms. They span the cell membrane four times (i.e., they have four transmembrane domains), and both their N- and C-termini are located in the cytoplasm. They have two highly conserved extracellular loops. The first extracellular loop (ECL1) is approximately 53 amino acids long, and the second extracellular loop (ECL2) is approximately 24 amino acids long. ECL1 controls paracellular ion selectivity, while ECL2 regulates homo- and heterodimerization with neighboring claudin proteins within tight junctions. The N-terminal end is usually short (e.g., 4–10 amino acids), while the C-terminal end is long (e.g., 21–63 amino acids), and is required for the localization of these proteins to tight junctions. Cysteines in individual or distinct claudins appear to form disulfide bonds. All human claudins (except claudin 12) contain a domain that allows them to bind to the PDZ domain of scaffolding proteins.

[0086] Junctional adhesion molecule (JAM) proteins are located within tight junctions between high endothelial cells and are not only involved in the formation of these junctions but also function as adhesive ligands for immune cells, and are also linked to endothelial cell polarity through their interactions with PAR-3 and JAM3.

[0087] In one embodiment, the cell junction protein is a member of the claudin family of proteins, e.g., selected from claudin-1, claudin-2, claudin-3, claudin-4, claudin-5, claudin-6, claudin-7, claudin-8, claudin-9, claudin-10a, claudin-10b, claudin-11, claudin-12, claudin-14, claudin-15, claudin-16, claudin-17, claudin-18, claudin-19, claudin-20, claudin-22, claudin-23, and claudin-25, or the related claudin domain containing 1, claudin domain containing 2, transmembrane protein 204, and peripheral myelin protein 22.

[0088] In still further embodiments, the cell-binding protein is claudin-3. In certain embodiments, the cell-binding protein is human claudin-3 (e.g., human claudin-3 set forth in SEQ ID NO: 13).

[0089] Claudin-3 (also known as CLDN3), encoded by the CLDN3 gene in humans, is not only an essential component of tight junctions but also a low-affinity receptor for Clostridium perfringens enterotoxin. Claudin-3 has been shown to interact with CLDN1 and CLDN5, and human claudin-3 has the following amino acid sequence:

[0090] [ka]

[0091] The underlined and bolded residues above represent N38 and E153. In further embodiments, the one or more epitopes are present in one or more extracellular loops of the cell-binding protein. In some embodiments, the extracellular loop has the following sequence: WSANTIIRDFYNPVVPEAQKREM (SEQ ID NO: 14) In some embodiments, the extracellular loop comprises extracellular loop 2 (ECL2) of human claudin-3, comprising the following sequence: RVSAFIGSNIITSQNIWEGLWMNCVVQSTGQMQCKVYDSLLALPQDLQAAR (SEQ ID NO: 26) The extracellular loop 1 (ECL1) of human claudin-3 comprises:

[0092] In yet further embodiments, one or more epitopes are uniquely present in claudin-3. The terms "unique" and "uniquely present" as used herein refer to the described characteristics not being found in other closely related proteins within the same protein family. For example, if one or more epitopes are uniquely present in claudin-3, the one or more epitopes are not found in another claudin family protein, such as the closely related proteins claudin-4, claudin-6, claudin-5, claudin-9, or claudin-17; in particular, the one or more epitopes are not found in claudin-4, claudin-6, claudin-5, or claudin-9, such as claudin-4, the closest known homolog of claudin-3. Such epitopes may be small or short, e.g., 4 to 10 amino acids in length, e.g., 4, 5, 6, 7, 8, 9, or 10 amino acids. Thus, in one embodiment, the one or more epitopes are 4 amino acids in length. An epitope unique to claudin-3 can be a linear epitope or a conformational epitope. A linear epitope is composed of consecutive residues in the primary protein sequence. For example, an epitope comprising the amino acid sequence PVVP (SEQ ID NO: 15) is present in claudin-3 but not in other closely related claudin family proteins, such as claudin-4, and is therefore a linear epitope that can be considered a unique epitope of claudin-3. A conformational epitope is composed of residues that are discontinuous in the primary protein sequence but come into close proximity to form an antigen surface in the three-dimensional structure of the protein / antigen. An epitope unique to claudin-3 can also be a conformational epitope.

[0093] In some embodiments, the one or more epitopes are conformational epitopes comprised of residues present in ECL1 and ECL2 of claudin-3. In some embodiments, the one or more epitopes are conformational epitopes comprising at least one residue present in ECL1 of claudin-3 and at least one residue present in ECL2 of claudin-3. In some embodiments, the one or more epitopes are conformational epitopes comprising at least N38 present in ECL1 of claudin-3 and at least E153 present in ECL2 of claudin-3, when numbered according to SEQ ID NO: 13. In some embodiments, the one or more epitopes comprise at least N38 and E153 of SEQ ID NO: 13. In some embodiments, the one or more epitopes consist essentially of N38 and E153 of SEQ ID NO: 13.

[0094] In one embodiment, an isolated claudin-3 binding protein is provided that binds to a discontinuous epitope of human claudin-3 comprising at least N38 and E153 of SEQ ID NO: 13. In one embodiment, an isolated claudin-3 binding protein is provided that binds to a discontinuous epitope of human claudin-3 consisting essentially of N38 and E153 of SEQ ID NO: 13. In one embodiment, the claudin-3 binding protein is chimeric or humanized; and / or the claudin-3 binding protein is selected from the group consisting of a monoclonal antibody, a human IgG1 isotype, camel Ig, Ig NAR, a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a F(ab)'3 fragment, an Fv, an scFv, a bis-scFv, an (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), and an sdAb. In yet another embodiment, a chimeric antigen receptor (CAR) is provided comprising a polypeptide comprising: a) an extracellular domain comprising an isolated claudin-3 binding protein that binds to a discontinuous epitope of human claudin-3 comprising at least N38 and E153 of SEQ ID NO: 13; b) a transmembrane domain; and c) one or more intracellular signaling domains. In another embodiment, the extracellular domain comprises an isolated claudin-3 binding protein that binds to a discontinuous epitope of human claudin-3 consisting essentially of N38 and E153 of SEQ ID NO: 13.

[0095] The terms "specific binding affinity," "specifically binds," "specifically bound," "specifically binding," or "specifically targets," as used herein, refer to the binding of an antigen / epitope binding domain (or a CAR comprising it) to an epitope that is accessible and / or available for binding only on cancer cells. In certain embodiments, the binding domain (or a CAR comprising the binding domain or a fusion protein containing the binding domain) binds to an epitope that is accessible and / or available for binding only on cancer cells. 6 M -1 , 10 7 M -1 , 108 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 or 10 12 M -1 A "high affinity" binding domain (or single-chain fusion protein thereof) binds to a target with a Ka of at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 or at least 10 12 M -1 A binding domain (or a CAR comprising a binding domain or a fusion protein containing a binding domain) may have a Ka of, for example, 10 3 M -1 ~10 10 M -1 A compound can be considered to "specifically bind" to claudin-3 if it binds or associates with claudin-3 with an affinity or Ka (i.e., the equilibrium association constant of a particular binding interaction in units of I / M).

[0096] Alternatively, affinity can be expressed as the equilibrium dissociation constant (Kd) of a particular binding interaction in units of M (e.g., 10 -5 M~10 -13M or lower). The affinity of a binding domain polypeptide and a CAR according to the present disclosure can be readily determined using conventional techniques, for example, by competitive ELISA (enzyme-linked immunosorbent assay), by binding association-dissociation assays using a labeled ligand, by binding association-dissociation assays using a surface plasmon resonance instrument such as a Biacore T 100 (available from Biacore, Inc., Piscataway, NJ), or by binding association-dissociation assays using optical biosensor technology such as the EPIC system or EnSpire (available from Corning and Perkin Elmer, respectively) (see also, e.g., Scatchard, Ann NY Acad Sci. 1949; 51(4): 660 and U.S. Pat. No. 5,283,173 or equivalents).

[0097] However, those skilled in the art will recognize that a CAR comprising an extracellular domain comprising an antigen / epitope binding protein disclosed herein may exhibit greater sensitivity and selectivity than an antibody comprising the same antigen / epitope binding domain. This means that even if binding cannot be detected directly (for example, by visualizing binding using a labeled antibody or CAR), binding can be determined indirectly (for example, by cell function assays and measurements). For example, binding can be detected directly by visualizing binding using a labeled antibody or CAR, for example, a fluorescently labeled soluble antibody, or indirectly through the function (such as activation) of cells expressing the antibody or CAR, such as by cytokine release assays (for example, measuring IFNγ release), measuring cell killing, or other functional measurements / techniques as described in detail in the Examples section below. Thus, in certain embodiments, binding is detected, e.g., by activation of the CAR-expressing cell, when the antibody or antigen / epitope-binding fragment is contained in the CAR and thus expressed in a cell in a non-soluble cellular format, but is not detected when the antibody or antigen / epitope-binding fragment is contained in a soluble, non-cellular format (e.g., a soluble antibody or antigen-binding fragment thereof).Furthermore, one skilled in the art will recognize that CARs as described herein may have higher selectivity for target antigens / epitopes, such as claudin-3, over other related proteins, e.g., other claudin family member proteins, including claudin-4, claudin-5, claudin-6, and / or claudin-9.

[0098] In certain embodiments, the extracellular binding domain of the CAR comprises an antigen binding protein, such as an anti-claudin-3 binding protein, wherein the antigen binding protein is selected from an antibody or an antigen-binding fragment thereof.

[0099] In certain embodiments, antigen-binding proteins, e.g., anti-claudin-3 antibodies or antigen-binding fragments thereof, include, but are not limited to, Camel Ig (Camelidae antibody (VHH)), Ig NAR, Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fv, scFv, bis-scFv, (scFv)2, minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins ("dsFv"), and sdAbs (also known as nanobodies).

[0100] In one embodiment, the antigen-binding protein, e.g., an anti-claudin-3 antibody or antigen-binding fragment thereof, is an scFv.

[0101] In some embodiments, the CAR extracellular domain comprises a claudin-3 binding protein. An exemplary claudin-3 binding protein is an immunoglobulin variable region specific for claudin-3, comprising at least one human framework region. A "human framework region" refers to a wild-type (i.e., naturally occurring) framework region of a human immunoglobulin variable region, an altered framework region of a human immunoglobulin variable region in which less than about 50% (e.g., preferably less than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%) of the amino acids in the region have been deleted or substituted (e.g., substituted with one or more amino acid residues from a non-human immunoglobulin framework region at corresponding positions), or an altered framework region of a non-human immunoglobulin variable region in which less than about 50% (e.g., preferably less than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%) of the amino acids in the region have been deleted or substituted (e.g., substituted with one or more amino acid residues from a human immunoglobulin framework region at exposed residue positions and / or at corresponding positions), resulting in, in one embodiment, reduced immunogenicity.

[0102] In certain embodiments, the human framework regions are wild-type framework regions of a human immunoglobulin variable region. In certain other embodiments, the human framework regions are altered framework regions of a human immunoglobulin variable region having amino acid deletions or substitutions at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more positions. In other embodiments, the human framework regions are altered framework regions of a non-human immunoglobulin variable region having amino acid deletions or substitutions at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more positions.

[0103] In certain embodiments, the claudin-3 binding protein comprises at least one, two, three, four, five, six, seven, or eight human framework regions (FRs) selected from human light chain framework region (FR) 1, human heavy chain FR1, human light chain FR2, human heavy chain FR2, human light chain FR3, human heavy chain FR3, human light chain FR4, and human heavy chain FR4.

[0104] Human FRs that may be present in the claudin-3 specific binding domain also include variants of the exemplary FRs provided herein in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids of the exemplary FRs have been substituted or deleted.

[0105] In certain embodiments, the claudin-3 binding protein comprises (a) a humanized light chain variable region comprising human light chain FR1, human light chain FR2, human light chain FR3, and human light chain FR4; and (b) a humanized heavy chain variable region comprising human heavy chain FR1, human heavy chain FR2, human heavy chain FR3, and human heavy chain FR4.

[0106] The claudin-3 binding proteins provided herein also comprise one, two, three, four, five, or six CDRs. Such CDRs may be non-human or altered non-human CDRs selected from CDRH1, CDRH2, and CDRH3 of the heavy chain variable region and CDRL1, CDRL2, and CDRL3 of the light chain variable region. In certain embodiments, the claudin-3 binding protein comprises a heavy chain variable region comprising a heavy chain CDRH1, a heavy chain CDRH1, and a heavy chain CDRH3. In certain embodiments, the claudin-3 binding protein comprises a light chain variable region comprising a light chain variable region comprising a light chain CDRL1, a light chain CDRL2, and a light chain CDRL3. In certain embodiments, the claudin-3 binding protein comprises (a) a heavy chain variable region comprising a heavy chain CDRH1, a heavy chain CDRH1, and a heavy chain CDRH3; and (b) a light chain variable region comprising a light chain CDRL1, a light chain CDRL2, and a light chain CDRL3.

[0107] Thus, in one embodiment, the claudin-3 binding protein comprises any one or combination of CDRs selected from CDRH1, CDRH2, and CDRH3 in SEQ ID NO: 7; and / or CDRL1, CDRL2, and CDRL3 in SEQ ID NO: 8. In a further embodiment, the claudin-3 binding protein comprises all six CDRs in SEQ ID NOs: 7 and 8.

[0108] In one embodiment, the claudin-3 binding protein comprises at least one heavy chain CDR sequence set forth in SEQ ID NOs: 1-3. In certain embodiments, the claudin-3 binding protein comprises at least one heavy chain CDR sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to a heavy chain CDR sequence set forth in SEQ ID NOs: 1-3. In further embodiments, the claudin-3 binding protein comprises at least one heavy chain CDR sequence at least 90% identical to a heavy chain CDR sequence set forth in SEQ ID NOs: 1-3. In certain embodiments, the claudin-3 binding protein comprises a CDRH1 at least 90% identical to SEQ ID NO: 1, a CDRH2 at least 90% identical to SEQ ID NO: 2, and / or a CDRH3 at least 90% identical to SEQ ID NO: 3. In other embodiments, the claudin-3 binding protein comprises a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, and / or a CDRH3 of SEQ ID NO: 3.

[0109] In one embodiment, the claudin-3 binding protein comprises at least one light chain CDR sequence set forth in SEQ ID NOs: 4-6. In certain embodiments, the claudin-3 binding protein comprises at least one light chain CDR sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the light chain CDR sequence set forth in SEQ ID NOs: 4-6. In further embodiments, the claudin-3 binding protein comprises at least one light chain CDR sequence at least 90% identical to the light chain CDR sequence set forth in SEQ ID NOs: 4-6. In certain embodiments, the claudin-3 binding protein comprises a CDRL1 at least 90% identical to SEQ ID NO: 4, a CDRL2 at least 90% identical to SEQ ID NO: 5, and / or a CDRL3 at least 90% identical to SEQ ID NO: 6. In other embodiments, the claudin-3 binding protein comprises a CDRL1 of SEQ ID NO:4, a CDRL2 of SEQ ID NO:5, and / or a CDRL3 of SEQ ID NO:6.

[0110] In some embodiments, the claudin-3 binding protein comprises a CDRH1 at least 90% identical to SEQ ID NO: 1, a CDRH2 at least 90% identical to SEQ ID NO: 2, a CDRH3 at least 90% identical to SEQ ID NO: 3, a CDRL1 at least 90% identical to SEQ ID NO: 4, a CDRL2 at least 90% identical to SEQ ID NO: 5, and a CDRL3 at least 90% identical to SEQ ID NO: 6.

[0111] In yet a further embodiment, the claudin-3 binding protein comprises a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, a CDRH3 of SEQ ID NO: 3, a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6.

[0112] "VH" refers to the variable region of an immunoglobulin heavy chain, including the variable region of an antibody, Fv, scFv, dsFv, Fab, sdAb, or other antibody fragment disclosed herein. Specific examples of heavy chain variable regions suitable for constructing the claudin-3 binding proteins contemplated herein include, but are not limited to, the heavy chain variable region sequence shown in SEQ ID NO:7.

[0113] "VL" refers to the variable region of an immunoglobulin light chain, including the variable region of an antibody, Fv, scFv, dsFv, Fab, or other antibody fragment disclosed herein. Specific examples of light chain variable regions suitable for constructing claudin-3 binding proteins contemplated herein include, but are not limited to, the light chain variable region sequence set forth in SEQ ID NO:8.

[0114] In one embodiment, the claudin-3 binding protein comprises a VH sequence at least 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 7. In another embodiment, the claudin-3 binding protein comprises a VH sequence at least 90% identical to the sequence of SEQ ID NO: 7. In an alternative embodiment, the claudin-3 binding protein comprises the VH sequence of SEQ ID NO: 7.

[0115] In further embodiments, the claudin-3 binding protein comprises a VL sequence at least 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 8. In another embodiment, the claudin-3 binding protein comprises a VL sequence at least 90% identical to the sequence of SEQ ID NO: 8. In an alternative embodiment, the claudin-3 binding protein comprises the VL sequence of SEQ ID NO: 8.

[0116] Thus, in one embodiment, the claudin-3 binding protein comprises a VH sequence at least 90% identical to the sequence of SEQ ID NO: 7 and a VL sequence at least 90% identical to the sequence of SEQ ID NO: 8. In a further embodiment, the claudin-3 binding domain comprises the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 8.

[0117] In certain embodiments, the claudin-3 binding protein is an sdAb comprising a VH sequence at least 90% identical to the sequence of SEQ ID NO: 7. In one embodiment, the claudin-3 binding protein is an sdAb comprising the VH sequence of SEQ ID NO: 7.

[0118] In certain embodiments, the claudin-3 binding protein is an scFv comprising a VH sequence at least 90% identical to the sequence of SEQ ID NO: 7 and a VL sequence at least 90% identical to the sequence of SEQ ID NO: 8, preferably comprising the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 8.

[0119] In some embodiments, the claudin-3 binding protein is an scFv comprising, from N-terminus to C-terminus, a VH sequence and a VL sequence, optionally separated by a linker sequence. In certain embodiments, the claudin-3 binding protein is an scFv comprising, from N-terminus to C-terminus, a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 8. In other embodiments, the claudin-3 binding protein is an scFv comprising, from N-terminus to C-terminus, a VL sequence and a VH sequence, optionally separated by a linker sequence. In certain embodiments, the claudin-3 binding protein is an scFv comprising, from N-terminus to C-terminus, a VL sequence of SEQ ID NO: 8 and a VH sequence of SEQ ID NO: 7.

[0120] In certain embodiments, the claudin-3 binding protein comprises a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 11. In further embodiments, the claudin-3 binding protein comprises a sequence at least 90% identical to SEQ ID NO: 11. In yet further embodiments, the claudin-3 binding protein comprises the sequence of SEQ ID NO: 11.

[0121] In certain embodiments, the claudin-3 binding protein comprises a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 18. In further embodiments, the claudin-3 binding protein comprises a sequence at least 90% identical to SEQ ID NO: 18. In yet further embodiments, the claudin-3 binding protein comprises the sequence of SEQ ID NO: 18.

[0122] Linker In certain embodiments, a CAR comprises linker residues added between various domains, for example, between the VH and VL domains, to ensure proper spacing and conformation of the molecule. In certain embodiments, the linker is a variable region linking sequence. A "variable region linking sequence" is an amino acid sequence that connects the VH and VL domains and provides a spacer function to accommodate the interaction of two sub-binding domains, such that the resulting polypeptide retains the same specific binding affinity for the same target molecule as an antibody comprising the same light and heavy chain variable regions. In certain embodiments, the linker separates one or more heavy or light chain variable domains, hinge domains, transmembrane domains, costimulatory domains, and / or intracellular signaling domains. A CAR may comprise one, two, three, four, five, or more linkers. In certain embodiments, the length of the linker is about 1 to about 25 amino acids, about 5 to about 20 amino acids, about 10 to about 20 amino acids, or any amino acid length therebetween. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids in length.

[0123] Specific examples of linkers include glycine polymers (G) n ; glycine-serine polymer (G 1-5 S 1-5 ) n wherein n is an integer of at least 1, 2, 3, 4, or 5; glycine-alanine polymers; alanine-serine polymers; and other flexible linkers known in the art. An exemplary linker is the glycine-serine polymer set forth in SEQ ID NO:9.

[0124] spacer domain In certain embodiments, the extracellular domain (i.e., the binding domain of the CAR) is followed by one or more "spacer domains," which refer to regions that distance the antigen-binding domain from the surface of the effector cell to allow proper cell-cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999; 6: 412-419). Spacer domains may be derived from natural, synthetic, semi-synthetic, or recombinant sources. In certain embodiments, the spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.

[0125] In one embodiment, the spacer domain comprises the CH2 and CH3 domains of IgG1, IgG4 or IgD.

[0126] Hinge domain Generally, the binding domain of a CAR is followed by one or more "hinge domains," which serve to distance the antigen-binding domain from the surface of the effector cell to allow proper cell-cell contact, antigen binding, and activation. CARs generally comprise one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be derived from natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.

[0127] Exemplary hinge domains suitable for use in the CARs described herein include hinge regions derived from the extracellular regions of type I membrane proteins, such as CD8α and CD4, which may be wild-type hinge regions derived from these molecules or may be altered. In certain embodiments, the hinge domain is derived from or comprises a CD8α hinge region.

[0128] Transmembrane domain In certain embodiments, the CAR further comprises a transmembrane domain. The "transmembrane domain" (TM) is the portion of the CAR that fuses the extracellular binding moiety with the costimulatory domain / intracellular signaling domain and anchors the CAR to the plasma membrane of the immune effector cell, e.g., by spanning the cell membrane. The TM domain may be derived from natural, synthetic, semi-synthetic, or recombinant sources. The TM domain may be derived from (e.g., comprise) at least the transmembrane region of the α or β chain of the T cell receptor, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137 (4-1BB), CD152, CD154, CD278 (ICOS), and PD1. In certain embodiments, the TM domain is synthetic and comprises primarily hydrophobic residues such as leucine and valine.

[0129] In one embodiment, the CAR comprises a TM domain derived from CD8α. In another embodiment, the CAR comprises a TM domain derived from CD8α and a short, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid long, oligopeptide or polypeptide linker that connects the TM domain and the costimulatory / intracellular signaling domain of the CAR. A glycine-serine based linker provides a particularly suitable linker. An exemplary TM domain derived from CD8α is set forth in SEQ ID NO: 19.

