Enhancing cytotoxicity of engineered immune effector cells by additional synapse stabilization receptors

Abstract

Aspects of the present disclosure include synapse-stabilizing receptors comprising a LAT domain that can be used in combination with a CAR, TCR, or BiTE. Another aspect of the present disclosure is immune cells comprising synapse-stabilizing receptors comprising a LAT domain that can be used in combination with a CAR, TCR, or BiTE, and methods of their use in treating a disease (e.g., cancer).

Description

[0001] Priority requirements

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 594,363, filed October 30, 2023, which is incorporated herein by reference in its entirety. Technical Field

[0003] This disclosure generally relates to the field of cancer treatment. More specifically, it relates to cellular immunotherapy using chimeric antigen receptors (CARs), T-cell receptors (TCRs), and connector-redirected immune effectors, and methods of using them. Background Technology

[0004] Chimeric antigen receptor (CAR) cell therapy is often considered an effective solution for relapsed or refractory tumors, particularly hematologic malignancies; however, such therapies may be resistant or ineffective against cancer cells that express low levels of the antigen. Antigen modulation is a major pathway leading to tumor resistance to targeted immunotherapy. CD19.CAR T-cells are frequently grown from CD19-negative B-cell leukemia clones, and relapse following CAR T-cell therapy targeting CD22 or BCMA is associated with partial downregulation of the target antigen. While this bottleneck can be overcome in B-cell malignancies by co-targeting multiple B-lineage antigens, extending this approach to non-B-cell tumors is challenging due to the often-absent surface antigens not present in key healthy tissues. One example of this limitation is acute myeloid leukemia (AML), which comprises dozens of different diseases and exhibits high inter- and even intra-patient heterogeneity in the expression of targetable antigens. Most of these antigens are present in peripheral blood cells as well as key bone marrow progenitor cells, complicating combined antigen targeting and increasing the risk of extratumor toxicity. Solid tumors are another example of diseases with high antigenic heterogeneity and resistance to conventional cell immunotherapy.

[0005] Besides CARs, T-cell receptor (TCR)-mediated target cell recognition can be ineffective due to low antigen or HLA expression. Furthermore, some TCRs (such as survivin-specific TCRs (Arber et al., JCI 2015)) may have low affinity for target peptide-HLA complexes to avoid extratumor toxicity. T cells expressing these TCRs will not produce sufficient cytotoxicity against cancer cells with suboptimal levels of peptide and HLA expression, thus limiting their therapeutic benefit. Therefore, selectively enhancing the activity of these TCR-expressing T cells against tumor cells would enhance their clinical activity and therapeutic benefit.

[0006] Bispecific T-cell adaptors (BiTEs) recognize target antigens and cross-link T-cell receptors, triggering the lysis of target cells. If the target antigen is expressed at low levels, BiTE-mediated T-cell activation is often insufficient to lyse tumor cells. Stabilizing the interaction between BiTE-promoted T cells and target cells can enhance tumor lysis.

[0007] While immunotherapy has yielded impressive results in some cancers, setbacks such as high relapse rates and drug resistance are common. There is a need to discover engineered components that enhance CAR-T and TCR-T cells targeting cancer cells with low antigen expression, as well as BiTE therapies, to help overcome drug resistance and low efficacy. Such components would enable more effective therapeutic uses and further improve patient outcomes. Summary of the Invention

[0008] This disclosure relates to methods and compositions relating to peptides comprising an antigen receptor domain, a hinge, a transmembrane domain, and a modified LAT domain. The methods and compositions may further comprise an antigen receptor for immune cell activation (AgRICA) having an antigen-binding domain, wherein the antigen-binding domain and the antigen receptor domain bind different antigens. The AgRICA may be a CAR, TCR, or BiTE.