[0130] Intracellular signaling domains In certain embodiments, the CAR further comprises an intracellular signaling domain. The "intracellular signaling domain" (also referred to as "intracellular effector domain" or "signaling domain") refers to the portion of the CAR that is involved in transmitting the message of effective binding of the extracellular domain to a target antigen (e.g., claudin-3 protein) (e.g., anti-claudin-3 CAR binding) into immune effector cells to initiate effector cell function. The intracellular signaling domain is responsible for activating at least one of the normal effector functions of the CAR-expressing immune cells, such as activation, cytokine production, proliferation, and / or cytotoxic activity (including release of cytotoxic factors toward CAR-bound target cells), or other cellular responses initiated by antigen binding to the extracellular CAR domain.

[0131] The term "effector function" refers to a specialized function of an immune effector cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transduces an effector function signal and causes the cell to perform a specialized function. Typically, the entire intracellular signaling domain can be used, but in many cases, it is not necessary to use the entire domain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the entire domain, so long as it transduces an effector function signal.

[0132] The term intracellular signaling domain is meant to include a truncated portion of the intracellular signaling domain sufficient to transduce an effector function signal.

[0133] It is known that signals generated by the T cell receptor (TCR) alone are insufficient for the complete activation of T cells, and that secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two different classes of signaling domains: intracellular signaling domains (e.g., TCR / CD3 complexes), which initiate antigen-dependent primary activation by the TCR; and costimulatory signaling domains, which act to provide secondary or costimulatory signals in an antigen-independent manner. In some embodiments, a CAR comprises at least one "costimulatory signaling domain" and at least one "intracellular signaling domain."

[0134] The intracellular signaling domain controls the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Intracellular signaling domains that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs.

[0135] Specific examples of ITAM-containing intracellular signaling domains suitable for use in certain embodiments of the CARs described herein include those derived from FcRγ, FcRβ, CD3γ, CD3ε, CD3δ, CD3ζ, CD22, CD66d, CD79a, and CD79b. In one embodiment, the one or more intracellular signaling domains are CD3ζ. An exemplary CD3ζ intracellular signaling domain is set forth in SEQ ID NO: 21. In certain preferred embodiments, the CAR comprises a CD3ζ intracellular signaling domain and one or more costimulatory signaling domains. The intracellular signaling and costimulatory signaling domains can be tandemly linked to the carboxyl terminus of the transmembrane domain in any order.

[0136] Costimulatory domain In certain embodiments, the CAR further comprises one or more costimulatory signaling domains to enhance the efficacy and expansion of CAR-expressing T cells. As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or an Fc receptor that provides a secondary signal required for efficient activation and function of T lymphocytes upon antigen binding. Specific examples of such costimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TRIM, and ZAP70.

[0137] In one embodiment, the CAR comprises one or more costimulatory signaling domains selected from the group consisting of CD28, CD134 (OX40), and CD137 (4-1BB). In a further embodiment, the one or more costimulatory domains are CD137 (4-1BB). An exemplary CD137 (4-1BB) costimulatory domain is set forth in SEQ ID NO: 20.

[0138] In another embodiment, the CAR comprises a CD137 (4-1BB) costimulatory signaling domain and a CD3ζ intracellular signaling domain.

[0139] In certain embodiments, the CAR further comprises a leader sequence. In certain embodiments, the leader sequence is a CD8 leader sequence. An exemplary CD8 leader sequence is set forth in SEQ ID NO: 10.

[0140] Thus, in certain embodiments, a CAR contemplated herein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the sequence of SEQ ID NO: 12, 34, 35, 36, 37, 38, or 39. In further embodiments, the CAR comprises a sequence that is at least 90% identical to SEQ ID NO: 12, 34, 35, 36, 37, 38, or 39. In yet further embodiments, the CAR comprises the sequence of SEQ ID NO: 12, 34, 35, 36, 37, 38, or 39.

[0141] In certain embodiments, a CAR contemplated herein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the sequence of SEQ ID NO: 25. In further embodiments, the CAR comprises a sequence that is at least 90% identical to SEQ ID NO: 25. In yet further embodiments, the CAR comprises the sequence of SEQ ID NO: 25.

[0142] In certain embodiments, a CAR contemplated herein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the sequence of SEQ ID NO: 27. In further embodiments, the CAR comprises a sequence that is at least 90% identical to SEQ ID NO: 27. In yet further embodiments, the CAR comprises the sequence of SEQ ID NO: 27.

[0143] In certain embodiments, a CAR contemplated herein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the sequence of SEQ ID NO: 28. In further embodiments, the CAR comprises a sequence that is at least 90% identical to SEQ ID NO: 28. In yet further embodiments, the CAR comprises the sequence of SEQ ID NO: 28.

[0144] In certain embodiments, a CAR contemplated herein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the sequence of SEQ ID NO: 29. In further embodiments, the CAR comprises a sequence that is at least 90% identical to SEQ ID NO: 29. In yet further embodiments, the CAR comprises the sequence of SEQ ID NO: 29.

[0145] In certain embodiments, a CAR contemplated herein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the sequence of SEQ ID NO: 30. In further embodiments, the CAR comprises a sequence that is at least 90% identical to SEQ ID NO: 30. In yet further embodiments, the CAR comprises the sequence of SEQ ID NO: 30.

[0146] In certain embodiments, the following domains: a) an extracellular domain comprising a claudin-3 binding protein according to any one of the embodiments disclosed herein; b) a transmembrane domain derived from CD8α; and c) CD3ζ-derived intracellular signaling domain A chimeric antigen receptor (CAR) comprising:

[0147] In another particular embodiment, the following domains: a) an extracellular domain comprising a claudin-3 binding protein comprising a CDRH1 sequence of SEQ ID NO: 1; a CDRH2 sequence of SEQ ID NO: 2; a CDRH3 sequence of SEQ ID NO: 3; a CDRL1 sequence of SEQ ID NO: 4; a CDRL2 sequence of SEQ ID NO: 5; and a CDRL3 sequence of SEQ ID NO: 6; b) the transmembrane domain derived from CD8α; c) an intracellular signaling domain derived from CD3ζ; and d) Costimulatory domain derived from CD137 (4-1BB) A chimeric antigen receptor (CAR) comprising:

[0148] In some embodiments, the claudin-3 binding protein of the extracellular domain is an sdAb. In other embodiments, the claudin-3 binding protein of the extracellular domain is an sdAb comprising the CDRH1 sequence of SEQ ID NO: 1; the CDRH2 sequence of SEQ ID NO: 2; and the CDRH3 sequence of SEQ ID NO: 3. In some embodiments, the claudin-3 binding protein of the extracellular domain is an scFv. In other embodiments, the claudin-3 binding protein of the extracellular domain is an scFv comprising the CDRH1 sequence of SEQ ID NO: 1; the CDRH2 sequence of SEQ ID NO: 2; the CDRH3 sequence of SEQ ID NO: 3; the CDRL1 sequence of SEQ ID NO: 4; the CDRL2 sequence of SEQ ID NO: 5; and the CDRL3 sequence of SEQ ID NO: 6. In still other embodiments, the claudin-3 binding protein of the extracellular domain is an scFv comprising the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 8.

[0149] Also provided herein are claudin-3 binding proteins or chimeric antigen receptors (CARs) that compete for binding with a CAR comprising an extracellular domain comprising a claudin-3 binding protein comprising the CDRH1 sequence of SEQ ID NO: 1, the CDRH2 sequence of SEQ ID NO: 2, the CDRH3 sequence of SEQ ID NO: 3, the CDRL1 sequence of SEQ ID NO: 4, the CDRL2 sequence of SEQ ID NO: 5, and the CDRL3 sequence of SEQ ID NO: 6. In certain embodiments, a CAR contemplated herein competes for binding with a CAR comprising an extracellular domain comprising a claudin-3 binding protein comprising the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 8. Preferably, the CAR comprises a claudin-3 binding protein that is an scFv.

[0150] Polypeptides Contemplated herein are various polypeptides, including but not limited to CAR polypeptides and fragments thereof, cells and compositions comprising the same, antibodies and vectors expressing the polypeptides. In preferred embodiments, a polypeptide comprising one or more CARs is provided. In certain embodiments, the CAR is a claudin-3 binding CAR that comprises at least 90% identical amino acid sequence with SEQ ID NO: 11, preferably at least 90% identical sequence with SEQ ID NO: 12, 34, 35, 36, 37, 38 or 39.

[0151] The terms "polypeptide," "polypeptide fragment," "peptide," and "protein" are used interchangeably according to their conventional meaning, i.e., as sequences of amino acids, unless otherwise specified. Polypeptides can be synthetically or recombinantly produced. Polypeptides are not limited to a particular length and can comprise, for example, a full-length protein sequence or a fragment of a full-length protein, and can include post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and other naturally occurring and non-naturally occurring modifications known in the art. In various embodiments, a CAR polypeptide comprises a signal (or leader) sequence at the N-terminus of the protein that co-translationally or post-translationally directs translocation of the protein. Specific examples of suitable signal sequences useful for the CARs contemplated herein include, but are not limited to, an IgG1 heavy chain signal polypeptide, a CD8α signal polypeptide, or a human GM-CSF receptor α signal polypeptide. Polypeptides can be produced using any of a variety of well-known recombinant and / or synthetic techniques. Polypeptides contemplated herein specifically encompass the CARs of the present disclosure, or sequences of the CARs contemplated herein that have one or more amino acid deletions, additions, and / or substitutions.

[0152] "Isolated peptide" or "isolated polypeptide," etc., as used herein, refer to the in vitro isolation and / or purification of a peptide or polypeptide molecule from its cellular environment and from association with other components of a cell, i.e., it is not significantly associated with in vivo materials. Similarly, an "isolated cell" refers to a cell obtained from an in vivo tissue or organ and is substantially free of extracellular matrix.

[0153] Polypeptides include "polypeptide variants." Polypeptide variants can differ from naturally occurring polypeptides by one or more substitutions, deletions, additions, and / or insertions. Such variants can be naturally occurring or can be synthetically produced, for example, by modifying one or more of the above polypeptide sequences.

[0154] It is recognized that a polypeptide comprising a CAR, such as a CAR comprising the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12, 34, 35, 36, 37, 38, or 39 provided herein, can comprise further and / or additional polypeptide sequences or elements. Such additional elements include, but are not limited to, ablation elements or control elements that can be used to control expression of the polypeptide sequence within a cell or to target cells containing the polypeptide. Elements or polypeptide sequences that control expression can comprise an internal ribosome entry site (IRES), a translation initiation sequence, and / or a cleavage site that allows for separation of elements of the polypeptide sequence after translation.

[0155] Thus, in one embodiment, the polypeptides contemplated herein further comprise an ablation element. As used herein, "ablation element" refers to a polypeptide sequence and / or protein that is expressed on the surface of a cell and can be used to target or detect the cell (also known as an "elimination marker"). For example, an ablation element can be a polypeptide sequence of a cell surface protein comprising an extracellular epitope or binding region of an antibody or antigen-binding fragment thereof. Thus, in one embodiment, the ablation element is a cell surface protein that is targeted for antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) using an antibody or antigen-binding fragment thereof specific for the cell surface protein. It is therefore recognized that by utilizing such mechanisms, cells expressing the polypeptides contemplated herein can be specifically labeled / detected and, for example, specifically selected or isolated from a mixed population of transduced and non-transduced cells. Furthermore, cells expressing a polypeptide comprising an ablation element can be specifically and selectively eliminated, e.g., eliminated / removed, from the circulation of a treated subject.

[0156] Examples of suitable ablation elements include, but are not limited to, truncated human EGFR polypeptide (huEGFRt) and CD20, which can be recognized by cetuximab and rituximab, respectively (Wang et al., Blood, 2011; 118(5): 1255-1263, Paszkiewicz et al., J Clin Invest, 2016; 126(11):4262-4272, Vogler et al., Mol Ther J Am Soc Gene Ther, 2010; 18:1330-8, Griffioen et al., Haematologica, 2009; 94:1316-20 and Philip et al., Blood, 2014; 124:1277-87). Another example of a suitable ablation element is a short polypeptide epitope tag (so-called "E-tag") that is incorporated into the extracellular domain of a CAR, against which an anti-epitope tagged CAR can then be generated (Koristka et al., Cancer Immunol Immunother CII, 2019; 68:1401-15).

[0157] Thus, in one embodiment, the ablation element is selected from the group consisting of a truncated human EGFR polypeptide (huEGFRt) and CD20. In a specific embodiment, the ablation element is CD20.

[0158] In some embodiments, the ablation element is cleaved from the CAR polypeptide sequence. Thus, in one embodiment, a polypeptide contemplated herein comprises a cleavage site, such as a P2A cleavage site.

[0159] In certain embodiments, the polypeptide comprises the sequence set forth in SEQ ID NO:24.

[0160] Polynucleotides In another aspect, provided is a polynucleotide encoding one or more CARs as described herein.As used herein, the term "polynucleotide" or "nucleic acid" refers to messenger RNA (mRNA), RNA, genomic RNA (gRNA), positive-strand RNA (RNA(+)), negative-strand RNA (RNA(-)), genomic DNA (gDNA), complementary DNA (cDNA) or recombinant DNA.Polynucleotide includes single-stranded and double-stranded polynucleotides.

[0161] In various exemplary embodiments, the polynucleotides include expression vectors, viral vectors, and transfer plasmids, as well as compositions and cells comprising them. In various exemplary embodiments, the polynucleotides encode a CAR or polypeptide contemplated herein, including, but not limited to, a CAR having the sequence of SEQ ID NO: 12, 34, 35, 36, 37, 38, or 39, or a polynucleotide sequence encoding SEQ ID NO: 12, 34, 35, 36, 37, 38, or 39, or the polynucleotide sequence set forth in SEQ ID NOs: 16 and 17.

[0162] Thus, also provided are polynucleotides encoding the antigen binding proteins disclosed herein, including polynucleotides comprising a sequence at least 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 16 and / or 17. In one embodiment, the polynucleotide comprises the sequence of SEQ ID NO: 16 and / or 17.

[0163] As used herein, an "isolated polynucleotide" refers to a polynucleotide that has been purified from sequences that flank it in its naturally occurring state, e.g., a DNA fragment that has been removed from sequences that normally flank the fragment. "Isolated polynucleotide" also refers to complementary DNA (cDNA), recombinant DNA, or other polynucleotides produced by the hand of man that do not occur in nature.

[0164] Polynucleotides can be created, manipulated, and / or expressed using any of a variety of well-established techniques known and available in the art. To express a desired polypeptide, a nucleotide sequence encoding that polypeptide can be inserted into an appropriate vector.

[0165] vector In another aspect, the present invention provides a vector comprising a polynucleotide encoding one or more CARs and / or polypeptides described herein.

[0166] The term "vector" is used herein to refer to a nucleic acid molecule that can introduce or deliver another nucleic acid molecule. The nucleic acid to be introduced is generally linked, for example, inserted, into the nucleic acid molecule of the vector. The vector may contain a sequence that directs autonomous replication in cells, or may contain a sequence sufficient to allow integration into the DNA of a host cell. Useful vectors include, for example, plasmids (for example, DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication-defective retroviruses and lentiviruses.

[0167] In certain embodiments, the vector is an expression vector. Expression vectors can be used to produce the CARs and polypeptides contemplated herein. Furthermore, expression vectors can contain additional components that allow for the production of viral vectors, which vectors comprise the polynucleotides contemplated herein. Viral vectors can be used to deliver the polynucleotides contemplated herein to a subject or a subject's cells. Examples of expression vectors include, but are not limited to, plasmids, autonomously replicating sequences, and transposable elements. Further exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, transposons, artificial chromosomes (e.g., yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or PI-derived artificial chromosomes (PACs)), bacteriophages (e.g., λ phage or M13 phage), and animal viruses.

[0168] Further examples of expression vectors include the pClneo vector (Promega) for expression in mammalian cells; pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentiviral-mediated gene transfer and expression in mammalian cells. In certain embodiments, the coding sequences for the CARs and polypeptides disclosed herein can be ligated into such expression vectors for expression of the CARs and / or polypeptides in mammalian cells.

[0169] In certain embodiments, the expression vector provided herein is a BAC comprising the polynucleotide described herein. In certain embodiments, the BAC further comprises one or more polynucleotides encoding proteins necessary for enabling the production of a viral vector when expressed in a producer cell line or a packaging cell line. For example, PCT applications WO2017 / 089307 and WO2017 / 089308 describe expression vectors used to produce retroviral vectors, particularly lentiviral vectors. In certain embodiments, the expression vectors described in WO2017 / 089307 and WO2017 / 089308 are provided, comprising the polynucleotide described herein.

[0170] "Control elements" or "regulatory sequences" present in expression vectors are those untranslated regions of the vector (origins of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno or Kozak sequences), introns, polyadenylation sequences, 5' and 3' untranslated regions) that interact with host cell proteins to effect transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements may be used, including ubiquitous and inducible promoters.

[0171] Delivery Vector Also provided is the vector for delivering the polynucleotide described herein to subject and / or subject's cell.The examples of such vector include but are not limited to plasmid, autonomously replicating sequence, transposable element, phagemid, cosmid, artificial chromosome (for example, yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or PI-derived artificial chromosome (PAC)), bacteriophage (for example, λ phage or M13 phage) and virus vector.

[0172] Examples of categories of animal viruses useful as viral vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses (AAV), herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). These vectors are referred to herein as "viral vectors."

[0173] As those skilled in the art will recognize, the term "viral vector" is used broadly to refer to a nucleic acid molecule (e.g., a transfer plasmid) that generally contains viral-derived nucleic acid elements that facilitate the introduction or integration of a nucleic acid molecule into the genome of a cell, or a viral particle that mediates nucleic acid transfer.

[0174] Retroviruses are common tools for gene delivery (Miller, 2000, Nature. 357: 455-460). In certain embodiments, retroviruses are used to deliver a polynucleotide encoding a CAR as described herein to cells. As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and then covalently integrates its genomic DNA into the host genome. Once a virus is integrated into the host genome, it is called a "provirus." The provirus serves as a template for RNA polymerase II, directing the expression of RNA molecules that encode the structural proteins and enzymes required to produce new viral particles.

[0175] Exemplary retroviruses suitable for use in certain embodiments include, but are not limited to, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV), and lentiviruses.

[0176] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include, but are not limited to, HIV (including human immunodeficiency virus, HIV type 1, and HIV type 2); visna-maedi virus (VMV); caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In one embodiment, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is preferred.

[0177] Retroviral vectors, and in particular lentiviral vectors, may be used in the practice of certain embodiments. Thus, the terms "retrovirus" or "retroviral vector," as used herein, are meant to include "lentivirus" and "lentiviral vector," respectively.

[0178] A viral particle generally contains various viral components and, optionally, host cellular components and nucleic acids.

[0179] The term viral vector can refer to a virus or viral particle capable of introducing a nucleic acid into a cell, or the introduced nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements primarily derived from viruses. The term "retroviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, primarily derived from retroviruses. The term "lentiviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof (including LTRs), primarily derived from lentiviruses. The term "hybrid vector" refers to a vector, LTR, or other nucleic acid containing both retroviral, e.g., lentiviral, and non-lentiviral viral sequences. In one embodiment, a hybrid vector refers to a vector or transfer plasmid comprising retroviral, e.g., lentiviral, sequences for reverse transcription, replication, integration, and / or packaging.

[0180] In certain embodiments, the terms "lentiviral vector" and "lentiviral expression vector" can be used to refer to lentiviral transfer plasmids and / or infectious lentiviral particles. When elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc. are described herein, it should be understood that the sequences of these elements are present in RNA form in lentiviral particles and in DNA form in DNA plasmids.

[0181] At each end of the provirus, there is a structure called a "long terminal repeat" or "LTR." The term "long terminal repeat (LTR)" refers to a domain of base pairs located at the end of retroviral DNA. In their natural sequence relationship, they are directly repeated sequences and include the U3, R, and U5 regions. LTRs generally provide essential functions for retroviral gene expression (e.g., promotion, initiation, and polyadenylation of gene transcripts) and viral replication. LTRs contain numerous regulatory signals, including transcriptional control elements, polyadenylation signals, and sequences required for viral genome replication and integration. Viral LTRs are divided into three regions, designated U3, R, and U5. The U3 region contains enhancer and promoter elements. The U5 region is located between the primer binding site and the R region and contains the polyadenylation sequence. The R (repeat) region is flanked by the U3 and U5 regions. LTRs comprise the U3, R, and U5 regions and are found at both the 5' and 3' ends of the viral genome. The 5'LTR is flanked by sequences required for reverse transcription of the genome (tRNA primer binding site) and for efficient packaging of viral RNA into particles (Psi site).

[0182] As used herein, the term "packaging signal" or "packaging sequence" refers to a sequence located within the retroviral genome that is necessary for the insertion of viral RNA into the viral capsid or particle (see, e.g., Clever et al., J Virol. 1995; 69(4): 2101-9). Some retroviral vectors use a minimal packaging signal (also called a psi[W] sequence) required for encapsidation of the viral genome. Thus, as used herein, the terms "packaging sequence," "packaging signal," "psi," and the symbol "W" are used to refer to non-coding sequences required for encapsidation of retroviral RNA strands during the formation of viral particles.

[0183] In various embodiments, the vector comprises a modified 5'LTR and / or 3'LTR. One or both of the LTRs may comprise one or more modifications, including, but not limited to, one or more deletions, insertions, or substitutions. Modification of the 3'LTR is often performed to improve the safety of lentiviral or retroviral systems by rendering them replication-deficient. As used herein, the term "replication-deficient" refers to a virus that is unable to replicate completely and efficiently and does not produce infectious virions (e.g., produces replication-deficient lentiviral progeny). The term "replication-competent" refers to a wild-type or mutant virus that is capable of replication and can produce infectious virions (e.g., replication-competent lentiviral progeny) through viral replication.