[0009] One embodiment of this disclosure is a composition comprising a polypeptide comprising an antigen receptor domain, a hinge, a transmembrane domain, and a modified LAT domain. The composition may further comprise a polypeptide comprising AgRICA having a second antigen receptor domain, wherein the second antigen receptor domain and the antigen receptor bind to different antigens. In some specific embodiments, the LAT domain has at least 90%, 95%, 97%, 98%, or 99% sequence similarity to SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, the LAT domain is SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, the antigen receptor domain is anti-CD38 or anti-EGFR. In a specific implementation scheme, the second antigen receptor domain binds to 5T4, 8H9, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR, EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, folate receptor-α, FAP, and FB. P, fetal AchR, FR, GD2, G250 / CAIX, GD3, phosphatidylinositol proteoglycan-3 (GPC3), Her2, IL-13Rα2, Lambda, Lewis-Y, Kappa, KDR, MAGE, MCSP, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, survivin, TAG72, TEMs, carcinoembryonic antigen, HMW-MAA, AFP, CA-125, ETA, tyrosinase, MAGE, laminin receptor, HPV E6, E7, BING-4, calcium-activated chloride channel 2, cyclin B1, 9D7, EphA3, telomerase, SAP-1, B melanoma antigen, cancer / testis antigen, melanoma-associated antigen, sarcoma antigen, CT antigen, NY-ESO-1 / LAGE-1, SSX-2, Melan-A / MART-1, GP100 / pmel17, TRP-1 / -2, P. peptide, MC1R, prostate-specific antigen, β-catenin, BRCA1 / 2, CML66, fibronectin, MART-2, TGF-βRII, CLL1, TdT, or VEGF. In the implementation scheme, the transmembrane domain or extracellular domain of the CAR or TCR does not dimerize with a similar domain of the SSR.The AgRICA can be CAR, TCR, or BiTE. In a specific embodiment, the hinge domain of the CAR is CD8a, and the hinge domain of the SSR is CD8a with a C164S mutation, which excludes heterodimerization with the CAR.

[0010] Another general embodiment of this disclosure is a composition comprising a polynucleotide encoding a polypeptide comprising an antigen receptor domain, a transmembrane domain, and a modified LAT domain. The composition may further comprise a second polynucleotide encoding a second polypeptide comprising AgRICA having a second antigen receptor domain, wherein the second antigen receptor domain does not bind the same antigen as the antigen receptor domain. The polynucleotide and the second polynucleotide may be contained on one or more vectors. In a specific embodiment, the LAT domain has at least 90%, 95%, 97%, 98%, or 99% sequence similarity to SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, the antigen receptor domain is anti-CD38 or anti-EGFR. In some specific embodiments, the second antigen receptor domain binds to 5T4, 8H9, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR, EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, folate receptor-α, FAP, and F. BP, fetal AchR, FR, GD2, G250 / CAIX, GD3, phosphatidylinositol proteoglycan-3 (GPC3), Her2, IL-13Rα2, Lambda, Lewis-Y, Kappa, KDR, MAGE, MCSP, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, survivin, TAG72, TEMs, carcinoembryonic antigen, HMW-MAA, AFP, CA-125, ETA, tyrosinase, MAGE, laminin receptor, HPV E6, E7, BING-4, calcium-activated chloride channel 2, cyclin B1, 9D7, EphA3, telomerase, SAP-1, B melanoma antigen, cancer / testis antigen, melanoma-associated antigen, sarcoma antigen, CT antigen, NY-ESO-1 / LAGE-1, SSX-2, Melan-A / MART-1, GP100 / pmel17, TRP-1 / -2, P. polypeptide, MC1R, prostate-specific antigen, β-catenin, BRCA1 / 2, CML66, fibronectin, MART-2, TGF-βRII, CLL1, TdT or VEGF.In some embodiments, the transmembrane domains or extracellular domains of the CAR or TCR do not dimerize with themselves or with other transmembrane domains. The AgRICA can be a CAR, TCR, or BiTE. In some specific embodiments, the transmembrane domain of the CAR or TCR is CD8a, and the transmembrane domain contains a C164S mutation.