[0184] A "self-inactivating" (SIN) vector refers to a replication-deficient vector, e.g., a retroviral or lentiviral vector in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription after the first round of viral replication. This is because the right (3') LTR U3 region is used as a template for the left (5') LTR U3 region during viral replication, and therefore viral transcripts cannot be made without the U3 enhancer-promoter. In a further embodiment, the 3' LTR has been modified so that the U5 region has been replaced with, for example, an ideal poly(A) sequence. Note that modifications to the LTRs are also included, such as modifications to the 3' LTR, the 5' LTR, or both the 3' and 5' LTRs.

[0185] Further safety improvements can be achieved by replacing the U3 region of the 5' LTR with a heterologous promoter to drive transcription of the viral genome during viral particle production. Examples of heterologous promoters that can be used include the simian virus 40 (SV40) promoter (e.g., early or late promoter), the cytomegalovirus (CMV) promoter (e.g., immediate-early promoter), the Moloney murine leukemia virus (MoMLV) promoter, the Rous sarcoma virus (RSV) promoter, and the herpes simplex virus (HSV) (thymidine kinase) promoter. Typical promoters are capable of driving high levels of transcription in a Tat-independent manner. Because the complete U3 sequence is absent in this viral production system, this substitution reduces the possibility of recombination to generate replicative virus. In certain embodiments, heterologous promoters offer the added advantage of controlling the manner in which the viral genome is transcribed. For example, heterologous promoters can be induced so that transcription of all or part of the viral genome occurs only in the presence of an inducer. Inducers include, but are not limited to, one or more chemicals or physiological conditions (e.g., the temperature or pH at which the host cells are cultured).

[0186] According to certain embodiments, most or all of the viral vector backbone sequences are derived from a lentivirus, such as HIV-1. However, it should be understood that many different sources of retroviral and / or lentiviral sequences can be used or combined, and that many substitutions and changes in a particular lentiviral sequence can be achieved without impairing the ability of the transfer vector to perform the functions described herein. Additionally, a variety of lentiviral vectors are known in the art (see Naldini et al., (Science. 1996; 272(5259): 263-7; Proc Natl Acad Sci USA. 1996; 93(21): 11382-8; Curr Opin Biotechnol. 1998; 9(5): 457-63); Zufferey et al., Nat Biotechnol. 1997; 15(9): 871-5; Dull et al., J Virol. 1998; 72(11): 8463-71; U.S. Patent Nos. 6,013,516; and 5,994,136), many of which can be adapted to produce viral vectors or transfer plasmids.

[0187] In various embodiments, the vector comprises a promoter operably linked to a polynucleotide encoding a CAR or a polypeptide as described herein.

[0188] In certain embodiments, the vector is a non-integrated vector, including, but not limited to, an episomal vector or a vector that is maintained extrachromosomally. As used herein, the term "episomal" refers to a vector that can replicate without being integrated into the host's chromosomal DNA and gradually lost from dividing host cells, which also means that the vector replicates extrachromosomally or episomally.

[0189] In some embodiments, the vectors described herein are viral vectors.

[0190] In some embodiments, the viral vectors described herein are retroviral vectors.

[0191] In some embodiments, the retroviral vectors described herein are lentiviral vectors.

[0192] In some embodiments, the retroviral vectors described herein are selected from the group consisting of human immunodeficiency virus I (HIV-I), human immunodeficiency virus 2 (HIV-2), visna-maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus.

[0193] In some embodiments, the vector comprises a nucleic acid comprising the sequence of SEQ ID NO: 17. In some embodiments, the vector is a viral vector comprising a nucleic acid sequence comprising the sequence of SEQ ID NO: 17. In some embodiments, the viral vector is a retroviral vector comprising a nucleic acid sequence comprising the sequence of SEQ ID NO: 17. In some embodiments, the retroviral vector is a lentiviral vector comprising a nucleic acid comprising the sequence of SEQ ID NO: 17. In some embodiments, the retroviral vector comprising a nucleic acid comprising the sequence of SEQ ID NO: 17 is a retroviral vector selected from the group consisting of human immunodeficiency virus I (HIV-I), human immunodeficiency virus 2 (HIV-2), visna-maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus.

[0194] Control Elements In certain embodiments, vectors, including but not limited to expression vectors and viral vectors, contain foreign, endogenous, or heterologous regulatory sequences, such as promoters and / or enhancers. An "endogenous" regulatory sequence is one that is naturally linked to a given gene in the genome. An "exogenous" regulatory sequence is one that has been placed in close proximity to that gene by genetic engineering (i.e., molecular biological techniques) such that transcription of the gene is directed by the linked enhancer / promoter. A "heterologous" regulatory sequence is an exogenous sequence that originates from a species different from the cell being genetically engineered.

[0195] The term "promoter," as used herein, refers to a recognition site in a polynucleotide (DNA or RNA) to which an RNA polymerase binds. The RNA polymerase initiates and transcribes a polynucleotide operably linked to the promoter. In certain embodiments, promoters that operate in mammalian cells comprise an AT-rich region located approximately 25-30 bases upstream from the site where transcription is initiated and / or another sequence, a CNCAAT region (where N can be any nucleotide), found 70-80 bases upstream from the transcription start site.

[0196] The term "enhancer" refers to a DNA segment containing sequences that can enhance transcription and can function in some cases independently of their orientation relative to another regulatory sequence. Enhancers can function cooperatively or additively with promoters and / or other enhancer elements. The term "promoter / enhancer" refers to a DNA segment containing sequences that can provide both promoter and enhancer functions.

[0197] The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. In one embodiment, the term refers to the functional linkage between a nucleic acid expression control sequence (such as a promoter and / or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide of interest, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0198] As used herein, the term "constitutive expression control sequence" refers to a promoter, enhancer, or promoter / enhancer that allows for continuous or persistent transcription of an operably linked sequence. A constitutive expression control sequence can be a "ubiquitous" promoter, enhancer, or promoter / enhancer that allows expression in a variety of cell and tissue types, or a "cell-specific," "cell type-specific," "cell line-specific," or "tissue-specific" promoter, enhancer, or promoter / enhancer that allows expression in limited types of cells and tissue types, respectively.

[0199] Exemplary ubiquitous expression control sequences suitable for use in certain embodiments include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, Simian virus 40 (SV40) (e.g., early or late), Moloney murine leukemia virus (MoN4LV) LTR promoter, Rous sarcoma virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, the H5, P7.5, and P11 promoters from vaccinia virus, the elongation factor 1-alpha (EF1a) promoter, the early growth response 1 (EGR1) promoter, the HIV-1 (HIV-2) (HIV-3) (HIV-4) (HIV-5) (HIV-6) (HIV-7) (HIV-8) (HIV-9) (HIV-10) (HIV-11) (HIV-12) (HIV-13) (HIV-14) (HIV-15) (HIV-16) (HIV-17) (HIV-18) (HIV-19) (HIV-20) (HIV-21) (HIV-22) (HIV-23) (HIV-24) (HIV-25) (HIV-25) (HIV-26) (HIV-27) (HIV-28) (HIV-29) (HIV-29) (HIV-21) (HIV-21) (HIV-22) 1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa β member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), β-kinesin (βKIN), and the human ROSA26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, β-actin promoter and myeloproliferative sarcoma virus enhancer, negative control region deletion, and d1587rev primer binding site substitution (MND) promoter (Challita et al., J Virol. 69(2)·748-55 (1995)).

[0200] In one embodiment, the vector comprises a PGK promoter.

[0201] In certain embodiments, it may be desirable to express the polynucleotide comprising the CAR from a T cell-specific promoter.

[0202] As used herein, "conditional expression" can refer to any type of conditional expression, including, but not limited to, inducible expression; repressible expression; expression in cells or tissues having a particular physiological state, biological state, or pathological condition, etc. This definition is not intended to exclude cell type-specific or tissue-specific expression. Certain embodiments provide conditional expression of a polynucleotide of interest, for example, expression is controlled by subjecting a cell, tissue, organism, etc. to a treatment or condition that causes expression of the polynucleotide or a treatment or condition that causes increased or decreased expression of a polynucleotide encoded by the polynucleotide of interest.

[0203] Examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters such as promoters of genes encoding glucocorticoid or estrogen receptors (induced by treatment with the corresponding hormones), the metallothionein promoter (induced by treatment with various heavy metals), the MX-I promoter (induced by interferon), the "GeneSwitch" mifepristone regulatory system (Sirin et al., 2003, Gene, 323:67), cumate-inducible gene switches (WO2002 / 088346), tetracycline-dependent regulatory systems, and the like.

[0204] In some embodiments, the polynucleotide or cells comprising the polynucleotide utilize a suicide gene, including an inducible suicide gene, to reduce the risk of direct toxicity and / or uncontrolled proliferation. In certain embodiments, the suicide gene is not immunogenic to a host comprising the polynucleotide or cells. Specific examples of suicide genes that can be used are caspase-9, caspase-8, or cytosine deaminase. Caspase-9 can be activated using certain protein dimerization-inducing compounds (CIDs).

[0205] In certain embodiments, the vector comprises a gene segment that renders immune effector cells, such as T cells, susceptible to negative selection in vivo. "Negative selection" means that the injected cells may be eliminated as a result of changes in the individual's in vivo conditions. A negatively selectable phenotype may be due to the insertion of a gene that confers sensitivity to an administered agent, e.g., a compound.

[0206] Negatively selectable genes are known in the art and include, among others: the herpes simplex virus type I thymidine kinase (HSV-I TK) gene, which confers ganciclovir sensitivity (Wigler et al., Cell I:223, 1977); the cellular hypoxanthine phosphoribosyltransferase (HPRT) gene; the cellular adenine phosphoribosyltransferase (APRT) gene; and bacterial cytosine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA, 1992; 89(33)).

[0207] In some embodiments, the genetically modified immune effector cells, such as T cells, comprise a polynucleotide that further comprises a positive marker that allows for the selection of cells with a phenotype amenable to negative selection in vitro. A positive selection marker can be a gene that, when introduced into a host cell, expresses a dominant phenotype that allows for the positive selection of cells carrying the gene. Genes of this type are known in the art and include, among others, the hygromycin-B phosphotransferase gene (hph), which confers resistance to hygromycin B, the aminoglycoside phosphotransferase gene (neo or aph) from Tn5, which encodes resistance to the antibiotic G418, the dihydrofolate reductase (DHFR) gene, the adenosine deaminase gene (ADA), and the multidrug resistance (MDR) gene.

[0208] Preferably, the positive and negative selectable markers are linked such that loss of the negative selectable element necessarily results in loss of the positive selectable marker. Even more preferably, the positive and negative selectable markers are fused such that loss of one forces loss of the other. An example of a fused polynucleotide whose expression product is a polypeptide that confers both the desired positive and negative selection characteristics is the hygromycin phosphotransferase thymidine kinase fusion gene (HyTK). Expression of this gene produces a polypeptide that confers hygromycin B resistance for in vitro positive selection and ganciclovir sensitivity for in vivo negative selection. See Lupton SD, et al., Mol. And Cell. Biology, 1991; 11:3374-3378. Furthermore, in preferred embodiments, the polynucleotide encoding the CAR is present within a retroviral vector that contains a fusion gene, particularly a fusion gene that confers hygromycin B resistance for in vitro positive selection and ganciclovir sensitivity for in vivo negative selection, such as the HyTK retroviral vector described in Lupton, SD et al. (1991), supra.

[0209] Vector production In certain embodiments, cells (e.g., immune effector cells) are transduced with a retroviral vector, such as a lentiviral vector, encoding a CAR. For example, immune effector cells are transduced with a vector encoding a CAR as described herein. These transduced cells can induce CAR-mediated cytotoxic responses.

[0210] "Host cells" include cells that have been electroporated, transfected, infected, or transduced with a vector or polynucleotide in vivo, ex vivo, or in vitro. Host cells may include packaging cells, producer cells, and viral vector-introduced cells. In certain embodiments, host cells into which a viral vector has been introduced are administered to a subject in need of treatment. In certain embodiments, the term "target cells" is used interchangeably with host cells and refers to transfected, infected, or transduced cells of a desired cell type. In a preferred embodiment, the target cells are T cells.

[0211] Large-scale production of viral vectors is often necessary to achieve adequate viral titers. Viral particles can be produced by transfecting transfer vectors into packaging cell lines comprising viral structural and / or accessory genes, such as the gag, POI, env, tat, rev, vif, vpr, vpu, vpx, or nef genes, or other retroviral genes.

[0212] As used herein, the term "packaging vector" refers to an expression or viral vector that lacks a packaging signal and comprises a polynucleotide encoding one, two, three, four, or more viral structural and / or accessory genes. Generally, packaging vectors are contained in packaging cells and introduced into cells via transfection, transduction, or infection. Methods for transfection, transduction, or infection are well known to those skilled in the art. In certain embodiments, a retroviral / lentiviral transfer vector is introduced into a packaging cell line via transfection, transduction, or infection to generate a producer cell or cell line. In certain embodiments, the packaging vector is introduced into human cells or cell lines by standard methods, including, for example, calcium phosphate transfection, lipofection, or electroporation. In some embodiments, the packaging vector is introduced into cells with a dominant selection marker, such as neomycin, hygromycin, puromycin, blasticidin, zeocin, thymidine kinase, DHFR, Gln synthetase, or ADA, followed by selection in the presence of the appropriate drug and isolation of clones. The selectable marker gene can be physically linked to the packaging vector, for example, a gene encoded by an IRES or a self-cleaving viral peptide.

[0213] As used herein, the term "packaging cell line" refers to a cell line that does not contain a packaging signal but stably or transiently expresses viral structural proteins and replication enzymes (e.g., gag, pol, and env) necessary for proper packaging of viral particles. Any suitable cell line can be used to generate packaging cells. Generally, these cells are mammalian cells. In certain embodiments, the cells used to generate packaging cell lines are human cells. Suitable cell lines that can be used include, for example, CHO cells, BHK cells, NOCK cells, C3H IOT1 / 2 cells, FLY cells, Psi2 cells, BOSC23 cells, PA317 cells, WEHI cells, COS cells, BSC1 cells, BSC40 cells, BMT10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, HEK293 cells, HEK293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 21 IA cells. In a preferred embodiment, the packaging cells are HEK293 cells or HEK293T cells. In another preferred embodiment, the cells are HEK293T cells.

[0214] As used herein, the term "producer cell line" refers to a cell line capable of producing recombinant retroviral particles comprising a packaging cell line and a vector construct comprising a packaging signal. Infectious viral particles and viral stocks can be produced using conventional techniques. Examples of producer cell lines include those described in WO2017 / 089307 and WO2017 / 089308, which contain all of the elements necessary for the production of a retroviral vector at a single location in the host cell genome.

[0215] Methods for preparing virus stock solutions are known in the art, and are described, for example, by Y. Soneoka et al., (1995) Nucl. Acids Res. 23:628-633 and NR Landau et al., (1992) J. Virol. 66:5110-5113. Infectious virus particles can be recovered from packaging cells using conventional techniques. For example, infectious particles can be recovered by cell lysis or by recovering the supernatant of cell culture, as known in the art. Optionally, the recovered virus particles can be purified as desired. Suitable purification techniques are well known to those skilled in the art.

[0216] Viral envelope proteins (env) determine the range of host cells that can ultimately be infected and transformed by recombinant retroviruses produced from these cell lines. In the case of lentiviruses such as HIV-1, HIV-2, SIV, FIV, and EIV, env proteins include gp41 and gp120.

[0217] The term "pseudotype" or "pseudotyping," as used herein, refers to a virus in which viral envelope proteins have been replaced with those of another virus having favorable characteristics. For example, HIV can be pseudotyped with the vesicular stomatitis virus G protein (VSV-G) envelope protein, such that the HIV envelope protein (encoded by the env gene) is normally encoded by the CD4 + Because the virus is targeted to presentation cells, HIV can infect a wider range of cells. In a preferred embodiment, lentivirus envelope protein is pseudotyped with VSV-G. In one embodiment, packaging cells produce recombinant retroviruses, such as lentiviruses, that are pseudotyped with VSV-G envelope glycoprotein.

[0218] In other embodiments, the viral vectors may be pseudotyped with an envelope protein from another retrovirus or an unrelated virus. Those skilled in the art will recognize that the viral vectors described herein can be pseudotyped with any suitable envelope protein.

[0219] Delivery of a gene or other polynucleotide sequence using a retroviral or lentiviral vector by viral infection rather than transfection is referred to as "transduction." In one embodiment, a retroviral vector transduces a cell by infection and proviral integration. In certain embodiments, a target cell, e.g., a T cell, is said to be "transduced" when it comprises a gene or other polynucleotide sequence delivered to the cell by infection with a viral or retroviral vector. In certain embodiments, a transduced cell comprises one or more genes or other polynucleotide sequences delivered by a retroviral or lentiviral vector within its cellular genome.

[0220] Immune effector cells In another aspect, immune effector cells are provided that comprise the CAR, polypeptide, polynucleotide and / or vector described herein. In various embodiments, genetically modified cells are provided that express the CAR contemplated herein for use in cancer treatment. As used herein, the term "genetically engineered" or "genetically modified" refers to the addition of foreign genetic material in the form of DNA or RNA to the total genetic material in a cell. The terms "genetically modified cells," "modified cells," and "redirected cells" are used interchangeably. As used herein, the term "gene therapy" refers to the introduction of foreign genetic material in the form of DNA or RNA into the total genetic material in a cell for the purpose of restoring, correcting, or modifying gene expression, or expressing a therapeutic polypeptide, such as a CAR.

[0221] In certain embodiments, a CAR contemplated herein is introduced into and expressed in an immune effector cell to redirect the specificity of the immune effector cell to a target antigen of interest located within a cell-cell junction, e.g., a cell junction protein, e.g., a member of the claudin family of proteins, particularly claudin-3.

[0222] An "immune effector cell" is any cell of the immune system that has one or more effector functions (e.g., cytotoxic killing activity, secretion of cytokines, induction of ADCC and / or CDC). Exemplary immune effector cells contemplated herein are T lymphocytes, particularly cytotoxic T cells (CTL; ​​CD8 + T cells), tumor-infiltrating lymphocytes (TILs) and helper T cells (HTLs; CD4 + In one embodiment, the immune effector cells include natural killer (NK) cells. In one embodiment, the immune effector cells include natural killer T cells. In another embodiment, the immune effector cells include macrophages. Immune effector cells can be autologous / autologous ("self") or "non-autologous" ("non-self"), e.g., allogeneic, syngeneic, or xenogeneic.

[0223] "Autologous," as used herein, refers to cells derived from the same subject.

[0224] "Allogeneic," as used herein, refers to cells of the same species that are genetically distinct from the cells to which they are being compared.

[0225] "Syngeneic," as used herein, refers to cells of a different subject that are genetically identical to the cells being compared.

[0226] "Xenogeneic," as used herein, refers to a cell of a different species than the cell to which it is being compared.

[0227] In preferred embodiments, the cells, eg, immune effector cells, are allogeneic.

[0228] Exemplary immune effector cells used in the CAR contemplated herein include T lymphocytes. The term "T cell" or "T lymphocyte" is art-recognized and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, such as T helper I (Th1) or T helper 2 (Th2) cells. T cells can be T helper cells (HT1; CD4 + T cells), cytotoxic T cells (CTL; ​​CD8 + T cells), CD4 + CD8 + T cells, CD4 - CD8 - They may be T cells or any other subset of T cells. Other exemplary populations of T cells suitable for use in certain embodiments include naive T cells and memory T cells.

[0229] In some embodiments, the immune effector cells are selected from the group consisting of T lymphocytes, natural killer T lymphocytes (NKT) cells, macrophages, and natural killer (NK) cells.

[0230] In one embodiment, the immune effector cells are cytotoxic T lymphocytes (CD8 + )

[0231] As will be understood by those skilled in the art, other cells can be used as immune effector cells in conjunction with CARs as described herein. In particular, immune effector cells also include NK cells, NKT cells, neutrophils, and macrophages. Immune effector cells also include precursors of effector cells, and such precursor cells can be induced to differentiate into immune effector cells in vivo or in vitro. Thus, in certain embodiments, immune effector cells include precursors of immune effector cells, such as hematopoietic stem cells (HSCs) contained in the CD34 population of cells derived from umbilical cord blood, bone marrow, or mobilized peripheral blood, which differentiate into mature immune effector cells when administered to a subject or can be induced to differentiate into mature immune effector cells in vitro.

[0232] As used herein, immune effector cells genetically engineered to contain, for example, a claudin-3-specific CAR may be referred to as "antigen-specific redirected immune effector cells" or "AG-specific redirected immune effector cells."

[0233] Methods for producing or generating immune effector cells that express a CAR described herein are provided in certain embodiments. In various embodiments, such methods comprise introducing a polynucleotide and / or vector as described herein into the immune effector cells. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from an individual so that the immune effector cells express one or more CARs contemplated herein. In certain embodiments, immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. Such cells can then be directly readministered to the individual. In further embodiments, immune effector cells are first activated and stimulated to proliferate in vitro before being genetically modified to express a CAR. In this regard, the immune effector cells can be cultured before and / or after being genetically modified (i.e., transduced or transfected to express a CAR contemplated herein). Thus, in certain embodiments, immune effector cells can be stimulated and induced to proliferate by contacting the cells with an antibody or antigen-binding fragment that binds CD3 and / or an antibody or antigen-binding fragment that binds CD28, thereby forming an immune effector cell population. In further embodiments, methods of generating immune effector cells contemplated herein comprise stimulating and inducing the immune effector cells to proliferate by contacting the cells with an antibody or antigen-binding fragment that binds CD3 and an antibody or antigen-binding fragment that binds CD28, thereby forming an immune effector cell population.

[0234] In certain embodiments, the immune effector cells are obtained from a subject prior to in vitro manipulation or genetic modification of the immune effector cells as described herein, hi certain embodiments, the CAR-modified immune effector cells comprise T cells.

[0235] In certain embodiments, peripheral blood mononuclear cells (PBMCs) can be directly genetically modified to express a CAR using the methods contemplated herein. In certain embodiments, following isolation of PBMCs, T lymphocytes are further isolated, and in certain embodiments, both cytotoxic and helper T lymphocytes can be sorted into subpopulations of naive, memory, and effector T cells before or after genetic modification and / or expansion.

[0236] Immune effector cells, such as T cells, can be genetically modified after isolation using known methods, or the immune effector cells can be activated and expanded in vitro (or differentiated, in the case of precursors) before being genetically modified. In certain embodiments, immune effector cells, such as T cells, are genetically modified with a CAR contemplated herein (e.g., transduced with a viral vector comprising a nucleic acid encoding the CAR) and then activated and expanded in vitro. In various embodiments, the T cells can be activated and expanded before or after genetic modification to express the CAR.