[0011] Embodiments of this disclosure are cells or cells comprising a polypeptide, said polypeptide comprising an antigen receptor domain, a hinge, a transmembrane domain, and a modified LAT domain. The cells or cells may additionally comprise a second polypeptide comprising an AgRICA having a second antigen receptor domain, such as CAR, TCR, or BiTE, wherein said second antigen receptor domain and said antigen receptor domain bind different antigens. Another general embodiment of this disclosure is cells or cells comprising a polynucleotide encoding a polypeptide, said polypeptide comprising an antigen receptor domain, a transmembrane domain, and a modified LAT domain. The composition may also comprise a second polynucleotide encoding a second polypeptide comprising an AgRICA having a second antigen receptor domain, wherein said second antigen receptor domain does not bind the same antigen as said antigen receptor domain. The polynucleotide and the second polynucleotide may be contained on one or more vectors. In embodiments, said cells or cells are immune effector cells. In some specific embodiments, the immune effector cells (one or more) are regulatory T cells, CD4+ T cells, CD8+ T cells, αβ T cells, γ-δ T cells, NK cells, invariant NKT cells, NKT cells, innate lymphocytes, B cells, dendritic cells, macrophages, cytotoxic T cells, MAIT cells, virus-specific T cells, or mixtures thereof. In some embodiments, the immune effector cells are T cells. Multiple cells may be lyophilized or frozen.

[0012] One embodiment of this disclosure is a method for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of a plurality of cells comprising a polypeptide, the polypeptide comprising an antigen receptor domain, a hinge, a transmembrane domain, and a modified LAT domain. The plurality of cells may additionally comprise a second polypeptide comprising an AgRICA having a second antigen receptor domain, such as CAR, TCR, or BiTE, wherein the second antigen receptor domain and the antigen receptor bind to different antigens. The cancer may be a hematologic malignancy or a solid tumor. In some embodiments, the method further comprises administering at least a second therapeutic agent to the subject. The second therapeutic agent may include chemotherapy, immunotherapy, surgery, radiation therapy, drug therapy, targeted therapy, hormone therapy, biological therapy, or combinations thereof.

[0013] Other objects, features, and advantages of the invention will become apparent from the following detailed description. However, it should be understood that while the detailed description and specific examples point to preferred embodiments of the invention, they are given by way of illustration only, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Attached Figure Description

[0014] The following figures form part of this specification and are included to further illustrate certain aspects of this disclosure. A better understanding of this disclosure can be achieved by referring to one or more of these figures in conjunction with the detailed description of the specific embodiments presented herein.

[0015] Figure 1 The concept and an example mechanism of action of the synaptic stable receptor (SSR) are illustrated.

[0016] Figure 2A -E indicates the expression of CD38 SSR in CLL1 CAR T cells. Figure 2A The configuration of the CLL1 CAR and CD38 SSR is shown, which is designed with a CD38-specific scFv, followed by a CD8α hinge and transmembrane region (TM) fused with the intracellular signal transduction domain. Cysteine ​​164 in CD8α is replaced with serine (C164S) to prevent heterodimerization with the CLL1 CAR, and IRES-NGFR is integrated for SSR detection. Figure 2B This is a representative flow cytometry plot showing the co-expression of CLL1 CAR and CD38 SSR with a specified intracellular domain or a truncated form (TC) of CD38 SSR without an intracellular domain on day 7 after cotransduction (TD). Figure 2C The expansion of CD38 SSR-armed CLL1 CAR T cells was demonstrated by comparison with CAR T cells and untransduced cells (NT). Figure 2D and 2E The CD4 (CD45RA and CCR7 staining was shown on day 7 after co-transduction.) Figure 2E ) and CD8 ( Figure 2D T cell phenotypes. Naïve T cells (TN, CD45RA+CCR7+), central memory T cells (TCM, CD45RA-CCR7+), effector memory T cells (TEM, CD45RA-CCR7-), and TEMRA (CD45RA+CCR7-). Subsets are shown as bar charts, including four donors from two independent experiments. P Values ​​were determined using one-way ANOVA and Tukey multiple comparisons. The bar chart shows the mean + SEM (mean + mean). P<0.05, P<0.01, P<0.001).