[0237] In certain embodiments, the population of engineered immune effector cells for the treatment of cancer comprises the CAR disclosed herein. For example, the population of engineered immune effector cells is prepared from peripheral blood mononuclear cells (PBMCs) obtained from a patient diagnosed with cancer (autologous donor). PBMCs are CD4 + , CD8 + , or CD4 + and CD8 + This results in the formation of a heterogeneous T lymphocyte population that may be

[0238] PBMCs may also contain other cytotoxic lymphocytes, such as NK cells or NKT cells. A vector carrying the coding sequence of the CAR described herein can be introduced into a population of human donor T cells, NK cells, or NKT cells. In certain embodiments, successfully transduced T cells carrying the expression vector are sorted using flow cytometry to isolate CD3-positive T cells, which are then further expanded to increase the number of these CAR protein-expressing T cells in addition to activating the cells using anti-CD3 antibody and / or anti-CD28 antibody and IL-2, or any other method known in the art, as described elsewhere herein.

[0239] Standard procedures are used for cryopreservation of CAR protein-expressing T cells for storage and / or preparation for use in human subjects.

[0240] In further embodiments, a mixture of, for example, one, two, three, four, five, or more different vectors can be used to genetically modify a population of donor immune effector cells, with each vector encoding a different chimeric antigen receptor protein as contemplated herein. The resulting modified immune effector cells form a mixed population of modified cells, with a proportion of the modified cells expressing two or more different CAR proteins.

[0241] T cell production method Methods for producing T cells for human therapy are known in the art. In preferred embodiments, T cells produced by the methods contemplated herein provide improved adoptive immunotherapy compositions. Without being bound by any particular theory, it is believed that T cell compositions produced by the methods of certain embodiments contemplated herein are endowed with superior properties, including increased survival, relatively differentiation-free expansion, in vivo persistence, and better anti-exhaustion properties. For example, T cells engineered to express anti-claudin-3 CARs exhibit lower binding kinetics to accessible claudin-3 and are less likely to become exhausted in the presence of accessible claudin-3 targets in vivo. The reduced propensity for exhaustion of anti-claudin-3 CAR-T cells preserves their effector function, reduces persistent expression of inhibitory receptors, and preserves the transcriptional state of functional effector or memory T cells. Exhaustion is an undesirable characteristic for CAR-T therapy because it prevents optimal control of infections and tumors.

[0242] In one embodiment, T cells are modified by transducing them with a viral vector comprising an anti-claudin-3 CAR contemplated herein. Anti-claudin-3 CAR-T cells exhibit low levels of basal CAR activation and interferon-gamma (IFNγ) secretion in the absence of antigen, a desirable characteristic of CAR-T therapy. The antigen-independent (basal) signaling tendency of CARs can exhibit self-aggregation, leading to antigen-independent CAR activation, which in turn can cause premature CAR exhaustion and reduced therapeutic efficacy (Ajina and Maher, 2018 and Long et al., 2015a). Basal activation of CAR-T cells can be determined by the level of activation marker CD69, exhaustion markers PD1 and TIM3, phosphorylation of the CD3ζ intracellular signaling domain, and the ability of CAR-T cells to secrete IFNγ in the absence of antigen. Humanized anti-claudin-3 CAR-T cells exhibited levels of IFNγ secretion, activation and exhaustion marker expression, and sustained CD3ζ signaling in vitro that were comparable to those of untransduced cells and lower than those of CAR-T cells transduced with a positive control CAR.

[0243] In one embodiment, T cells are engineered by transducing them with a viral vector comprising an anti-claudin-3 CAR as contemplated herein, which requires a higher target threshold to be activated, making it a "safer" CAR. It has been shown that high-affinity CARs can result in collateral targeting of healthy tissues, resulting in on- and off-target, extratumoral toxicity (Johnson et al., 2015; Park et al., 2017; Watanabe et al., 2018).

[0244] Pharmaceutical Composition The immune effector cells described herein can be formulated into pharmaceutical compositions for use in treating the human diseases described herein. In one embodiment, the pharmaceutical composition comprises the immune effector cells, optionally in combination with one or more pharmaceutically acceptable carriers and / or excipients.

[0245] Such compositions will comprise pharmaceutically acceptable carriers as required by accepted pharmaceutical practice, as is known (see, e.g., Remington's Pharmaceutical Sciences, 16 th edition (1980) Mack Publishing Co.).

[0246] The pharmaceutical composition may be administered by injection or continuous infusion (examples include, but are not limited to, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, intraocular, and intraportal). In one embodiment, the composition is suitable for intravenous administration.

[0247] The "therapeutically effective amount" of genetically modified therapeutic cells can vary according to factors such as the disease state, the age, sex, and weight of the individual, and the ability of the stem and progenitor cells to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or adverse effects of the virus or transduced therapeutic cells are outweighed by the therapeutically beneficial effects. The term "therapeutically effective amount" includes an amount effective to treat a subject (e.g., a patient). When a therapeutic amount is indicated, the exact amount of the composition to be administered can be determined by a physician, taking into account differences in the individual's age, weight, tumor size, infection or metastasis, and the extent of the patient's (subject's) disease state. Generally, pharmaceutical compositions comprising the T cells described herein are administered in an amount of 10 2 ~10 10 cells / kg body weight, preferably 10 5 ~10 6 It can be said that the composition can be administered at a dose of 10 cells / kg body weight (including all integer values ​​within these ranges). The number of cells will vary depending on the intended end use of the composition and the type of cells contained therein. For the uses provided herein, the cells will generally be in a volume of 1 liter or less, which may be 500 mL or less, or even 250 mL or 100 mL or less. Thus, the desired cell density is generally 10 6 More than 10 cells / mL, e.g. 6 , 10 7 , 108 or 10 9 More than cells / mL.

[0248] Cumulatively 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 or 10 12 Multiple infusions of a clinically relevant number of immune cells equivalent to or exceeding 10 cells can be allocated. In some embodiments, particularly since all infused cells are redirected to a specific target antigen, 10 6 / kilogram (10 per patient) 6 ~10 11 ) may be administered. In certain embodiments, less than 1 x 10 7 ~9×10 7 The CAR-expressing cell composition can be administered in doses of 100-200 mg / kg / day or 100-200 mg / kg / day. The CAR-expressing cell composition can be administered multiple times within these ranges. For example, the CAR-expressing cell composition can be administered every 7 days. Alternatively, the CAR-expressing cell composition can be administered as a single dose. The cells can be allogeneic, syngeneic, xenogeneic, or autologous to the patient receiving therapy. Optionally, treatment can also include administration of a mitogen (e.g., PHA), or lymphokines, cytokines, and / or chemokines (e.g., IFNγ, IL-2, IL-12, TNFα, IL-18, TNFβ, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) as described herein to enhance induction of an immune response.

[0249] In general, compositions comprising cells activated and expanded as described herein can be used in the treatment and prevention of diseases occurring in immunocompromised individuals. In particular, compositions comprising CAR-modified T cells as contemplated herein are used in the treatment of cancer. In certain embodiments, CAR-modified T cells can be administered alone or as a pharmaceutical composition in combination with a carrier, diluent, excipient, and / or other components such as IL-2 or other cytokines or cell populations. In certain embodiments, the pharmaceutical composition comprises a quantity of genetically modified T cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0250] Pharmaceutical compositions comprising a population of CAR-expressing immune effector cells, such as T cells, may comprise a buffer such as neutral buffered saline or phosphate buffered saline; a sugar such as glucose, mannose, sucrose, or dextran, mannitol; a protein; an amino acid such as a polypeptide or glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. In certain embodiments, the composition is preferably formulated for parenteral administration, for example, intravascular (intravenous or intraarterial), intraperitoneal, or intramuscular administration.

[0251] Liquid pharmaceutical compositions, whether in a solution, suspension, or other similar form, may contain one or more of the following: sterile diluents, such as water for injection, saline, preferably saline, Ringer's solution, isotonic sodium chloride, hydrogenated oils, such as synthetic mono- or diglycerides, polyethylene glycol, glycerin, propylene glycol, or other solvents that can function as solvents or suspending media; antibacterial agents, such as benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates, and agents for adjusting tonicity, such as sodium chloride or dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic. Pharmaceutical compositions for injection are preferably sterile.

[0252] In one embodiment, the T cell composition contemplated herein is formulated in a pharmaceutically acceptable cell culture medium. Such a composition is suitable for administration to human subjects. In certain embodiments, the pharmaceutically acceptable cell culture medium is a serum-free medium.

[0253] In another preferred embodiment, compositions comprising T cells contemplated herein are formulated in a solution comprising a cryopreservation medium. For example, a cryopreservation medium containing a cryopreservative can be used to maintain high cellular viability after thawing. Specific examples of cryopreservation media for use in certain compositions include, but are not limited to, CRYOSTOR CS10, CRYOSTOR CS5, and CRYOSTOR CS2.

[0254] In certain embodiments, the composition comprises an effective amount of CAR-expressing immune effector cells, alone or in combination with one or more therapeutic agents. Thus, the CAR-expressing immune effector cell composition can be administered alone or in combination with other known cancer treatments, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormonal therapy, or photodynamic therapy. The composition can also be administered in combination with antibiotics. Such therapeutic agents may be recognized in the art as standard treatments for certain conditions described herein, such as certain cancers. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory drugs, chemotherapeutic agents, radiotherapeutic agents, therapeutic antibodies, or other active and adjuvant agents.

[0255] In certain embodiments, the compositions comprising the CAR-expressing immune effector cells disclosed herein can be administered with any number of chemotherapeutic agents known in the art.

[0256] Various other therapeutic agents can be used together with the compositions described herein. In one embodiment, the composition comprising CAR-expressing immune effector cells is administered together with an anti-inflammatory agent. Anti-inflammatory agents or drugs are known in the art.

[0257] In certain embodiments, the compositions described herein are administered with cytokines. As used herein, "cytokine" refers to a general term that refers to a protein released by one cell population that acts as an intercellular mediator on another cell. Examples of such cytokines are known in the art.

[0258] In further embodiments, the compositions and CARs contemplated herein are administered together with other CARs or CAR-expressing cells and / or compositions. For example, an anti-claudin-3 CAR or composition can be administered together with an anti-cell surface-associated mucin 1 (MUC1) CAR or CAR-expressing cell composition. In one embodiment, the anti-MUC1 CAR targets aberrantly glycosylated MUC1 proteins ("AG-MUC1," e.g., TnMUC1, STnMUC1, etc.), such as those expressed by cancer cells. Alternatively, an anti-claudin-3 CAR or composition contemplated herein can be administered together with an anti-New York esophageal squamous cell carcinoma 1 (NY-ESO-1) T-cell receptor (TCR) or TCR-expressing cell composition.

[0259] The pharmaceutical composition may be included in a kit of parts containing CAR-expressing immune effector cells together with other agents, optionally and / or instructions for use. For convenience, the kit may comprise a predetermined amount of reagents together with instructions for use. The kit may also include a device used to administer the pharmaceutical composition.

[0260] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to any animal exhibiting symptoms of a disease, disorder, or condition treatable by the CARs, gene therapy vectors, cell-based therapeutics, and methods contemplated elsewhere herein. In a preferred embodiment, the subject includes any animal exhibiting symptoms of a cancer-related disease, disorder, or condition treatable by the CARs, gene therapy vectors, cell-based therapeutics, and methods contemplated elsewhere herein. Suitable subjects (e.g., patients) include laboratory animals (such as mice, rats, rabbits, or guinea pigs), farm animals, and domestic animals or pets (such as cats or dogs). Non-human primates, preferably human patients, are included. Typical subjects include human patients diagnosed with or at risk of having cancer that expresses an accessible claudin-3 protein. Thus, in one embodiment, the subject is a mammal, such as a primate, for example, a marmoset or monkey. In another embodiment, the subject is a human. In a further embodiment, the subject is a mouse.

[0261] As used herein, the term "patient" refers to a subject diagnosed with a particular disease, disorder, or condition treatable with the CARs, gene therapy vectors, cell-based therapeutics, and methods contemplated elsewhere herein.

[0262] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathology of a disease or condition, and may include showing a minimal reduction in one or more measurable markers of the disease or condition being treated. Treatment may optionally include regression of the disease or condition, or slowing of the progression of the disease or condition, e.g., slowing of tumor growth. "Treatment" does not necessarily indicate a complete eradication or cure of the disease or condition, or its associated symptoms.

[0263] As used herein, "prevention" and similar terms such as "prevented," "preventing," and the like refer to an approach to avoid, inhibit, or reduce the likelihood of the occurrence or recurrence of a disease or condition. The term also refers to delaying the occurrence or recurrence of a disease or condition, or delaying the occurrence or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar terms also include reducing the intensity, impact, symptoms, and / or burden of a disease or condition before the occurrence or recurrence of the disease or condition.

[0264] cancer As used herein, the terms "cancer," "neoplasm," and "tumor" are used interchangeably and, in the singular or plural, refer to cells that have undergone malignant transformation or cellular changes that result in abnormal or unregulated growth or excessive proliferation. Such changes or malignant transformation typically render such cells pathological to the host organism, and thus also include precancerous or precancerous cells that are pathological or have the potential to become pathological and require or benefit from intervention. Primary cancer cells (i.e., cells obtained from the vicinity of the site of malignant transformation) can be readily distinguished from noncancerous cells by well-established techniques such as histological examination. For example, such histological examination can distinguish cancerous cells from noncancerous cells by identifying disruption or damage to intercellular junctions, such as tight junctions.

[0265] Thus, in certain embodiments, the cancer cells comprise disrupted or damaged cell-cell junctions. In further embodiments, the cancer cells comprise disrupted or damaged tight junctions. In such embodiments, cell-cell junctions, e.g., cell junction proteins located within tight junctions, are improperly localized and become accessible and / or available for binding to the CAR and CAR-expressing cells described herein. In other embodiments, the cancer cells comprise improperly localized cell junction proteins, e.g., located at the cell-cell junctions, on their surface. Thus, in some embodiments, the cancer cells comprise improperly localized cell junction proteins exposed on their surface.

[0266] The definition of cancer cell as used herein includes not only primary cancer cell but also any cell derived from precancerous cell.This includes metastatic cancer cell, and in vitro culture and cell line derived from cancer cell.When referring to the type of cancer that usually manifests as solid tumor, "clinically detectable" tumor is based on tumor mass, for example, by means such as CAT scan, MR imaging, X-ray, ultrasound or palpation, and / or by the expression of one or more cancer-specific antigens in samples that can be obtained from patients.In other words, these terms herein include any stage of cell, neoplasm, cancer and tumor, including what clinicians call precancerous, tumor, localized growth and late metastatic growth.

[0267] The terms "treat," "treatment," and derivatives thereof, as used herein, refer to therapeutic therapy. With respect to a particular condition, treat or treatment means (1) ameliorating the condition or one or more of the biological manifestations of the condition; (2) inhibiting (a) one or more points in the biological cascade leading to or causing the condition, or (b) one or more of the biological manifestations of the condition; (3) alleviating one or more symptoms, effects, or side effects associated with the condition or one or more symptoms, effects, or side effects associated with the condition or its treatment; or (4) slowing the progression of the condition or one or more of the biological manifestations of the condition.

[0268] As used herein, "prevention" refers to the prophylactic administration of a drug, such as a pharmaceutical agent, to substantially reduce the likelihood or severity of a pathological condition or its biological manifestation, or to delay the onset of such a pathological condition or its biological manifestation.Those skilled in the art will recognize that "prevention" is not an absolute term.Preventive therapy is appropriate when a subject is considered to be at high risk of developing cancer, for example, when the subject has a strong family history of cancer, or when the subject has been exposed to a carcinogen.

[0269] As used herein, the term "malignant" refers to a cancer in which a group of tumor cells exhibit one or more of the following: uncontrolled growth (e.g., dividing beyond normal limits), invasion (e.g., invading and destroying adjacent tissue), and metastasis (e.g., spreading to other parts of the body via the lymphatics or blood).

[0270] "Cancer cell" refers to an individual cell of a cancerous growth or tissue. Cancer cells include both solid and liquid cancers. "Tumor" or "tumor cell" generally refers to a swelling or lesion formed by abnormal cell growth, which may be benign, pre-malignant, or malignant. Most cancers form tumors, but liquid cancers, such as leukemia, do not necessarily form tumors. In the case of cancers that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably. The amount of tumor in an individual is the "tumor burden," which can be measured as the number, volume, or weight of the tumor.

[0271] In one embodiment, the target cell (e.g., a cancer cell) expresses a cell-binding protein comprising an antigen or epitope that is also expressed on healthy, non-cancerous cells, sometimes at the same level as on healthy, non-cancerous cells. In a further embodiment, the antigen or epitope of the cell-binding protein is available and / or accessible only when expressed by the cancer cell. Thus, in certain embodiments, the antigen or epitope of the cell-binding protein is unavailable or inaccessible (e.g., hidden) when expressed by healthy, non-cancerous cells.

[0272] In certain embodiments, the cancer comprises or is characterized by inappropriate localization of claudin-3 outside of tight junctions and / or disruption of tight junctions, such that claudin-3 is accessible for binding by a CAR as described herein.

[0273] In one embodiment, the target cell is a bone cell, osteocyte, osteoblast, adipocyte, chondrocyte, chondroblast, muscle cell, skeletal muscle cell, myoblast, myocyte, smooth muscle cell, bladder cell, bone marrow cell, central nervous system (CNS) cell, peripheral nervous system (PNS) cell, glial cell, astrocyte, neuron, pigment cell, epithelial cell, skin cell, endothelial cell, vascular endothelial cell, breast cell, colon cell, esophageal cell, gastric cell, colon cell, head cell, cervical cell, gingival cell, tongue cell, kidney cell, liver cell, lung cell, nasopharyngeal cell, ovarian cell, follicular cell, cervical cell, vaginal cell, uterine cell, pancreatic cell, pancreatic parenchymal cell, pancreatic duct cell, pancreatic islet cell, prostate cell, penile cell, gonadal cell, testicular cell, hematopoietic cell, lymphoid cell, or bone marrow cell.

[0274] In one embodiment, the target cell expresses claudin-3 protein. In one embodiment, the target cell is a hematopoietic cell, an esophageal cell, a lung cell, an ovarian cell, a cervical cell, a pancreatic cell, a gallbladder or bile duct cell, a stomach cell, a colon cell, a breast cell, a goblet cell, an intestinal cell, a stem cell, an endothelial cell, an epithelial cell, or any cell that expresses claudin-3 protein. In a specific embodiment, the target cell is an endothelial cell or an epithelial cell.

[0275] Specific examples of cells that may be targeted by the compositions and methods contemplated in certain embodiments include, but are not limited to, the following solid cancers: adrenal carcinoma, adrenocortical carcinoma, anal carcinoma, appendix carcinoma, astrocytoma, atypical teratoma / rhabdoid tumor, basal cell carcinoma, bile duct carcinoma, bladder cancer, bone cancer, brain / CNS cancer, breast cancer, bronchial tumor, cardiac tumor, cervical cancer, bile duct carcinoma, chondrosarcoma, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma in situ (DCIS), endometrial carcinoma, ependymoma, esophageal carcinoma, olfactory neuroblastoma, Ewing's disease Sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, fibrous tissue sarcoma, fibrosarcoma, bladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, glioma, glioblastoma, head and neck cancer, hemangioblastoma, hepatocellular carcinoma, hypopharyngeal cancer, intraocular melanoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, lip cancer, liposarcoma, liver cancer, lung cancer, non-small cell lung cancer, pulmonary carcinoid tumor, malignant mesothelioma, medullary carcinoma, medulloblastoma, meningioma, melanoma, Merkel cell carcinoma, midline duct carcinoma, oral cancer cancer), myxosarcoma, myelodysplastic syndrome, myeloproliferative neoplasms, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, oligodendroglioma, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic islet cell tumor, papillary carcinoma, paraganglioma, parathyroid carcinoma, penile cancer, pharyngeal cancer, pheochromocytoma, pinealoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, prostate cancer, rectal cancer, retinoblastoma, renal cell carcinoma, renal pelvis and ureter cancer, rhabdomyosarcoma, salivary gland cancer, sebaceous gland cancer, skin cancer, soft tissue sarcoma, squamous cell carcinoma, small cell lung cancer, small intestine cancer, gastric cancer, sweat gland carcinoma, synovial tumor, testicular cancer, throat cancer, thymus cancer, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular cancer, vulvar cancer, and Wilms' tumor.

[0276] In another embodiment, the cell is a solid cancer cell that expresses an accessible claudin-3 protein. In yet a further embodiment, the cancer is a solid cancer. Examples of solid cancer cells that express an accessible claudin-3 protein that can be prevented, treated, or improved with the CAR, CAR-expressing cells, and compositions described herein include, but are not limited to, esophageal cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), ovarian cancer, cervical cancer, pancreatic cancer, bile duct cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, kidney cancer, and breast cancer (e.g., triple-negative breast cancer (TNBC)) cells.

[0277] In certain embodiments, the cancer cells are colon cancer, pancreatic cancer, breast cancer, ovarian cancer, or lung cancer. In some embodiments, the breast cancer is triple-negative breast cancer (TNBC). In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC). Thus, in further embodiments, the cancer is selected from colon cancer, pancreatic cancer, triple-negative breast cancer (TNBC), ovarian cancer, and non-small cell lung cancer (NSCLC).

[0278] In other embodiments, the cells are epithelial cells. In yet other embodiments, the cancer is an epithelial cancer. Examples of epithelial cancers include, but are not limited to, solid cancers such as those listed above.

[0279] Specific examples of liquid or blood cancers that can be prevented, treated, or ameliorated with the CARs, CAR-expressing cells, and compositions contemplated herein include, but are not limited to, leukemia, lymphoma, and multiple myeloma. Specific examples of cells that can be targeted by the CARs and compositions contemplated herein include, but are not limited to, the following leukemias: acute lymphocytic leukemia (ALL), T-cell acute lymphoblastic leukemia, acute myeloid leukemia (AML), myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, hairy cell leukemia (HCL), chronic lymphocytic leukemia (CLL), and cells of chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CNNL), and polycythemia vera.