[0017] Figure 3A -I shows that CD38 SSR-LAT177 improves CAR-mediated specific killing of low-antigen leukemia cells. Figure 3A B and B are indicators of CLL1 in the specified leukemia cell line. Figure 3A ) and CD38 ( Figure 3B A horizontal histogram. Figure 3C -F represents the difference between SSR-armed CLL1 CAR T cells and CLL1-low AML cells (Molm13). Figure 3C and 3D ) or CLL1 negative cells CCRF-CEM ( Figure 3E and Figure 3F Residual tumor counts after 3 days of culture at a low E:T ratio of 1:4 were plotted, and the fold change in T cell expansion during co-culture was plotted against the T cell count normalized to day 0. A subset is shown as a bar chart, including four donors from two independent experiments. Figure 3G -I is a histogram showing the levels of CLL1 and CD38 in THP1 with low CLL1 before and after sorting. Figure 3G ), and the residual tumor count and T cell expansion fold after 3 days of culture at an E:T ratio of 1:4 (). Figure 3H -I). P The values ​​were determined using one-way ANOVA and Tukey correction for multiple comparisons, and in ( Figure 3D In this study, a paired Student t-test was used to determine the difference between CAR and CAR+SSR-LAT177. The bar chart shows the mean + SEM (…). P<0.05, P<0.01, P < 0.001 (ns, not significant).

[0018] Figure 4A -F showed that SSR-LAT177 improved cytokine production and degranulation of CAR T cells. Compared with CLL1CAR T cells alone, it improved cytokine production and degranulation of target cells Molm13 ( Figure 4A and 4C ) or not with target cells Molm13 ( Figure 4B and 4D After co-culturing for 4 hours at an E:T ratio of 1:1, IFN-γ-positive T cells in SSR-armed CAR T cells ( Figure 4A and B), IL2-positive T cells ( Figure 4C and 4D) and granzyme B / CD107a double-positive T cells ( Figure 4E The percentage of F). P Values ​​were determined using one-way ANOVA and Tukey correction for multiple comparisons. The bar chart shows the mean + SEM (mean + mean). P<0.05, P<0.01, P < 0.001 (ns, not significant).

[0019] Figure 5 AI showed that CAR T cells with CD38 SSR-LAT177 inhibited the development of systemic leukemia and prolonged the survival of AML mice. Figure 5 A shows a schematic timeline of the experiment. Figure 5 B shows the monitoring of leukemia progression in peripheral blood by flow cytometry (hCD45+GFP+). Figure 5 C shows the overall survival of mice in the Molm13 AML model, NT, n=6, CAR, n=6, and CAR.SSR (LAT177), n=5. Statistical significance was determined by the Log-Rank test. (D) Absolute CAR T cell counts (hCD45+hCD3+CAR+) in the blood at specified days after CAR T cell infusion and AUC from day 0 to day 14. Figure 5 E represents the frequency of the initial cell population (CD45RA+CD62L+) in the T cells infused on day 19 post-infusion. Figure 5 F is a schematic timeline for the GFP.FFluc model with low THP1-CLL1. Figure 5 G is the progression of systemic leukemia quantified by bioluminescence (BLI) via IVIS at a specified time point. Figure 5 H is the average AUC from day 0 to day 47. Figure 5 I shows the overall survival of mice in the THP1-CLL low model, with n=5 in each group. Statistical significance was determined by the Log-Rank test. P Values ​​were obtained using one-way ANOVA and Tukey correction for multiple comparisons. Figure 5 D) Determined, or in Figure 5 E and Figure 5 The unpaired Student's t-test in H is used to determine the differences between the two groups. The bar chart shows the mean + SEM (mean + mean + SEM). P<0.05, P<0.01, P<0.001).

[0020] Figure 6 AD showed that SSR co-expression did not produce toxicity against CD38+ hematopoietic cells. Figure 6 A is a representative flow cytometry plot showing the expression of CD38 and CLL1 in PBMCs including CD14+ monocytes, CD3+ T cells, CD3-CD56+ NK cells and CD19+ B cells. Figure 6 B and Figure 6 C represents residual PBMCs after 24 hours of co-culturing with autologous and allogeneic T cells at E:T ratios of 1:4 and 1:1, respectively. Figure 6 B) or purified NK cells ( Figure 6 C). The subset is displayed as a bar chart, including 3 donors. Figure 6 D shows the quantification of burst erythroid formation units (BFU-E) and granulocyte / macrophage colony-forming units (CFU-GM) from hematopoietic stem cells / progenitor cells after co-culturing designated CAR T cells with CD34+ umbilical cord blood cells at an E:T ratio of 10:1 for 5 hours and expanding in semi-solid methylcellulose for 12 days. P Values ​​were determined using one-way ANOVA and Tukey correction for multiple comparisons. The bar chart shows the mean + SEM (mean + mean). P<0.05, P<0.01, P < 0.001 (ns, not significant).

[0021] Figure 7 AC demonstrated the induction of CAR-independent cross-linking apoptosis in leukemia cells via SSR binding to CD38. Figure 7 Figure A shows CAR-independent apoptosis induced by CD38-CD38 SSR crosslinking. Figure 7 B is a representative flow cytometry plot and Figure 7 C is a representative bar chart showing the increase in early apoptosis (7-AAD-annexin V+) and late apoptosis (7-AAD+annexin V+) populations after co-culturing CD38-overexpressing leukemia cells and CD38 knockout (KO)-derived cells with SSR-TC T cells at E:T ratios of 1:1 to 4:1 for 24 hours. Data were generated from two individual donors. The bar chart shows the mean + SEM.

[0022] Figure 8 AC demonstrated protection of CAR T cells from daratumumab-induced toxicity by masking CD38 via SSR. Figure 8 Figure A illustrates how CAR T cells are protected from the CD38 monoclonal antibody daratumumab by masking CD38 via SSR. Figure 8 B is a representative flow cytometry and dot plot showing the detection of CD38 in SSR-armed CLL1 CAR T cells compared to unarmed CLL CAR-T cells. Figure 8 C is a schematic model of antibody-dependent cell-mediated cytotoxicity (ADCC) established by co-culturing purified NK cells with CD38 SSR CLL1 CAR T cells at an E:T ratio of 3:1 in the presence of 10 µg / ml daratumumab, allotype control IgG1, and untreated control (Un). The bar chart shows the percentage of apoptotic cells 4 hours after treatment. The bar chart shows the mean + SEM (…). P<0.05, P < 0.01 (paired t-test).

[0023] Figure 9 AK demonstrated that CD38 SSR improves the cytolytic effect of survivin-specific T cells on leukemia cells. Figure 9 Aa is a schematic diagram showing the enhanced cytotoxicity of survivin-specific TCRs (Sur-TCRs) via CD38.SSR. Figure 9 B is a representative flow cytometry plot showing the co-expression of Sur-TCR and SSR on day 7 after transduction (TD), and Figure 9 C represents the expansion of TCR T cells derived from both HLA-A2- (n=3) and HLA-A2+ donors (n=3) on day 10. Figure 9 D is a histogram showing the levels of intracellular survivin, surface HLA-A2, and CD38 in leukemia cell lines BV173, THP1, and TALL1. Figure 9 E shows the residual tumor count (n=3-6) after 3 days of co-culturing with Sur-TCR T cells at an E:T ratio of 1:4. After 4 hours of co-culturing with designated leukemia cells at an E:T ratio of 1:1... Figure 9 The percentage of F cytokine-positive T cells and Figure 9 Percentage of G-granzyme B / CD107a double-positive T cells. Subsets are shown as bar charts with 3-6 donors. P Values ​​were determined using one-way ANOVA and Tukey correction for multiple comparisons. Figure 9 H is a schematic timeline for in vivo experiments. In Figure 9 In Figure I, total tumor progression was measured by IVIS imaging, and the mean area under the curve (AUC) from day 0 to day 36 is plotted on the right. Figure 9 J shows the quantitative distribution of circulating leukemia cells and T cells in peripheral blood by flow cytometry. P Values ​​were determined using an unpaired Student's t-test. The bar chart shows the mean + SEM (mean + mean). P<0.05, P<0.01, P<0.001). Figure 9K shows the Kaplan-Meier curves, which represent the overall survival for each experimental group. Tumor only, n=5; TCR, n=6; and TCR+SSR (LAT177), n=6. Statistical significance was determined by the Log-Rank test.