[0280] Therapeutic and cytotoxicity enhancement methods The CAR molecules contemplated herein are intended for use in compositions, cells, and methods for treating the cancers described herein, thereby preventing, treating, or ameliorating at least one symptom associated with said cancer. In certain embodiments, the invention relates to improved cellular therapy of cancer using genetically modified immune effector cells that express an epitope accessible and / or available for CAR binding only in said cancer.

[0281] The improved adoptive cell therapy compositions and methods contemplated herein provide genetically engineered immune effectors that are readily expandable, exhibit long-term persistence in vivo, and exhibit antigen-dependent cytotoxicity against cancer cells expressing the epitopes described herein.

[0282] The terms "individual," "subject," and "patient" are used interchangeably herein. In one embodiment, the subject is an animal. In another embodiment, the subject is a mammal, such as a primate, for example, a marmoset or monkey. In another embodiment, the subject is a human.

[0283] Thus, in another aspect, methods for treating cancer using the CARs, polypeptides, vectors, immune effector cells, and compositions described herein are provided. The genetically modified immune effector cells contemplated herein provide improved methods of adoptive immunotherapy for use in the prevention, treatment, and amelioration of cancers that express accessible claudin-3 proteins, or for preventing, treating, or ameliorating at least one symptom associated with accessible claudin-3 protein-expressing cancers.

[0284] In various embodiments, the genetically modified immune effector cells contemplated herein provide improved methods of adoptive immunotherapy for use in enhancing cytotoxicity against cancer cells or reducing the number of cancer cells in a subject with cancer. In some embodiments, the cancer or cancer cells express a claudin-3 protein that is accessible and / or available for binding by the CARs and CAR-expressing cells described herein.

[0285] In certain embodiments, the specificity of primary immune effector cells is redirected to cells expressing an accessible claudin-3 protein, e.g., cancer cells, by genetically modifying the primary immune effector cells with a CAR contemplated herein. In various embodiments, a viral vector is used to genetically modify the immune effector cells with a specific polynucleotide encoding a CAR comprising a claudin-3 binding protein, e.g., an anti-claudin-3 antibody or antigen-binding domain; a hinge domain; a transmembrane (TM) domain; one or more costimulatory domains; and one or more intracellular signaling domains.

[0286] In one embodiment, a type of cell therapy is provided in which T cells are genetically modified to express a CAR as described herein to provide CAR-T cells, which are then infused into a recipient or subject in need thereof. The infused cells can kill disease-causing cells in the recipient. Unlike antibody therapy, CAR-T cells can replicate in vivo and produce long-term persistence that can lead to sustained cancer therapy. Furthermore, the CAR-T cell therapy contemplated herein can exhibit greater sensitivity and selectivity than antibody therapy, as described herein above. For example, a CAR comprising an extracellular domain comprising an antigen / epitope-binding domain as described herein can exhibit greater sensitivity and selectivity than an antibody comprising the same antigen / epitope-binding domain; therefore, activation of CAR-expressing cells can be detected (i.e., when the antibody or antigen / epitope-binding domain is included in the CAR and is therefore expressed by cells in a non-soluble cell format), but binding of a soluble antibody cannot be detected by direct visualization methods.

[0287] In one embodiment, CAR-T cells can undergo robust in vivo T cell expansion and are sustainable for long periods of time. In another embodiment, CAR-T cells evolve into specific memory T cells that can be reactivated to inhibit further tumor formation or growth.

[0288] In some embodiments, compositions comprising immune effector cells comprising a CAR contemplated herein are used in the treatment of conditions associated with cancer cells or cancer stem cells that express accessible claudin-3 protein.

[0289] As used herein, the phrase "ameliorate at least one symptom" refers to alleviating one or more symptoms of the disease or condition that the subject is being treated for.In certain embodiments, the disease or condition that is being treated for is cancer, and the one or more symptoms that are being ameliorated include, but are not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, enlarged lymph nodes, abdominal distension or pain (due to enlargement of abdominal organs), bone or joint pain, fractures, unplanned weight loss, loss of appetite, night sweats, persistent low-grade fever, and decreased urination (due to decreased kidney function).

[0290] As used herein, the terms "enhancement," "promotion," "augmentation," or "expansion" generally refer to the ability of a composition contemplated herein, e.g., a genetically modified T cell or a CAR-encoding vector, to generate, elicit, or cause a physiological response (i.e., a downstream effect) that is greater than the response caused by a control molecule / composition or in a control disease state. A measurable physiological response can include, among other things, an increase in T cell expansion, activation, persistence, and / or increased cancer cell killing capacity, as understood in the art and described herein. An "enhanced" or "enhanced" amount is generally a "statistically significant" amount and can include 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50, 100, 200, 500, or more times the response generated by a control composition or control cell line. For example, such increased or enhanced levels can be compared to the response seen in healthy, non-cancerous cells that express inaccessible / unavailable claudin-3 protein and / or comprise intact or intact (e.g., undisrupted) cell-cell junctions. Thus, in some embodiments, such increased or enhanced responses are seen in cancer cells that express accessible claudin-3 protein and / or comprise impaired / disrupted cell-cell junctions.

[0291] The terms "reduce," "lower," "attenuate," "reduce," or "mitigate" generally refer to the ability of a composition contemplated herein to generate, elicit, or cause a physiological response (i.e., a downstream effect) that is less than the response caused by a control molecule / composition or in a control condition. A "reduced" or "decreased" amount is generally a "statistically significant" amount and can include a 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50, 100, 200, 500-fold or greater reduction in the response (reference response) produced by a control composition or a particular cell line.

[0292] In one aspect, a CAR, polypeptide, polynucleotide, vector, immune effector cell, or composition contemplated herein is provided for use in therapy, for example, for use in cancer therapy and / or treatment. In a further aspect, a CAR, polypeptide, polynucleotide, vector, immune effector cell, or composition contemplated herein is provided for use as a medicament, such as an anti-cancer agent. In some embodiments, a CAR, polypeptide, polynucleotide, vector, immune effector cell, or composition contemplated herein for use in therapy is for use in a method of therapy and / or treatment, such as a method of cancer therapy and / or a method of treating cancer.

[0293] In one aspect, a CAR, polypeptide, polynucleotide, vector, immune effector cell, or composition contemplated herein is provided for use in treating cancer, wherein the cancer comprises disrupted and / or impaired cell-cell junctions. In another aspect, a method for treating a subject suffering from cancer is provided, comprising administering to the subject a therapeutically effective amount of a CAR, polypeptide, polynucleotide, vector, immune effector cell, or pharmaceutical composition contemplated herein, wherein the cancer comprises disrupted and / or impaired cell-cell junctions. Thus, in some embodiments, a method for treating cancer in a subject in need thereof comprises administering an effective amount, e.g., a therapeutically effective amount, of a composition comprising a genetically modified immune effector cell contemplated herein. Appropriate dosages can be determined by clinical trials, but dosage and frequency will depend on factors such as the patient's condition and the type and severity of the patient's disease.

[0294] Those skilled in the art will recognize that the compositions contemplated herein may need to be administered multiple times to achieve desired therapeutic effect.For example, the compositions can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times for 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5 years, 10 years, or more.Alternatively, only one administration of the compositions contemplated herein is required.

[0295] In one embodiment, a subject in need thereof is administered an effective amount of the composition to enhance a cellular immune response against cancer in the subject. Thus, in a further aspect, there is provided a method for enhancing cytotoxicity against cancer cells comprising disrupted cell-cell junctions in a subject in need thereof, such as a subject suffering from cancer, the method comprising administering to the subject an amount of a CAR, polypeptide, polynucleotide, vector, immune effector cell, or composition contemplated herein.

[0296] The immune response may include a cellular immune response mediated by cytotoxic T cells, regulatory T cells, and helper T cell responses, which can kill infected cells. A humoral immune response, primarily mediated by helper T cells, which can activate B cells and thus lead to antibody production, may also be induced. Various techniques can be used to analyze the type of immune response induced by a composition, and these are well described in the art, for example, Current Protocols in Immunology, John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (eds.) (2001) John Wiley & Sons, NY, NY.

[0297] In the case of T cell-mediated killing, CAR-ligand binding induces CAR signaling to the T cell, resulting in the activation of various T cell signaling pathways that induce the T cell to produce or release proteins that can induce apoptosis of target cells by various mechanisms. These T cell-mediated mechanisms include (but are not limited to) the transfer of intracellular cytotoxic granules from the T cell to the target cell, T cell secretion of proinflammatory cytokines that can induce target cell killing directly (or indirectly by recruiting other killer effector cells), and upregulation of death receptor ligands (e.g., FasL) on the surface of T cells, which induce apoptosis of target cells after their cognate death receptors (e.g., Fas) bind to the target cells. Thus, in one aspect, a method is provided for reducing the number of cancer cells comprising disrupted cell-cell junctions in a subject suffering from cancer, the method comprising administering to the subject a therapeutically effective amount of a CAR, polypeptide, polynucleotide, vector, immune effector cell, or composition as contemplated herein. In one embodiment, the reduction in the number of cancer cells comprising disrupted cell-cell junctions comprises T cell-mediated killing.

[0298] In one embodiment, an "effective amount," which may include a therapeutically effective amount, is sufficient to enhance cytotoxicity against cancer cells, e.g., cancer cells comprising disrupted and / or impaired intercellular junctions, compared to the cytotoxicity against cancer cells comprising disrupted and / or impaired intercellular junctions prior to administration. In another embodiment, an "effective amount" is sufficient to reduce the number of cancer cells, e.g., cancer cells comprising disrupted and / or impaired intercellular junctions, compared to the number of cancer cells comprising disrupted and / or impaired intercellular junctions prior to administration.

[0299] In certain embodiments, the methods contemplated herein further comprise administering an ablation element activator or binder. Such activators or binders include, but are not limited to, antibodies (e.g., clinically approved antibodies), such as those that recognize and bind to huEGFRt or CD20, i.e., cetuximab or rituximab, respectively, small molecule antagonists of CD20, and the like. Administration of the ablation element activator or binder can occur once the subject's treatment is deemed complete, such as after a complete response of the subject or cancer. It will be appreciated that administration of the ablation element activator or binder thus prevents chronic CAR-expressing T-cell activity in the subject. In further embodiments, the methods contemplated herein further comprise utilizing an ablation element to target CAR-expressing cells for antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). Thus, in certain embodiments, the methods contemplated herein further comprise elimination of CAR-expressing cells, such as CAR-expressing T-cells. In other embodiments, it may be desirable to suppress acute CAR-expressing T cell activity in a subject, for example to treat a cytokine storm, such as by administering steroids.

[0300] In a further aspect, there is provided a use of a CAR, polypeptide, polynucleotide, vector, immune effector cell, or composition contemplated herein for the manufacture of a medicament, such as an anti-cancer agent. In one embodiment, the use contemplated herein for the manufacture of a medicament is the manufacture of a medicament for the treatment of cancer.

[0301] In one embodiment, a chimeric antigen receptor (CAR) comprises: a) an extracellular domain comprising an antigen-binding protein that binds to at least one epitope of a cell-binding protein, said cell-binding protein being located within a cell-cell junction, and said at least one epitope of said cell-binding protein being accessible for binding by said CAR extracellular domain only in cancer cells; b) a transmembrane domain; and c) one or more intracellular signaling domains. In one embodiment, the CAR further comprises one or more costimulatory domains. In one embodiment, in a CAR according to any one of the embodiments disclosed herein, the cell junction protein is a tight junction protein, and / or the at least one epitope is inaccessible for binding by the CAR extracellular domain when cell junctions exist between cells in an ordered tissue; and / or when the cell junctions are intact; and / or the at least one epitope is accessible for binding by the CAR extracellular domain when cell junctions exist between cancer cells, between cancer cells and non-cancerous cells, when the cell junctions are impaired, and / or when the cell junction protein is inappropriately localized outside the cell junction.

[0302] In one embodiment, in the CAR according to any one of the preceding embodiments, the cell-binding protein is a member of the claudin family of proteins; and / or at least one epitope is present in one or more extracellular loops of the cell-binding protein; and / or the cell-binding protein is claudin-3; and / or claudin-3 is exposed on the cell surface in solid cancers with disrupted or disorganized tight junctions; and / or claudin-3 is not exposed on the cell surface but is located within cell junctions in normal or non-cancerous cells.

[0303] In one embodiment, in a CAR according to any one of the preceding embodiments, at least one epitope is uniquely present within claudin-3; and / or at least one epitope is four amino acids in length; and / or at least one epitope is a discontinuous epitope.

[0304] In one embodiment, in the CAR according to any one of the preceding embodiments, the antigen binding protein is selected from an antibody or an antigen-binding fragment thereof; and / or the antigen binding protein is selected from the group consisting of a monoclonal antibody, camel Ig, Ig NAR, a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a F(ab)'3 fragment, an Fv, an scFv, a bis-scFv, an (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide-stabilized Fv protein ("dsFv"), and an sdAb; and / or the antigen binding protein is an scFv.

[0305] In one embodiment, in the CAR according to any one of the preceding embodiments, the antigen binding protein comprises any one or combination of CDRs selected from CDRH1, CDRH2 and CDRH3 in SEQ ID NO: 7 and / or CDRL1, CDRL2 and CDRL3 in SEQ ID NO: 8; or the antigen binding protein comprises all six CDRs from SEQ ID NOs: 7 and 8; or the antigen binding protein comprises the CDRH1 sequence of SEQ ID NO: 1; the CDRH2 sequence of SEQ ID NO: 2; the CDRH3 sequence of SEQ ID NO: 3; the CDRL1 sequence of SEQ ID NO: 4; the CDRL2 sequence of SEQ ID NO: 5; and the CDRL3 sequence of SEQ ID NO: 6. Consistent with these embodiments, the antigen binding protein comprises a variable heavy chain (VH) sequence that is at least 90% identical to the sequence of SEQ ID NO: 7, and a variable light chain (VL) sequence that is at least 90% identical to the sequence of SEQ ID NO: 8; or the antigen binding protein comprises the variable heavy chain (VH) sequence of SEQ ID NO: 7, and the variable light chain (VL) sequence of SEQ ID NO: 8; or the antigen binding protein comprises, from N-terminus to C-terminus, the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 8; or the antigen binding protein comprises, from N-terminus to C-terminus, the VL sequence of SEQ ID NO: 8 and the VH sequence of SEQ ID NO: 7.

[0306] In one embodiment, in the CAR according to any one of the preceding embodiments, the transmembrane domain is derived from a polypeptide selected from the group consisting of the alpha or beta chain of the T cell receptor, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137 (4-1BB), CD152, CD154, CD278 (ICOS), and PD1; or the transmembrane domain is derived from CD8 alpha. In one embodiment, in a CAR according to any one of the preceding embodiments, the one or more intracellular signaling domains are derived from an intracellular signaling molecule selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3ε, CD3δ, CD3ζ, CD22, CD66d, CD79a, and CD79b; or the one or more intracellular signaling domains are CD3ζ. In one embodiment, in a CAR according to any one of the preceding embodiments, the CAR further comprises one or more costimulatory domains derived from a costimulatory molecule selected from the group consisting of CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TRIM, and ZAP70; or the one or more costimulatory domains are CD137 (4-1BB).

[0307] In one embodiment, in a CAR according to any one of the preceding embodiments, the extracellular domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 11; or the CAR comprises the amino sequence of SEQ ID NO: 11. In one embodiment, in a CAR according to any one of the preceding embodiments, the extracellular domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 18; or the CAR comprises the amino sequence of SEQ ID NO: 18.

[0308] In one embodiment, in a CAR according to any one of the preceding embodiments, the CAR comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 12, 24, 25, 27, 28, 29 or 30; or the CAR comprises the amino acid sequence of SEQ ID NO: 12, 24, 25, 27, 28, 29 or 30; or the CAR comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 12, 24, 25, 27, 28, 29 or 30, but does not include the CD8 leader sequence of SEQ ID NO: 10. Those skilled in the art will recognize that the CD8 leader sequence of SEQ ID NO: 10 introduced into a CAR according to any one of the above embodiments can be modified or deleted using standard techniques known in the art without affecting the function of the CAR. Consistent with these embodiments, in a CAR according to any one of the above embodiments, the CAR comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 34, 35, 36, 37, 38 or 39.

[0309] In one embodiment, in a CAR according to any one of the preceding embodiments, the CAR comprises: a) an extracellular domain comprising a claudin-3 binding protein comprising the CDRH1 sequence of SEQ ID NO: 1; the CDRH2 sequence of SEQ ID NO: 2; the CDRH3 sequence of SEQ ID NO: 3; the CDRL1 sequence of SEQ ID NO: 4; the CDRL2 sequence of SEQ ID NO: 5; and the CDRL3 sequence of SEQ ID NO: 6; b) a transmembrane domain derived from CD8α; c) a costimulatory domain derived from CD137 (4-1BB); and d) an intracellular signaling domain derived from CD3ζ. In one embodiment, a CAR is provided that competes for binding with a CAR according to any one of the preceding embodiments.

[0310] In one embodiment, a polypeptide is provided comprising the amino acid sequence of a CAR according to any one of the preceding embodiments. In yet another embodiment, the polypeptide further comprises an ablation element. Consistent with these embodiments, the ablation element is a cell surface protein targeted for antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) using an antibody or antigen-binding fragment; and / or the ablation element is derived from a polypeptide selected from the group consisting of a truncated human EGFR polypeptide (huEGFRt) and CD20; or the ablation element is CD20. In one embodiment, a vector is provided comprising a polynucleotide according to any one of the preceding embodiments. In yet another embodiment, the vector is a viral vector; and / or the viral vector is a retroviral vector, such as a lentiviral vector; and / or the retroviral vector is selected from the group consisting of human immunodeficiency virus I (HIV-I); human immunodeficiency virus 2 (HIV-2), visna-maedi virus (VMV); caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus. In one embodiment, there is provided a vector-producing cell comprising a polynucleotide sequence according to any one of the embodiments disclosed herein and / or a vector according to any one of said embodiments.

[0311] In one embodiment, an immune effector cell is provided comprising a CAR, polypeptide, polynucleotide, and / or vector according to any one of the preceding embodiments. Consistent with these embodiments, the immune effector cell is selected from the group consisting of T lymphocytes, natural killer T lymphocytes (NKT) cells, macrophages, and natural killer (NK) cells; or the immune effector cell is a cytotoxic T lymphocyte (CD8 +). In one embodiment, a pharmaceutical composition is provided comprising immune effector cells according to any one of the preceding embodiments and a pharmaceutically acceptable excipient. Also provided is a method of generating immune effector cells comprising a CAR according to any one of the preceding embodiments, the method comprising introducing into the immune effector cells a polynucleotide and / or vector according to any one of the preceding embodiments. Consistent with these embodiments, the method further comprises stimulating the immune effector cells and inducing the cells to proliferate by contacting the cells with an antibody, or antigen-binding fragment thereof, that binds CD3 and an antibody, or antigen-binding fragment thereof, that binds CD28, thereby generating an immune effector cell population. In one embodiment, the stimulation of the immune effector cells occurs prior to introducing into the cells a vector according to any one of the preceding embodiments; and / or the immune effector cells comprise T lymphocytes.

[0312] In one embodiment, a CAR, polypeptide, polynucleotide, vector, immune effector cell, or pharmaceutical composition according to any one of the preceding embodiments is provided for use in treating cancer. In one embodiment, a method of treating cancer in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of a CAR, polypeptide, polynucleotide, vector, immune effector cell, or pharmaceutical composition according to any one of the preceding embodiments. In yet another embodiment, a method of enhancing cytotoxicity against cancer cells in a subject having cancer is provided, comprising administering to the subject an effective amount of a CAR, polypeptide, polynucleotide, vector, immune effector cell, or pharmaceutical composition according to any one of the preceding embodiments. In one embodiment, a method of enhancing cytotoxicity against cancer cells in a subject having cancer is provided, comprising administering to the subject an effective amount of a CAR, polypeptide, polynucleotide, vector, immune effector cell, or pharmaceutical composition according to any one of the preceding embodiments. In other embodiments, a method for reducing the number of cancer cells in a subject with cancer is provided, the method comprising administering to the subject an effective amount of a CAR, polypeptide, polynucleotide, vector, immune effector cell, or pharmaceutical composition according to any one of the preceding embodiments. Consistent with these embodiments, the cancer is characterized by inappropriate localization of claudin-3 outside of tight junctions, making claudin-3 accessible for binding, and / or disruption of tight junctions; or the cancer is characterized by claudin-3 exposed on the cell surface due to disruption of tight junctions. In one embodiment, the cancer is a solid cancer; or the solid cancer is colon cancer, pancreatic cancer, breast cancer (e.g., triple-negative breast cancer (TNBC)), ovarian cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), or prostate cancer; or the cancer is an epithelial cancer.

[0313] In one embodiment, there is provided a use of a CAR, polypeptide, polynucleotide, vector, immune effector cell, or pharmaceutical composition according to any one of the preceding embodiments in the manufacture of a medicament for the treatment of cancer. In yet another embodiment, there is provided a CAR, polypeptide, polynucleotide, vector, immune effector cell, or pharmaceutical composition according to any one of the preceding embodiments as described in claim 48 for use in therapy.

[0314] Although the foregoing embodiments have been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings contemplated herein that certain changes and modifications can be made without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only, and not by way of limitation. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield essentially similar results. [Example]

[0315] Example 1: Generation of CAR-T cells CD4 derived from peripheral blood of healthy humans + and CD8 + CD4 T cells were isolated and then transduced with lentiviral vectors encoding either an anti-claudin-3 CAR construct or a control CAR construct (the control CAR was an anti-CD19 CAR). + and CD8 + All T cells were successfully transduced with lentiviral vectors encoding either the anti-claudin-3 CAR or the control anti-CD19 CAR. CAR T cells were generated from cells isolated from multiple donors, expanded in culture, and frozen as needed for subsequent in vitro and in vivo functional assays.

[0316] material and method CD4 + and CD8 + T cell isolation and activation Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral whole blood and NHS Blood and Transplant (NHSBT) cones using Histopaque (Sigma, Cat. No. 10771) according to the manufacturer's instructions as follows: Cells were resuspended in AutoMACS running buffer and 10 7 FcR blocking reagent, CD4 microbeads, and CD8 microbeads (all Miltenyi Biotec) were added per cell. The cells were mixed and incubated at 4°C for 15 minutes. The cells were then washed, centrifuged, and collected in 10 ml tubes. 8 The cells were resuspended in cold AutoMACS running buffer. The cell solution was run on an AutoMACS pro-separator (Miltenyi Biotec) using the Possel_S separation protocol. Because EDTA can affect T cell activation, magnetically labeled CD4 + and CD8 + The positive fraction containing T cells was washed three times with PBS. After the final PBS wash, the cell pellet was resuspended in an appropriate volume of TEXMACS medium (Miltenyi Biotec) and a sample was removed for counting in an NC-250 Nucleocounter (ChemoMetec).

[0317] The cells were resuspended in TEXMACS medium. TransAct T cell activation reagent (Miltenyi Biotec) and IL-7 and IL-15 were added to the cells to a final concentration of 10 ng / mL for each cytokine. The cell solution was seeded onto cell culture plates (1 × 10 6 cells / mL), and then the cells were incubated for 24 hours at 37°C in a humidified incubator with 5% CO2.

[0318] Transduction of T cells with lentiviral vectors and expansion of CAR-T cells Cells were transduced at an MOI of 3 with a lentiviral vector encoding an anti-claudin-3 CAR (906_009) along with a reporter gene (LNGFR) (designated 906_009-LNGFR) or an anti-CD19 CAR vector (control). The CAR construct contained an LNGFR marker to enable detection and / or enrichment of CAR-expressing T cells. The LNGFR marker system uses a transiently expressed truncated human low-affinity nerve growth factor receptor (LNGFR) molecule as a surface marker to detect and / or select transfected cells. Cells were incubated at 37°C and 5% CO2 in a humidified incubator. Cells were maintained in TEXMAC medium and IL-17 and IL-15 at 10 ng / mL of each cytokine throughout the culture period. In some batches, cells were cultured with IL-2 instead of IL-7 and IL-15. When IL-2 was used, the same culture procedure was followed, except that 100 international units (IU) / mL of IL-2 was added instead of IL-7 and IL-15. T cells were harvested 12 days after transduction and 1 × 10 7 ~1×10 8 Cells were frozen in CS5 freezing medium (Sigma, #C2999) at a cell density of 1000 cells / mL.

[0319] Transduction efficiency was determined by flow cytometry (MACSQuant Analyser 10) using a PE-conjugated anti-LNGFR antibody (Ab) to detect the expression of the truncated human low-affinity nerve growth factor receptor (LNGFR; CD271). Data were analyzed using FlowJo v10.1.

[0320] For a particular CAR-T batch, the lowest transduction efficiency was required to be uniform across all CAR-T populations. To achieve accurate uniformity across CAR-T cell populations, LNGFR + The cell frequency was analyzed and the cells were counted. + The volume of non-transduced T cells required to reduce the cell frequency to a defined level was calculated and added to each cell population accordingly.

[0321] Two cell preparations transduced with the anti-claudin-3 CAR vector (906_009-LNGFR) were generated: one suspension cell (designated "Vector 1") and one adherent cell (designated "Vector 3"). Other than differences in transduction efficiency between the two different cell preparations (see discussion below), no significant differences were observed between the two different cell preparation methods.

[0322] Enrichment of T cells for the generation of pure CAR-T cell populations For some CAR-T batches, on day 12 post-induction, T cells were enriched by positive selection using AutoMACs Pro-Separator enrichment or EasySep enrichment as described below to generate pure CAR-T cell populations.

[0323] For AutoMACs Pro-Separator concentration, 10 7 LNGFR microbeads (Miltenyi Biotec) were added per cell, and the cells were mixed thoroughly and then incubated for 15 minutes at 4°C. The cells were washed, and the cell solution was run on an AutoMACS pro-separator using the Possel_S separation protocol. Because EDTA can affect T cell activation, magnetically labeled LNGFR microbeads were added to ensure the cell solution contained up to 200-fold less EDTA. + The positive fraction containing T cells was washed three times with PBS.

[0324] EasySep enrichment was performed either on day 9 post-transduction or day 12 post-transduction, depending on the assay requirements and total cell number of the culture. T cells transduced with various LNGFR-tagged CARs were positively selected using the EASYSEP Human CD271 Positive Selection Kit II (STEMCELL Technologies UK Ltd) and EASYSEP rapidshere beads (StemCell Technologies). Depending on the number of transduced T cells to be enriched, either an EASYPLATE magnet (StemCell Technologies) or an EASYEIGHT magnet (StemCell Technologies) was used. Freshly thawed or cultured transduced T cells were cultured at a concentration of 1 × 10 in TEXMACS medium supplemented with EASYSEP Human FcR Blocker (25 μL / mL) and EASYSEP Human CD271 Positive Selection Kit (50 μL / mL). 7 ~2×10 7 The cells were resuspended at a density of 1000 and incubated at room temperature for 15 minutes.

[0325] At higher cell densities, 1 x 10 8 ~2×10 8Cells were resuspended at the indicated concentrations in TEXMACS medium supplemented with EASYSEP Human FcR Blocker and EASYSEP Human CD271 Positive Selection Cocktail. 50 μL / mL of EASYSEP rapidshere beads were added to each sample, and the cells were incubated at room temperature for 15 minutes. After incubation, wash buffer (PBS containing 2% fetal bovine serum (FBS) and 2 mM EDTA) was added to the samples, which were then transferred to an EASYPLATE or EASYEIGHT EASYSEP magnet and incubated for 10 minutes. The supernatant was carefully removed to avoid disturbing the positively selected cells attached to the beads. After three additional washes, the cells were resuspended in TEXMACS medium, and samples were removed for counting on an NC-250 nucleocounter and post-enrichment LNGFR analysis to confirm successful enrichment.

[0326] If enrichment was performed on day 9 post-transduction, enriched CAR-T cells were replated in TEXMACS medium containing IL-7 and IL-15 at 10 ng / mL. Cells were incubated for 72 hours at 37°C in a humidified incubator with 5% CO2 and then frozen as described above on day 12 post-transduction.

[0327] If enrichment was performed 12 days post-transduction, the enriched cells were either used immediately for functional assays or frozen.

[0328] result T cell expansion, transduction efficiency, and enrichment and homogenization of CAR-T cell populations All T cell populations were successfully expanded, with expansion folds ranging from 14-fold to 178-fold depending on the specific donor. The mean expansion fold for all T cell populations was 76.

[0329] The transduction efficiency (based on LNGFR-positive cell frequency) of CAR-T cells transduced with anti-claudin-3 CAR vector 1 at an MOI of 3 ranged from 41 to 60% across multiple donors and vector batches. CAR-T cells transduced with anti-claudin-3 CAR vector 3 at an MOI of 3 had lower transduction efficiencies of 29 to 37% across the three donors used. All control CAR T cells (anti-CD19 CARs) had transduction efficiencies of 48 to 75% across multiple donors and vector batches using an MOI of 3.

[0330] 100% LNGFR + LNGFR for generating CAR-T cell populations + CAR-T cell enrichment was successful for all CAR-T cell batches produced, both by AutoMACS pro-separator enrichment (data not shown) and EasySep LNGFR enrichment, as shown in Figure 2A.

[0331] Homogenization among CAR-T cell populations to the required LNGFR-expressing T cell frequency was successful as shown in Figure 2B for the produced CAR-T batches.

[0332] CD4 and CD8 positive selection using the AutoMACS Pro-Separator resulted in >95% CD3 + This allowed efficient transduction of the desired cell population with the vector. Monocyte contamination after CD4 / CD8 positive selection was minimal (<5%), and remaining monocytes died during the culture period, resulting in pure CD3+ cells by day 12 post-transduction. + A cell population was obtained.

[0333] CD4 isolated from healthy donors + and CD8 +All T cells were successfully transduced with lentiviral vectors encoding either an anti-claudin-3 CAR or a control anti-CD19 CAR. Differences in transduction efficiency were observed between lentiviral vectors 1 and 3. All T cell populations were successfully expanded, with some aberrant expansion observed within donors for certain CAR constructs.

[0334] CAR-T cell enrichment using both the AutoMACs pro-separator and EasySep LNGFR microbeads was successful, with 100% LNGFR for subsequent functional assays. + The CAR-T population was available. In addition, the required LNGFR + We also succeeded in achieving uniformity among CAR-T cell populations in terms of cell frequency.

[0335] All CAR-T cells produced could be used in functional assays to test the anti-claudin-3 CAR vector 1. CAR-T cells produced in suspension were used in subsequent experiments.

[0336] Example 2: Effect of CAR expression and T cell phenotype The purpose of this study was to evaluate the effects of tonic signaling (antigen-independent signaling) of anti-claudin-3 CAR-T cells in vitro. CAR-T cells exhibiting tonic signaling result in impaired T cell function and exhaustion in vitro and reduced efficacy in vivo. Tonic signaling is influenced by a combination of characteristics of the CAR structure, linker or hinge, signaling domain, and surface expression location and level. Tonic signaling was assessed by measuring basal levels of cytokines (IFNγ) secreted into the cell supernatant, differentiation into a sustained T cell phenotype by measuring activation markers (CD69) and exhaustion markers (PD-1 and TIM-3), and enhanced antigen-independent signaling (pCD3ζ). Responses were evaluated against a negative control anti-CD19 CAR and a positive control (GD2-28ζ) CAR, which exhibit low and high levels of tonic signaling, respectively.

[0337] These results indicate that both the anti-CD19 CAR negative control and the anti-claudin-3 CAR conferred low levels of tonic signaling compared to the positive control (GD2-28ζ) CAR-T cells, indicating low levels of antigen-independent activation in vitro for the claudin-3 CAR construct.

[0338] material and method Preparation of negative and positive control CARs A negative control anti-CD19 CAR was generated using the FMC62 ScFv with the 4-1BB-CD3ζ cytoplasmic signaling domain and was used herein as a control to evaluate low-level tonic signaling responses. A positive control CAR (GD2-28ζ) was generated using the 14g2a scFv with the CH2-CH3 IgG1 linker and the CD28-CD3ζ transmembrane and cytosolic spanning domain. Here, the EF1a promoter was used to increase the transduction efficiency of the CAR, which should enhance its propensity to promote tonic signaling responses. The CD28ζ transmembrane and cytosolic domain should increase tonic signaling levels compared to the 4-1BBζ cytoplasmic domain, which is independent of the lentivector transduction promoter used. Furthermore, the 14g2a anti-GD2 scFv clone has a tendency to oligomerize, a feature characteristic of the GD2-28ζ CAR structure, leading to intrinsic activation of CAR-dependent signaling. The IgG1CH2-CH3 extracellular linker used in the positive control CAR may also contribute to the observed tonic signaling levels.

[0339] Thawing and culture of CAR T cells CAR-T cells (thawed from deep-frozen stock or fresh cells) were resuspended in TEXMACS medium and the cell density was adjusted to 2 × 10 in TEXMACS medium containing 10 ng / mL of IL-7 / IL-15. 6 The resuspended cells were placed in a humidified incubator at 37°C with 5% CO2 for 24 hours before LNGFR enrichment.

[0340] Thawed CAR-T cells LNGFR enrichment LNGFR-expressing CAR-T cells were positively selected using the EASYSEP Human CD271 Positive Selection Kit and EASYSEP Dextran RAPIDSPHERES. CAR-T cells were harvested and cultured in non-tissue culture-treated 96-well plates at 10–20 × 10 cells / well in TEXMACS medium supplemented with EASYSEP Human FcR Blocker and EASYSEP Human CD271 Positive Selection Cocktail. 6 The cells were resuspended at a density of 100x100 and incubated at room temperature for 15 minutes. EASYSEP Dextran RAPIDSPHERES were added to the cell suspension and incubated at room temperature for 15 minutes. LNGFR-expressing cells were then selected using an EASYPLATE EASYSEP magnet, resuspended in TEXMACS medium supplemented with 10 ng / mL human IL-7 / IL-15, and placed in a humidified incubator at 37°C and 5% CO2 for 72 hours before further assay or cryopreservation. Cryopreserved LNGFR-enriched cells were thawed and resuspended at a density of 2.5x10 in TEXMACS medium supplemented with 10 U / mL IL-2. 6 The cells were seeded at a density of 1000 / well and placed in a humidified incubator at 37°C and 5% CO for 24 hours. The supernatant and cells were used for subsequent assays.

[0341] Lysate preparation and protein quantification CAR-T and non-transduced T cells were harvested from the cultures and 2 × 10 6 Cells were resuspended in cold dPBS (containing calcium and magnesium), centrifuged, and the resulting cell pellet was lysed by repeated pipetting of cold lysis buffer. The lysate was centrifuged, aliquoted, flash-frozen, and stored at -80°C for long-term storage. Protein levels in the lysate were quantified using the bicinchoninic acid (BCA) assay.

[0342] Determination of IFNγ cytokine secretion in CAR-T cell supernatants Test samples were placed in human IFNγ Mesoscale Discovery (MSD) plates. These plates were then sealed and incubated on a plate shaker at room temperature for 90 minutes. The plates were washed, and detection antibody was added to each well. These plates were sealed and incubated on a plate shaker at room temperature for 2 hours. After this, the plates were washed and read on an MSD Sector 600 imager. The mean and standard error of the mean of IFNγ secretion levels of test CAR T cells from six donors were calculated, and the data were plotted using GraphPAD PRISM (Bonferroni one-way analysis of variance).

[0343] Determination of CAR-T cell phenotype (CD69, TIM-3, PD-1) Untransduced T cells and CAR-T cells were thawed and counted. 2.5 × 10 5 Cells were dispensed into a 96-well plate. The cells were then washed, and the appropriate antibody mixture (containing antibodies against CD3, CD8, CD69, TIM3, and PD1) was added to each well. The cells were incubated in the dark at room temperature for 15 minutes before being resuspended in medium containing DAPI live / dead dye at a final concentration of 1 μg / mL. Samples were analyzed by flow cytometry, and flow cytometry data were analyzed using FLOWJO V10 software.

[0344] Expression and co-expression of activation / exhaustion markers CD69, PD-1 and TIM-3 were measured by CD4+ / CD69-associated immunoglobulins (IgE) assay. + and CD8 + Generated by stratifying by population. CD4 + Cell population or CD8 + After gating as a cell population, only those cells with a single positive result for the activation / exhaustion marker were identified. Subsequently, Boolean gating logic was applied to characterize single-, double-, and triple-positive / negative cell populations for the three activation / exhaustion markers.

[0345] For data analysis, the mean percentages of triple positive (PD-1, TIM-3, and CD69), double positive (CD69 and TIM-3, or CD69 and PD-1, or TIM-3 and PD-1), and single positive (PD-1 or TIM-3 or CD69) responses for the negative control (anti-CD19 CAR), positive control (GD2-28ζ CAR), and anti-claudin-3 CAR were calculated for six PBMC donors. Data were analyzed using GraphPAD PRISM (Bonferroni one-way ANOVA).

[0346] Determining downstream signaling via CAR-specific CD3ζ phosphorylation 2×10 6 Lysates were obtained from 100 donors, homogenized to a concentration of 300 μg / mL, and heated. Anti-pCD3ζ, anti-CD3ζ, GAPDH, and secondary antibodies were added to these lysates prior to loading. Protein levels were assessed using PEGY-SUE high-throughput capillary Western technology. Normalized levels of phosphorylated CD3ζ (pCD3ζ) were calculated based on total CD3ζ and GAPDH loading controls from up to six donors.

[0347] Antigen-independent signaling data analysis was performed using Compass for SW software (PEGGY-SUE) to generate key metrics. The area under the peak (AuP) of each staining was determined using the software, and responses were normalized based on the AuP of GAPDH (total protein loading) levels. Normalized pCD3ζ levels of test CAR-T cells (positive control (GD2-28ζ) and anti-claudin-3 CAR-T cells) were divided by the normalized pCD3ζ levels detected in negative control CAR-T cells (anti-CD19 CAR). The mean and standard error of the mean of CAR-specific phosphorylation (pCD3ζ) levels for test CAR T cells from six donors were calculated. Data were plotted using GraphPAD PRISM (Bonferroni one-way analysis of variance).

[0348] result Basal levels of IFNγ secretion from CAR T cells IFNγ secretion by T cells is an important indicator of T cell activation, and antigen-independent signaling can be assessed, in part, by the secretion of this cytokine. Data shown in Figure 3A demonstrate that IFNγ secretion from anti-claudin-3 CAR T cells (611.8 ± 755.1 pg / mL) was significantly lower than that from positive control (GD2-28ζ) CAR-T cells (22,557 ± 12,903 pg / mL). No significant differences were observed between non-transduced T cells (123.7 ± 103.0 pg / mL), the negative control (anti-CD19 CAR; 666.4 ± 725.1 pg / mL), and anti-claudin-3 CAR-T cells (Figure 3A).

[0349] Baseline T cell activation (CD69 + ) and exhaustion (TIM-3 + and PD-1 + )phenotype Activation marker (CD69 + ) and exhaustion marker (TIM-3 + and PD-1 + The differentiation of basal T cells into a sustained phenotype, which demonstrates an increase in CD4 expression, can complement some of the assays used to detect tonic signaling. The data shown in Figure 3B demonstrate a significant increase in the activated and exhausted phenotype in the positive control (GD2-28ζ CAR) compared to the negative control (anti-CD19 CAR) and anti-claudin-3 CAR-T cells. The positive control (GD2-28ζ CAR) showed a significant increase in the activated and exhausted phenotype compared to the negative control (anti-CD19 CAR) and anti-claudin-3 CAR-T cells. + T cells (triple positive: 2.05±1.57%, double positive: 6.89±3.61, single positive: 14.59±7.36%) were CD8 + Anti-claudin-3 CAR-T cells showed a higher increase in activated and exhausted phenotypes compared to CD4 T cells (triple positive: 0.42 ± 0.4%, double positive: 3.9 ± 2.31%, single positive: 14.18 ± 4.75%). + T cells (triple positive: 0.06 ± 0.06%, double positive: 0.71 ± 0.44, single positive: 2.99 ± 0.96%) had significantly lower CD8 +showed a higher increase in activated and exhausted phenotypes compared to T cells (triple positive: 0.54 ± 0.5%, double positive: 0.61 ± 0.25%, single positive: 3.51 ± 1.37%) ( Figure 3B-G ).

[0350] CAR-specific CD3ζ phosphorylation Analysis of the results showed that the normalized level of CAR pCD3ζ for the anti-claudin-3 CAR (0.68 ± 0.39) was significantly lower than that for the positive control (GD2-28ζ) CAR (7.32 ± 3.84), but was not significantly different from that for the negative control (anti-CD19) CAR pCD3ζ ( Figure 3H ).

[0351] IFNγ secretion, activation (CD69 + ) and exhaustion (TIM-3 + and PD-1 + Based on the results obtained from the phenotype and CAR pCD3ζ levels, both anti-CD19 CAR and anti-claudin-3 CAR induced lower levels of tonic signaling compared with the positive control (GD2-28ζ) CAR-T cells. The CAR transduction levels of T cells, assessed by total CD3ζ staining, showed differences based on the promoter used. The positive control (GD2-28ζ) CAR-T cells transduced with the EF1a promoter induced higher levels of CAR-specific total CD3ζ compared with the anti-CD19 CAR and anti-claudin-3 CARs expressed with the PGK promoter. Consequently, the anti-CD19 CAR and anti-claudin-3 CAR also showed lower levels of phosphorylated CD3ζ, cytokine release, and differentiation toward activated and exhausted phenotypes. This reaffirms that vector efficiency can be a contributing factor in inducing tonic signaling.

[0352] In addition to the promoter, unlike the positive control (GD2-28ζ) CAR-T cells, anti-CD19 and anti-claudin-3 CAR-T cells were also engineered with the cytoplasmic domain of 4-1BBζ without the IgG1 CH2-CH3 linker to improve tonic signaling efficacy. This data reaffirms that anti-claudin-3 CAR-T cells have low levels of tonic signaling and antigen-independent activation that adversely affect CAR-T cell function in vitro.

[0353] Example 3: Specificity of anti-claudin-3 CAR-T cells to claudin-expressing cell lines The purpose of these studies was to generate claudin-3-expressing cell lines for validation of anti-claudin-3 CAR-T cell specificity and evaluation of functional activity. Specifically, the cytotoxicity of anti-claudin-3 CAR-T cells was measured using the CYTOTOX Red assay and target cell confluency. Furthermore, CAR-T cell activation was assessed by measuring IFNγ release using the mesoscale discovery (MSD) assay after 24 hours of coculture with claudin-3-expressing cells. Results indicate that anti-claudin-3 CAR-T cells kill primarily in response to hCLDN3, with little or no cytotoxic cross-reactivity with other human claudins.

[0354] material and method Generation and characterization of RKO-KO cell lines, RKO-KO human CLDN3 GFP cell lines, and RKO-KO mouse CLDN3 GFP cell lines The RKO-KO cell line was generated by knocking out the CLDN3 gene in a colon cancer (RKO) cell line using CRISPR-CAS editing technology.

[0355] The selected RKO-KO clone was further tested by flow cytometry using an anti-CLDN3 antibody to confirm the lack of detectable extracellular CLDN3 expression. RKO-KO clone 26.1 was selected as the primary clone for further generation of cell lines overexpressing hCLDN3, mCLDN3, and other human claudins.

[0356] The RKO-KO cell lines overexpressing human and mouse CLDN3 were generated by transducing the RKO-KO clone 26.1 cell line with a commercially available lentiviral vector (LV) containing either (i) the human CLDN3 gene expressed as a C-terminal monomeric GFP-tagged protein or (ii) the mouse CLDN3 gene expressed as a C-terminal monomeric GFP-tagged protein, along with a puromycin selection marker, at an MOI of 5. Transduction efficiency (GFP expression) was assessed using flow cytometry.

[0357] Monoclonal cell lines were established by single-cell sorting from heterogeneous populations to select cells expressing high, medium, and low levels of GFP.

[0358] Polyclonal RKO-KO cell lines expressing the human CLDN family members CLDN4, CLDN5, CLDN6, CLDN8, CLDN9, or CLDN17 were generated by transducing the RKO-KO clone 26.1 cell line with commercially available lentiviruses encoding either the CLDN4, CLDN5, CLDN6, CLDN8, CLDN9, or CLDN17 gene expressed as a C-terminally monomeric GFP-tagged protein, along with a puromycin selection marker, using the same method as described above.

[0359] Characterization of RKO-KO and RKO-KO transduced cell lines by qPCR The mRNA expression of human claudins 3, 4, 5, 6, 9, and 17 and mouse CLDN3 genes was assessed relative to the housekeeping gene ACTB in untransduced RKO-KO cells and RKO-KO cells overexpressing CLDN3, 4, 6, 9, 17, and mCLDN3, respectively. mRNA expression was detected by real-time quantitative PCR (RT-qPCR). RT-qPCR results indicated that transduced CLDNs were overexpressed in each RKO-KO cell line (data not shown).

[0360] Thawing and culturing of CAR-T cells When deep-frozen CAR-T cells were used, the cells were thawed and resuspended in TEXMACS. In some experiments, CAR-T cells were enriched as described elsewhere herein.

[0361] Co-culture setup for the INCUCYTE killing assay 2 x 10 target cells in cell culture medium 5 After resuspending at a density of 2 × 10 cells / mL, transfer to each well of the assay plate. 4 The assay culture plates were then transferred to humidified INCUCYTE S3 at 36.5°C / 5% CO2, and co-culture with CAR-T cells was performed after 24 hours.

[0362] The cell supernatant was removed from each assay well and replaced with fresh cell culture medium containing 500 nM CYTOTOX Red reagent. The plate was then transferred to a humidified INCUCYTE S3 chamber at 36.5°C / 5% CO2 before the addition of effector cells (CAR-T cells). CAR-T cells and non-transduced T cells were resuspended in cell culture medium to a density of 2 × 10 5 Cells / mL were added to the wells. The assay plate was placed in humidified INCUCYTE S3 at 37°C / 5% CO2. Images were acquired at 2-hour intervals over a 6-day period. Image analysis was performed to confirm the specific increase in total red area visualization, indicative of target cell killing, and a total red area mask was generated to represent the total area (μm 2 / image) was determined. Normalization was performed within each donor.

[0363] Measurement of cytokine concentrations by MSD Homogenized or enriched T cells were mixed with target cells at a 1:1 E:T ratio (effector cells:target cells, where "effectors" are transduced CAR-T cells) and co-cultured at 37° C., 5% CO. After 24 hours, plates were centrifuged and supernatants were collected for quantification of cytokine secretion using appropriate detection antibodies (sulfo-tagged anti-hIFNγ, sulfo-tagged anti-hTNFα, and sulfo-tagged anti-hIL2 Abs) using methods similar to those described above in Example 2.

[0364] result Activation response of CAR T cells to claudin-expressing RKO KO Non-transduced cells, anti-CD19 CAR-T cells (control), or anti-claudin-3 CAR-T cells were co-cultured with the RKO KO cell line, which expresses claudin proteins closely related to claudin-3. Supernatants were then collected from these co-cultures, and cytokines of interest were quantified. Data from these experiments are shown in Figure 4A-4D and Table 3.

[0365] We first examined the secretion of IL-2, IFNγ, and TNFα by CAR-T cells in response to the entire panel of cell lines. The data shown in Figure 4A indicate that, apart from the dramatic response of anti-claudin-3 CAR-T cells to hCLDN3, the increase in secretion of all three cytokines in response to hCLDN4 was small.

[0366] Next, we focused on the core panel of cell lines (hCLDN4, hCLDN6, and hCLDN9) in an experiment examining IFNγ responses from six donors (shown in Figures 4B and 4C). Again, anti-claudin-3 CAR-T cells responded to hCLDN4, and in this experiment, responses to hCLDN9 were also observed. The fold change in IFNγ secreted by anti-claudin-3 CAR-T cells in response to hCLDN4 and hCLDN9 relative to anti-CD19 control CAR-T cells was 25 and 14.3, respectively. While this response is significant, it is put into perspective when compared to the 2495-fold higher response in the hCLDN3 coculture.

[0367] The data from these two experiments is further supported by the information shown in Figure 4D, which shows cytokine secretion from seven additional donors. Only one of these donors showed a response to hCLDN9, while a more consistent response was seen with hCLDN4 (again, this was minimal). Some of the donors shown in this figure were also used subsequently (12031, 92024, and C1700657). In each of these cases, the data confirmed the previous results.

[0368] Besides the robust response of anti-claudin-3 CAR-T cells to hCLDN3, the most consistent response was to mCLDN3, which was seen in the form of upregulation of not only IFNγ but also IL-2 and TNFα (Figures 4A, 4B, and 4C). The significance of this response is also shown in Figures 4B and 4C and Table 3, where the IFNγ response of anti-claudin-3 CAR-T cells was 439-fold higher than that of anti-CD19 control CAR-T cells.

[0369] [Table 3]

[0370] Killing of RKO cells by CAR-T cells To specifically examine target cell viability when cultured with CAR-T cells, several killing assays were performed to determine how T cell activation (measured by cytokine response) translates into cytotoxicity and subsequent target cell apoptosis.

[0371] The data collected from this series of experiments are summarized in Table 4 (reported as % viability at the assay endpoint). No experiments resulted in a decrease in viability of hCLDN4, hCLDN6, or hCLDN9, but responses to mCLDN3 were observed in some cases. This was most pronounced at 96 hours, with RKO KO mCLDN3 reaching 71% and 86%, respectively, or maximal responses when cultured with anti-claudin-3 CAR-T cells from two donors.

[0372] To confirm the CLDN3-specific cytotoxic response of anti-claudin-3 CAR-T cells, experiments were performed with six donors and a core panel of cell lines. Two measurements were used: % confluency (Figures 5A and 5B) and % CYTOTOX color development or viability (Figures 6A-C). The first of these, % confluency, indicates cell killing through changes in target cell number specific to a particular coculture. The second measurement quantifies the red color development that occurs when a decrease in viability leads to influx of CYTOTOX dye. Increased cell death was associated with a higher CYTOTOX Red response and a lower % viability. An example of an image used to collect this data is shown in Figure 5A.

[0373] Changes in % confluency were particularly evident when anti-claudin-3 CAR-T cells were cocultured with RKO KO expressing either hCLDN3 or mCLDN3, but as with IFNγ secretion, the magnitude of the response to hCLDN3 was much greater. This was confirmed by measurements of % viability, which showed a significant cytotoxic effect when anti-claudin-3 CAR-T cells were cocultured with RKO KO mCLDN3 or RKO KO hCLDN3 compared to controls.

[0374] [Table 4]

[0375] Cross-reactivity of anti-claudin-3 CAR-T cells with mCLDN3-expressing cells Several relevant experiments can be performed using mouse tissues, which is useful for understanding how anti-claudin-3 CAR-T cells react with mCLDN3. The experiments performed here consistently show that anti-claudin-3 CAR-T cells are activated by mCLDN3 expression, resulting in the secretion of IFNγ, TNFα, and IL-2. Although not as strong as the response to hCLDN3, activated T cells show a significant cytotoxic response, as indicated by the elimination of target cells.

[0376] While the majority of this data was generated in cell lines expressing different levels of mCLDN3, we also used a few cell lines expressing high or low levels of mCLDN3 to further understand the response. Under these conditions, an activating response was only seen when anti-claudin-3 CAR-T cells were cultured with RKO KO cells, which express high levels of mCLDN3.

[0377] Anti-claudin-3 CAR-T cells do not respond to and kill other human claudin proteins In none of the experiments presented herein was anti-claudin-3 CAR-T cell activation observed in response to hCLDN6, hCLDN5, hCLDN8, or hCLDN17. However, there was some reactivity to hCLDN4 and hCLDN9, as indicated by cytokine secretion. While a significant activation response is shown in Figure 4A, it is not comparable to the anti-claudin-3 CAR-T cell responses to hCLDN3 and mCLDN3. Clearly, this was not a killing response, indicating that anti-claudin-3 CAR-T cells did not significantly kill in response to human claudins other than hCLDN3.

[0378] The data presented herein demonstrate that anti-claudin-3 CAR-T cells are activated, at least in part, by hCLDN4 and hCLDN9. This response is significantly lower than the response to hCLDN3 and does not result in cytotoxic effects or cell death. However, anti-claudin-3 CAR-T cells cross-react with mCLDN3 and demonstrate partial target cell killing. This data indicates that anti-claudin-3 CAR-T cells primarily kill in response to hCLDN3, with little or no cytotoxic cross-reactivity with other human claudins.

[0379] Example 4: Cytotoxicity of anti-claudin-3 CAR-T cells against claudin-3-positive tumor cell lines The purpose of this study was to demonstrate the expression and cytotoxicity of an anti-claudin-3 CAR based on the propensity of T cells expressing this construct to specifically kill human claudin-3 (hCLDN3)-expressing target cells. These data demonstrate that anti-claudin-3 CAR-T cells secrete IFNγ and can kill hCLDN3-expressing cancer cell lines derived from colon, breast, and pancreatic cancers.

[0380] material and method Enrichment of LNGFR-positive CAR-T cells by MACSQuant Tyto sorting Cells were washed with buffer and stained with a 1:50 dilution of LNGFR PE antibody for 30 minutes at 4°C, after which the cells were sorted using MACSQuant Tyto sorting according to the manufacturer's instructions. All CAR-T cells used in the experiments described herein were at least 90% LNGFR positive.

[0381] Quantification of CAR molecules on the surface of T cells 1×10 5Cells and 50 μL of Bangs Labs Quantum Simply Cellular beads were resuspended in anti-LNGFR PE at a 1:20 dilution or anti-hCLDN3 PE at a 1:50 dilution and incubated for 30 minutes at 4°C. After washing the cells and beads twice, the PE signals of the cells and beads were measured on a CytoFLEX S machine.

[0382] Co-culture setup for cell killing assays Co-cultures for the INCUCYTE killing assay were set up as described above in Example 3, except that INCUCYTE Zoom was used instead of INCUCYTE S3.

[0383] Co-cultures for the XCELLIGENCE killing assay were set up by seeding target cells into cell culture plates at a density of 25,000 cells / well and culturing them in the cell culture incubator of the XCELLIGENCE Real-Time Cell Analysis (RTCA) instrument. Approximately 20 hours after seeding, effector cells were added at a 0.5:1 or 1:1 CAR-T cell:target cell ratio and returned to the cell culture incubator. The target cells used were the cancer cell lines listed in Table 5 below.

[0384] [Table 5]

[0385] Measurement of cytokine concentrations by MSD After resuspending the target cell line, 2 x 10 4 or 2.5 x 10 4 Cells were seeded into 96-well plates. Homogenized or enriched T cells were then added to the plates at a 1:1 E:T ratio (effector cells:target cells, where "effector" refers to the transduced CAR-T cells) and co-cultured for 24-48 hours at 37°C and 5% CO2. After co-culture, the plates were centrifuged, and the supernatants were collected for quantification of cytokine secretion using appropriate detection antibodies, as described above in Example 2.

[0386] result Quantification of CAR expression and hCLDN3 expression Expression of CAR molecules on the T cell surface is a prerequisite for CAR function. LNGFR expression was measured as a surrogate for CAR expression. Although LNGFR and CAR molecules should be converted in a 1:1 ratio, quantification of LNGFR is only an estimate of the number of CAR molecules due to potential differences in protein stability.

[0387] Untransduced cells (UT) showed only a very low signal of LNGFR expression, 10,000-20,000 (FIG. 7A), which was considered to be a background signal.

[0388] Donors 12031 and 92024 had an average of 190,000 and 166,000 LNGFR molecules on their surface, while donor D5 had 301,000 molecules on its surface, a difference likely due to variations in T cell generation.

[0389] As previously described herein, an hCLDN3 knockout RKO cell line (RKO-KO) was generated and used as a negative control for functional assays. Killing of RKO-KO cells expressing various levels of hCLDN3 (polyclonal RKO-KO hCLDN3 cell line; non-single-cell sorted) or RKO-KO cells sorted for high expression (RKO-KO hCLDN3 H12) or low expression (RKO-KO hCLDN3 L14) is an indicator of CAR efficacy. Differences in hCLDN3 expression were confirmed by quantitating expression with Quantum Simply Cellular beads and anti-CLDN3 PE (Figure 7B). Because RKO-KO cells showed a signal below the bead level, their hCLDN3 level was considered to be zero. RKO-KO hCLDN3 L14-low cells had an average of 80,000 hCLDN3 molecules on their surface, whereas RKO-KO hCLDN3 H12-high cells had an average of 800,000 hCLDN3 molecules on their surface, representing a 10-fold difference in hCLDN3 expression between these two cell lines.

[0390] CAR-T cell cytotoxicity Figures 8A-8D show example IncuCyte killing assays with a 90-hour incubation. Example images of LNGFR-enriched anti-CD19 control CAR-T cells (Figure 8A) or anti-claudin-3 CAR-T cells (Figure 8B) incubated with RKO-KO cells or RKO-KO cells expressing low levels of hCLDN3 are shown, and the corresponding killing curves demonstrate cytotoxicity for the anti-claudin-3 CAR-T cells but not the control (Figure 8C). Incubation of anti-claudin-3 CAR-T cells with the target cell line resulted in signal generation, measured by the area of ​​red dye. Quantification and normalization data are shown in Figure 8C, demonstrating that anti-claudin-3 CAR-T cells incubated with target-negative cells did not result in cell lysis, whereas anti-claudin-3 CAR-T cells incubated with hCLDN3-expressing RKO cells achieved complete target cell killing, whereas the anti-CD19 control did not.

[0391] Table 6 shows the time required to reach 50% of the maximum response, indicating that 50% of the target cells were killed. These values ​​revealed differences between assays that may be explained by differences in hCLDN3 expression, but this was inconsistent between experiments. In some experiments, 50% of the maximum response was achieved after 24–41 hours, while in other experiments, 50% of the maximum response was achieved after 51 hours or by 74 hours. Other data (not shown), in which T cells and target cells were treated equally in all six donors, showed a decrease in proliferation between 80–86 hours in four of the six donors and at 68 hours in one donor, with one donor not reaching 50% cytotoxicity within the 96-hour experiment. Importantly, complete target cell lysis occurred in all experiments, although differences in killing rates could not be explained.

[0392] Similar cytotoxicity results were obtained with the xCELLigence method against the claudin-3-expressing cell line HT-29-LUC (Figure 8D). 100% cytotoxicity was observed in three donors at a 1:1 ratio, with 100% cytotoxicity achieved after 80, 30, and 50 hours. At a 1:2 ratio (effector:target), 100% cytotoxicity was achieved after 40 and 60 hours. Cocultures performed similarly to the xCELLigence experiments were analyzed for cytokine secretion by MSD (data not shown). Interferon-γ (IFNγ) was detected in frozen and thawed donors and freshly used donors when T cells were cocultured with HT-29-LUC, but not when incubated without the cell line.

[0393] IFNγ secretion by untransduced or anti-CD19 control CAR-T cells in response to claudin-3-expressing cells was comparable to that in response to cells that did not express claudin-3. These results suggest that nonspecific cytokine secretion was not observed. Secretion by anti-claudin-3 CAR-T cells in response to target cells that did not express claudin-3 resulted in background cytokine secretion comparable to that of the negative control, indicating that the anti-claudin-3 CAR does not recognize other molecules on the target cells.

[0394] Incubation of anti-claudin-3 CAR-T cells with claudin-3-expressing target cells resulted in IFNγ secretion in all eight experiments performed. Although the actual cytokine levels measured varied between experiments, in all cases, specific cytokine secretion by anti-claudin-3 CAR-T cells in response to that target was at least 100-fold higher than that of anti-claudin-3 CAR-T cells in response to target-negative cells.

[0395] [Table 6]

[0396] Titering of RKO-KO CLDN3-expressing target cells Although the maximum response (complete target cell killing) varied between cell lines, partial target cell killing could also be observed. This may be due to insufficient cytotoxic effector function and may occur when not all tumor cells express the target. To determine differences in maximum responses and confirm that partial killing is achievable with the IncuCyte assay, target cell titration was performed. RKO-KO and RKO-KO hCLDN3 polyclonal cells were mixed at various ratios and incubated with anti-claudin-3 CAR-T cells. No signal was observed when only 2% of RKO-KO cells expressed hCLDN3, but an increased signal was observed with a higher percentage of hCLDN3-expressing cells (Figure 9). These results indicate that partial killing, as evidenced by differences in maximum responses, can be achieved when comparing the same target cell lines.

[0397] The relationship between hCLDN3 expression and CAR-T activation Understanding how CAR-T cells respond to target density may help predict the effects of target expression in other settings. Experiments were conducted to examine the relationship between T cell activation and target expression.

[0398] hCLDN3 expression was tightly controlled by nucleofection of an hCLDN3 knockout (KO) cell line (RKO-KO) with hCLDN3 mRNA (generated in vitro from a linearized plasmid using the mMESSAGE mMACHINE T7 Ultra kit) to create a gradient of hCLDN3 expression (Figure 10A). This allowed us to conduct studies in a well-defined system independent of variables (e.g., other biomarkers) that may affect T cell responses to hCLDN3. After confirming hCLDN3 expression, target cells were cultured with CAR-T cells, and activation responses were assessed by either CD69 expression (measured by flow cytometry as described in previous examples) or cytokine secretion (IFNγ / granzyme B).

[0399] In each experiment, we observed activation specific to anti-claudin-3 CAR-T cells and hCLDN3 expression. This hCLDN3-dependent activation demonstrated a correlation between target expression and IFNγ and granzyme B secretion (Figures 10C and 10D and Table 7). Although statistical analysis was not performed, there was also a clear relationship between the size of the hCLDN3-positive population and CD69 expression, which plateaued at 100% target expression (Figure 10B). Overall, this demonstrates a relationship between target expression and CAR-T activation.

[0400] In the experiments described above, RKO-KO were nucleofected with an optimized gradient to create a target gradient that gradually decreased the hCLDN3-positive population. In a separate experiment, the effect of gradually decreasing hCLDN3 expression across the cell population was examined (Table 8). In this case, the data suggested that gradually increasing hCLDN3 expression resulted in gradually decreasing T cell activation (as indicated by IFNγ secretion). CD69 expression was consistent across each expression level.

[0401] [Table 7]

[0402] Values ​​are reported as fold change in IFNγ levels between anti-claudin-3 and anti-CD19 control CAR-T cells. CL = confidence interval.

[0403] [Table 8]

[0404] Screening of cancer cell lines from three different indications for hCLDN3 expression and T cell activation In the above study, anti-claudin-3 CAR-T cell responses were examined in only one colon cancer cell line expressing various levels of exogenous hCLDN3. Here, we evaluated the anti-claudin-3 CAR-T cell responses to a panel of 31 cancer cell lines from three major indications (colon, pancreatic, and breast cancer) expressing endogenous hCLDN3, including the HT-29-LUC and RKO-KO cell lines as positive and negative controls, respectively. First, cell lines derived from colon cancer (Figure 11A), pancreatic cancer (Figure 11B), and breast cancer (Figure 11C) were screened for hCLDN3 expression by flow cytometry and real-time quantitative PCR (RT-qPCR). Various levels of hCLDN3 expression, ranging from 0% to 100%, were detected in all cell lines (Figures 11A–11C, left). Interestingly, in partially positive cell lines such as SW403 or SW480, a shift in the overall population was observed when cells were incubated with hCLDN3 antibody instead of the well-defined positive and negative populations. When hCLDN3 gene expression levels were quantified by RT-qPCR in the same cell line panel (Figures 11A-11C, center), a similar pattern of CLDN3 mRNA abundance was observed. Using the CRISPR methodology, low levels of hCLDN3 mRNA were also detectable in the negative cell line control (RKO-KO). Comparing the RT-qPCR and flow cytometry data, both assays revealed that HT-29-LUC, T84, and SW403 were the highest claudin-3 expressers, while RKO-KO and COLO320-DM were the lowest expressers.

[0405] The activation of anti-claudin-3 CAR-T cells was also examined after co-culture with select cancer cell lines using anti-CD19 as a negative CAR control (Figures 11A-11C, right). Importantly, all cell lines expressing hCLDN3 activated anti-claudin-3 CAR-T cells, as indicated by high levels of IFNγ, while no response was observed from anti-claudin-3 CAR-T cells co-cultured with RKO-KO. Nevertheless, several cell lines that did not exhibit detectable hCLDN3 expression by flow cytometry, including COLO320-DM (0.068%), DLD-1 (0.347%), HC1954 (0.55%), and BxPC3 (1.95%), were also able to activate anti-claudin-3 CAR-T cells. One possible explanation is the detection limit of the commercially available antibody used.

[0406] All hCLDN3-positive cell lines were able to activate anti-claudin-3, but anti-CD19 control CAR-T cells were not. However, no clear correlation was found between anti-claudin-3 CAR-T activation and hCLDN3 expression levels by flow cytometry or RT-qPCR.

[0407] Killing of cancer cell lines from three indications To investigate the functionality of anti-claudin-3 CAR-T cells against a wide range of tumors, we selected several cancer cell lines with different hCLDN3 expression levels for killing assays. Table 9 summarizes three IncuCyte experiments performed with three donors each. Because the results were highly consistent between donors, Table 9 summarizes the three donors for each experiment. Cell lines from three indications were killed by anti-claudin-3 CAR-T cells, indicating the potential for this CAR to be used in several indications. Complete killing of HT-29-LUC was also confirmed (data not shown). Only one colon cancer line, COLO-320DM, was not killed by anti-claudin-3 CAR-T cells. Three cell lines, HCC1954, BxPC3, and HPAC, were partially killed. Partial killing was confirmed in microscopic images as apoptotic cells or holes in the cell layer, but the resulting signal was extremely low or absent, as confirmed in the raw data (Figure 12).

[0408] [Table 9]

[0409] Quantitation of LNGFR molecules indicates robust CAR expression LNGFR counts ranging from 166,000 to 301,000 were detected. Assuming that CAR expression and LNGFR expression are comparable, we compared normal CD8 + Considering that T cells have 50,000 T cell receptors on their surface, the abundance of hCLDN3 CAR on the T cell surface is estimated to be sufficient for T cell activation.

[0410] Anti-claudin-3 CAR-T cell killing kinetics differ between experiments One aspect of determining the efficacy of CAR-T cells is how quickly cytotoxicity is induced. This was analyzed by determining after how long 50% of the maximum response was achieved. These results varied between experiments, making interpretation difficult. However, in all cases, achieving complete target cell killing is an important aspect for determining CAR efficacy.

[0411] Anti-claudin-3 CAR-T cells specifically kill CLDN3-expressing cells Anti-claudin-3 CAR-T cells are specific for target cells expressing hCLDN3. This evidence comes from experiments in which RKO cells (RKO-KO), in which endogenous hCLDN3 was knocked out, were not killed by anti-claudin-3 CAR-T cells. In contrast, cell lines that showed hCLDN3 expression were killed by anti-claudin-3 CAR-T cells. Furthermore, when RKO-KO cells and RKO-KO cells overexpressing exogenous hCLDN3 were mixed, only partial cytotoxicity was detected, indicating that T cells were specifically activated only by target cells expressing hCLDN3. These results suggest that anti-claudin-3 CAR-T cell activity was specific for hCLDN3.

[0412] Enhanced anti-claudin-3 CAR-T cell activation correlates with increased claudin-3 expression To determine which patient populations may respond to anti-claudin-3 CAR-T cell treatment, it is useful to understand the activation threshold of CARs. As described above, we used a control model to specifically examine the relationship between target expression and T cell activation. The goal was to define the target expression level required to activate anti-claudin-3 CAR-T cells by reducing target expression to a point where it was no longer detectable by flow cytometry.

[0413] IFNγ secretion is an important measure of T cell activation responses, and CD69 is a commonly used activation marker. Quantification of granzyme B, an essential inducer of target cell apoptosis, was also used to provide clear evidence of the relationship between target expression and CAR-T cell cytotoxicity. The upregulation of these indicators after nucleofection of RKO-KO with 1 ng of claudin-3 mRNA (resulting in a 5% positive population) clearly demonstrates the sensitivity of anti-claudin-3 CAR-T cells. Even with low antigen availability, the CAR-T cell response is efficient. The presence of granzyme B in the culture medium suggests that target cell apoptosis has occurred, but this cannot be determined definitively without examining the target cells themselves.

[0414] Due to the detection limit of the antibody, it is difficult to say whether the observed expression represents a small population of claudin-3-expressing cells or a low-expressing, 100% target-positive population. The observed target cell apoptosis could not be correlated with precise quantification of the claudin-3-positive population.

[0415] Although a clear relationship between antigen expression and anti-claudin-3 CAR-T cell activation was observed, a threshold for activation could not be established, primarily due to limitations in antigen detection. Importantly, however, there was a dose-response of anti-claudin-3 CAR-T cells to claudin-3 expression in this artificial system.

[0416] Anti-claudin-3 CAR-T cells were activated when co-cultured with a panel of cell lines from various indications To test the efficacy of anti-claudin-3 CAR-T cells in a broader panel of target cells, we screened 31 cancer cell lines, including colorectal, pancreatic, and breast cancers, for claudin-3 expression at both the mRNA and protein levels. Notably, all cell lines expressing hCLDN3 were able to activate anti-claudin-3 CAR-T cells, expanding the efficacy of this CAR to a broader panel of cancer indications.

[0417] Anti-claudin-3 CAR-T cells have the potential to kill tumor cells from various indications Because activation of anti-claudin-3 CAR-T cells may not result in target cell killing, we tested their cytotoxicity in three tumor indications. Complete or partial killing was observed in most of the target cell lines tested, consistent with IFNγ release by CAR-T cells (Figures 11A-11C). Only the colon cancer line COLO-320DM was not killed by anti-claudin-3 CAR-T cells, likely because it did not express hCLDN3, even though it could partially activate CAR-T cells.

[0418] Anti-claudin-3 CARs were expressed on T cells at levels suggesting they could redirect T cell activity toward hCLDN3-expressing tumor cells. They specifically killed target cells exogenously expressing hCLDN3 while sparing cells depleted of antigen by CRISPR / Cas9 technology. Anti-claudin-3 CAR T cells secreted IFNγ and were able to kill hCLDN3-expressing cancer cell lines derived from colorectal, breast, and pancreatic cancers, although the activation threshold could not be defined due to limitations in the detection reagent. This suggests that CARs can redirect T cells toward several types of cancer. Finally, no clear correlation was found between hCLDN3 expression and IFNγ release levels in these cell lines, likely due to differences in their biological characteristics.

[0419] Example 5: Repeated antigen-dependent stimulation to assess the long-term antitumor activity of anti-claudin-3 CAR-T cells in vitro The objective of this study was to analyze the expression, cytokine secretion, and cytotoxic potency of six anti-claudin-3 CAR T cell constructs based on the same scFv variant. Furthermore, the long-term functionality of anti-claudin-3 CAR T cells was assessed by repeated antigen stimulation.

[0420] material and method Cloning of scFv against claudin-3 into three scaffolds (L, S, XS) of the Miltenyi Biotec CAR spacer library Plasmids encoding scFv variants were generated in two different orientations: heavy chain-light chain (V H -V L ) and light chain-heavy chain (V L -V H The scFvs were cloned into three scaffolds from the Miltenyi Biotec CAR spacer library with different spacer lengths: long spacer (L, hIgG4 H-CH2-CH3), short spacer (S, hCD8), and ultrashort spacer (XS, hIgG4 hinge), to generate six different constructs.

[0421] [Table 10]

[0422] Preparation of PBMCs and isolation of Pan T cells PBMCs were isolated from buffy coats obtained from two healthy donors. T cells were directly isolated using a Pan T cell human isolation kit. Pan T cell isolation was performed using 2 × 10 8 The procedure was performed using 1 × 10 leukocytes according to the manufacturer's protocol. T cells were resuspended in TEXMACS medium containing IL-7 (10 ng / mL), IL-15 (10 ng / mL), and T CELL TRANSACT Human (1:100). 6 The concentration was adjusted to cells / mL.

[0423] Generation and expansion of CAR-T cells 1 × 10 Pan T cells 6T cells were seeded into 24-well plates at a concentration of 1000 cells / mL and 2 mL / well (see above). Transduction was performed 1 day after activation with TRANSACT. Lentiviral vectors were added to T cells at a multiplicity of infection (MOI) of 5. 24–48 hours after transduction, the supernatant from each well was removed and fresh TEXMACS containing IL-7 and IL-15 was added. Depending on the T cell density, T cell cultures were split 1:2 or 1:3 every 2–3 days to obtain 0.5 × 10 cells. 6 ~2×10 6 The concentration was maintained at 100 cells / mL.

[0424] Determination of transduction efficiency (by LNGFR) and expression of CAR Because the generated CAR construct contains LNGFR as a marker gene, transduction efficiency was analyzed via anti-LNGFR staining with anti-LNGFR-PE by flow cytometry (MACSQuant Analyzer 10) as previously described.

[0425] To analyze CAR expression, CAR was stained via the variable light chain (κ chain) using protein L. Cells were resuspended in a buffer containing protein L-biotin (5 μg / 1000 μL) and incubated for 45 minutes at 4°C. After 45 minutes, the cells were washed and resuspended in a buffer containing anti-biotin-PE. Cells were then washed and analyzed by flow cytometry (MACSQuant Analyzer 10).

[0426] Long-term co-culture: INCUCYTE The target cells used in this assay were RKO-KO CLDN3 H1 (human claudin-3 knockout by lentiviral transduction plus introduction of human claudin-3 and GFP marker). T cells were thawed 72 hours before assay setup (see above) and collected in TEXMACS containing IL-7 and IL-15. On the day of the assay, T cells were thoroughly resuspended and pooled under the same conditions (same donor and expressing the same CAR construct). T cells were adjusted for their transduction efficiency, taking into account cell concentration and the frequency of LNGFR-positive T cells, to 4 × 10.4 and 3 × 10 4 A co-culture was set up with 1x10 target cells at an effector:target ratio of 2.5:1. The T cell suspension was added, and the co-culture was incubated in INCUCYTE. Target cell proliferation was monitored via green confluency. On day 3, fresh target cell medium was added. On day 4, fresh target cells were seeded onto a new cell culture plate at the same concentration as the first time, followed by T cell addition approximately 4-5 hours later. T cells from the same conditions were pooled, and the conditioned T cells were added to the freshly seeded target cells at an E:T ratio of 2.5:1. The cell culture plate was incubated in INCUCYTE, and target cell proliferation was monitored via green confluency. On day 7, fresh target cells were added to a new cell culture plate at 3x10 4 The target cells were seeded at a concentration of 100 μg / ml, and T cells were added approximately 4-5 hours later. All T cells from the same conditions were pooled.

[0427] Repeated antigen stimulation: antigen spike-in Target cells RKO-KO CLDN3 H1 (human claudin-3 knockout + human claudin-3 and marker GFP) were co-cultured with T cells. Co-cultures were set up 96 hours after thawing T cells from donors H5 and P and allowed to recover in TEXMACS containing IL-7 and IL-15.

[0428] The following conjugates were used to stain for exhaustion markers, transduction efficiency, CD4, and CD8: LAG3 (CD223)-VioBlue, PD-1 (CD279)-PE-Vio770, TIM3 (CD366)-APC, CD8-APC-Vio770, CD4-VioGreen, LNGFR-PE, and 7-AAD. A master mix of these conjugates was prepared. Cells were resuspended in the master mix and then incubated at 4°C for 10 minutes (in the dark). Cells were resuspended in PEB (CliniMACS containing 0.5% BSA), and samples were measured on a MACSQuant Analyzer 10. The volume of T cell suspension required to reach an E:T ratio of 2:1 based on the transduced T cells was resuspended in the appropriate volume of target cell medium. The T cell suspension was added to the target cells, and the coculture was incubated in a humidified incubator (37°C and 5% CO). Every 24 hours, T cells were stained for exhaustion markers and fresh target cells were added to the co-cultures, with the last addition of fresh target cells on day 3.

[0429] result Staining of T cells to analyze transduction efficiency (by LNGFR) and CAR expression To analyze the transduction efficiency of CAR T cells generated from donor D5 (see above), LNGFR marker gene expression was measured by flow cytometry on day 7. Data obtained from staining indicated that transduction was successful, achieving frequencies of 39% to 50% LNGFR-positive T cells (see Figure 13). Furthermore, to calibrate T cells for their transduction efficiency for functional testing, LNGFR expression was analyzed again on day 15 (see Figure 13). Expression of CAR molecules on the T cell surface is a prerequisite for CAR function. Therefore, CAR expression was determined by protein L staining (see above). The obtained data indicated that all resulting CAR variants were expressed, achieving frequencies of CAR-positive T cell populations ranging from 35% to 43% (see Figure 13). Additionally, CAR-T cells expressing various scFvs against claudin-3 were included as positive controls.

[0430] Luciferase killing assay To evaluate the cytotoxicity of anti-claudin-3 CAR-T cells, a luciferase-based killing assay was performed. The breast cancer cell line T-47D was used for co-culture. This target cell line was engineered to express both luciferase and eGFP by transducing it with a lentiviral vector encoding both markers. T cells expressing various anti-claudin-3 CARs or untransduced T cells were generated, expanded, and cultured without cytokines for 48 hours before co-culture. These T cells were then added to the target cell line at three different effector:target (E:T) ratios, adjusted according to the lowest transduction efficiency (LNGFR-positive cell frequency), namely, 5:1, 1:1, and 0.2:1. The co-cultures were incubated (humidified, 37°C, 5% CO2). The supernatant was removed 20 hours after co-culture setup and stored until cytokines were measured using the MACSPlex assay. D-luciferin solution was added to the cells, and after 5 minutes of incubation, luminescence was read with a luminometer (VICTOR, PerkinElmer).

[0431] CAR T cells expressing various scFvs against claudin-3 were used as positive controls. The graph (Figure 14) shows the frequency of target cells killed after 20 hours of coculture. No increase in the frequency of target cells was observed when cocultured with untransduced T cells. T cells expressing various anti-claudin-3 CAR constructs cocultured with T-47D cells expressing human claudin-3 showed increased frequency of target cells killed and cytotoxicity depending on the E:T ratio. At an E:T ratio of 5:1, the frequency of target cells killed ranged from 96% to 99.7%, and the frequency of target cells killed decreased with lower E:T ratios. Furthermore, the frequency of target cells killed was similar among the seven CAR constructs tested.

[0432] Determination of cytokine secretion in the supernatant by MACSPlex assay Coculture supernatants (see above) were analyzed for concentrations of the cytokines IL-2, IFNγ, and TNF-α using the MACSPlex assay. Only samples from cocultures with an E:T ratio of 1:1 were analyzed. Samples and MACSPlex assays were prepared according to the manufacturer's protocol for the MACSPlex Cytokine 12 Kit (human) and analyzed on a MACSQuant Analyzer 10. The resulting concentrations are expressed in pg / mL (Figures 15A–15C). Supernatants used for analysis were undiluted. No elevated levels of IL-2, IFNγ, or TNF-α were detected in supernatants from conditions in which untransduced T cells were cocultured with the cancer cell line T-47D. Furthermore, no cytokines were detectable in samples from the "target cell alone" condition. CAR T cells expressing various anti-claudin-3 CARs based on the 906 variant were cocultured with T-47D cells in all conditions, demonstrating elevated levels of IL-2 and IFNγ compared to mock T cells. In the presence of T-47D, the highest amounts of IL-2 were secreted by 906_004, 906_009, and the positive control claudin-3 CAR. These constructs, including 906_005, also secreted the highest amounts of IFN-γ. Only low levels of TNF-α were detected, with the highest concentrations detected in sample 906_009 and the positive control claudin-3 CAR.

[0433] Long-term co-culture of T cells with repeated antigen stimulation Target cell proliferation in coculture with T cells expressing the 906_002 (long spacer), 906_004 (short spacer), or 906_005 (ultrashort spacer) CAR variants was monitored using INCUCYTE. Results showed that for both donors (G5 and H5), target cells were efficiently eliminated by all three CAR constructs at the first target cell exposure (Figures 16A and 16D). In contrast, proliferation was observed in the control, where target cells RKO-KO CLDN3 H1 were cultured alone. Differences between CAR variants became apparent after CAR T cells were transferred to fresh target cells for the second and third target cell exposures. T cells expressing CAR variants with long spacers were less efficient at controlling target cell pr...

Claims

1. The following domains: a) an extracellular domain comprising a claudin-3 binding domain comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (CDRH1) sequence of SEQ ID NO: 1; the heavy chain complementarity determining region 2 (CDRH2) sequence of SEQ ID NO: 2; and the heavy chain complementarity determining region 3 (CDRH3) sequence of SEQ ID NO: 3; b) a transmembrane domain; and c) A chimeric antigen receptor comprising a polypeptide comprising one or more intracellular signaling domains.

2. The chimeric antigen receptor of claim 1, wherein the extracellular domain further comprises a claudin-3 binding domain comprising a light chain variable region (VL) comprising: a light chain complementarity determining region 1 (CDRL1) sequence of SEQ ID NO: 4; a light chain complementarity determining region 2 (CDRL2) sequence of SEQ ID NO: 5; and a light chain complementarity determining region 3 (CDRL3) sequence of SEQ ID NO:

6.

3. The chimeric antigen receptor of claim 2, wherein the VL is located at the N-terminus of the VH, or the VH is located at the N-terminus of the VL.

4. The chimeric antigen receptor of claim 2, wherein the VL and the VH are directly fused to each other via a peptide bond or are linked to each other via a peptide linker.

5. The chimeric antigen receptor of claim 1, wherein the claudin-3 binding domain is selected from the group consisting of Camel Ig, Ig NAR, Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single chain variable fragment (scFv), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide stabilized Fv protein (dsFv) and single domain antibody (sdAb).

6. The chimeric antigen receptor of claim 1, wherein the VH comprises an amino acid sequence having at least 90% or 95% sequence identity to SEQ ID NO:7, or an amino acid sequence having one, two, three, four or five amino acid substitutions, insertions or deletions relative to SEQ ID NO:7, and / or the VL comprises an amino acid sequence having at least 90% or 95% sequence identity to SEQ ID NO:8, or an amino acid sequence having one, two, three, four or five amino acid substitutions, insertions or deletions relative to SEQ ID NO:

8.

7. The chimeric antigen receptor of claim 1, wherein the VH comprises the amino acid sequence of SEQ ID NO: 7 and / or the VL comprises the amino acid sequence of SEQ ID NO:

8.

8. 2. The chimeric antigen receptor of claim 1, wherein the extracellular domain comprises an amino acid sequence having at least 90% or 95% sequence identity to SEQ ID NO: 11, or an amino acid sequence having one, two, three, four or five amino acid substitutions, insertions or deletions relative to SEQ ID NO: 11; or wherein the extracellular domain comprises an amino acid sequence having at least 90% or 95% sequence identity to SEQ ID NO: 18, or an amino acid sequence having one, two, three, four or five amino acid substitutions, insertions or deletions relative to SEQ ID NO:

18.

9. 2. The chimeric antigen receptor of claim 1, wherein the transmembrane domain is derived from a polypeptide selected from the group consisting of the alpha or beta chain of the T cell receptor, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137 (4-1BB), CD152, CD154, CD278 (ICOS), and PD1.

10. The chimeric antigen receptor of claim 9, wherein the transmembrane domain is derived from CD8α.

11. The chimeric antigen receptor of claim 1, wherein the one or more intracellular signaling domains are derived from an intracellular signaling molecule selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3ε, CD3δ, CD3ζ, CD22, CD66d, CD79a and CD79b, or the one or more intracellular signaling domains are CD3ζ.

12. The chimeric antigen receptor of claim 1, further comprising a costimulatory signaling domain.

13. 13. The chimeric antigen receptor of claim 12, wherein the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TRIM, and ZAP70.

14. The following domains: a) an extracellular domain comprising a claudin-3 binding protein comprising a CDRH1 sequence of SEQ ID NO:1; a CDRH2 sequence of SEQ ID NO:2; a CDRH3 sequence of SEQ ID NO:3; a CDRL1 sequence of SEQ ID NO:4; a CDRL2 sequence of SEQ ID NO:5; and a CDRL3 sequence of SEQ ID NO:6; b) a transmembrane domain derived from CD8α; c) a costimulatory domain derived from CD137 (4-1BB); and d) A chimeric antigen receptor comprising an intracellular signaling domain derived from CD3ζ.

15. 2. The chimeric antigen receptor of claim 1, wherein the chimeric antigen receptor comprises an amino acid sequence having at least 90% or 95% sequence identity to SEQ ID NO: 12, 34, 35, 36, 37, 38 or 39, or an amino acid sequence having one, two, three, four or five amino acid substitutions, insertions or deletions relative to SEQ ID NO: 12, 34, 35, 36, 37, 38 or 39, or wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 12, 34, 35, 36, 37, 38 or 39.

16. A chimeric antigen receptor that competes with the chimeric antigen receptor of claim 1 for binding.

17. 2. An engineered immune effector cell comprising the chimeric antigen receptor of claim 1.

18. 18. The immune effector cell of claim 17, wherein the immune effector cell is selected from the group consisting of T cells, cytotoxic T lymphocytes, natural killer T lymphocytes, macrophages and natural killer cells.

19. The immune effector cell of claim 17, further comprising an ablation element.

20. 20. The immune effector cell of claim 19, wherein the ablation element is derived from a polypeptide selected from the group consisting of a truncated human EGFR polypeptide and CD20, or wherein the ablation element is CD20.

21. A polynucleotide encoding the chimeric antigen receptor of claim 1.

22. A vector comprising the polynucleotide of claim 21.

23. 23. The vector of claim 22, wherein the vector is selected from the group consisting of a retroviral vector, a lentiviral vector, an adeno-associated virus (AAV) vector, human immunodeficiency virus I (HIV-I), human immunodeficiency virus 2 (HIV-2), Visna-Maedi virus (VMV), Caprine Arthritis-Encephalitis Virus (CAEV), Equine Infectious Anemia Virus (EIAV), Feline Immunodeficiency Virus (FIV), Bovine Immunodeficiency Virus (BIV), and Simian Immunodeficiency Virus.

24. 20. A pharmaceutical composition comprising the engineered immune effector cells of claim 17 and a pharma- ceutically acceptable excipient.

25. The pharmaceutical composition of claim 24 for use in treating cancer.

26. 26. The pharmaceutical composition of claim 25, wherein the cancer is a solid cancer, an epithelial cancer, a colon cancer, a pancreatic cancer, a breast cancer, a triple-negative breast cancer (TNBC), an ovarian cancer, a lung cancer, a non-small cell lung cancer (NSCLC) or a prostate cancer.

27. The pharmaceutical composition of claim 26, wherein the cancer is colon cancer.

28. A claudin-3 binding protein comprising a heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (CDRH1) sequence of SEQ ID NO: 1; the heavy chain complementarity determining region 2 (CDRH2) sequence of SEQ ID NO: 2; and the heavy chain complementarity determining region 3 (CDRH3) sequence of SEQ ID NO: 3, and a light chain variable region (VL) comprising the light chain complementarity determining region 1 (CDRL1) sequence of SEQ ID NO: 4; the light chain complementarity determining region 2 (CDRL2) sequence of SEQ ID NO: 5; and the light chain complementarity determining region 3 (CDRL3) sequence of SEQ ID NO:

6.

29. A polynucleotide encoding the claudin-3 binding protein of claim 28.

30. A vector comprising the polynucleotide of claim 29.

31. A pharmaceutical composition comprising the claudin-3 binding protein of claim 28 and a pharma- ceutical acceptable excipient.

32. The pharmaceutical composition of claim 31 for use in treating cancer.

33. An isolated claudin-3 binding protein that binds to a discontinuous epitope of human claudin-3 comprising at least N38 and E153 of SEQ ID NO:

13.

34. The following domains: a) an extracellular domain comprising the isolated claudin-3 binding protein of claim 33; b) a transmembrane domain; and c) one or more intracellular signaling domains A chimeric antigen receptor comprising a polypeptide comprising:

35. 35. An engineered immune effector cell comprising the chimeric antigen receptor of claim 34.

36. 34. A pharmaceutical composition comprising the isolated claudin-3 binding protein of claim 33 and a pharma- ceutically acceptable excipient.

37. The pharmaceutical composition of claim 36 for use in treating cancer.

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