[0024] Figure 10 AB demonstrated that SSR-LAT177 enhances the cytotoxicity of redirected T cells targeting TdT+ leukemia via cTCR or BiTE. Figure 11A Residual live tumor cells were observed after co-culturing with unarmed or SSR-armed T cells expressing TdT-specific cTCR B1. Figure 10 B shows the cytotoxicity of unarmed (NT) or SSR-expressing T cells against T-ALL cell lines TALL-1 and P12-ICHIKAWA in the presence of soluble TdT BiTE.

[0025] Figure 11A -D indicates that truncation of LAT after 177 amino acids improves SSR function and T cell phenotype. Figure 11A An exemplary construction of a CD38 SSR with truncated (aa 28-177) and full-length (aa 28-233) LATs is shown, in which phosphorylation sites for recruiting PLCγ-1, GADS and Grb2 are shown. Figure 11B This is a diagram showing the expansion of SSR-armed CLL1 CAR T cells, and Figure 11C The CD4 and CD8 T cell phenotypes, characterized by CD45RA and CD62L staining, are shown on day 7 post-co-transduction. Naïve T cells (TN, CD45RA+CD62L+), central memory T cells (TCM, CD45RA-CD62L+), effector memory T cells (TEM, CD45RA-CD62L-), and TEMRA (CD45RA+CD62L-). Figure 11D This shows the residual tumor count of Molm13 or CCRF-CEM cells after 3 days of culture with SSR T cells at a low E:T ratio of 1:4, compared to untransduced (NT) T cells. P Values ​​were determined using one-way ANOVA and Tukey correction for multiple comparisons. The bar chart shows the mean + SEM (mean + mean). P<0.05, P<0.01, P < 0.001 (ns, not significant). Detailed Implementation

[0026] II. Definition

[0027] As used herein, "substantially free of" with respect to the specified component means that the specified component is not intentionally formulated into the composition and / or is present only as a contaminant or in trace amounts. Therefore, the total amount of the specified component resulting from any accidental contamination of the composition is well below 0.05%, preferably below 0.01%. Most preferably, the composition in which no amount of the specified component is detectable using standard analytical methods is preferred.

[0028] As used herein, “an” or “a” may mean one (a type) or a plurality (multiple types). As used herein in the claims, when used in conjunction with the word “comprising,” the word “an” or “a” may mean one (a type) or more than one (more than one type).

[0029] The use of the term "or" in the claims is intended to mean "and / or" unless explicitly indicated as referring only to alternatives or that the alternatives are mutually exclusive, although this disclosure supports the definition of "and / or" referring only to alternatives. As used herein, "another" may mean at least a second or more. The terms "about," "substantially," and "approximately" generally mean a specified value ±5%.

[0030] "Treatment" or "management" of a disease or condition refers to the implementation of a protocol that may include administering one or more medications to a patient in an effort to reduce the signs or symptoms of the disease. Ideal therapeutic effects include slowing the rate of disease progression, improving or reducing the disease state, and alleviating or improving prognosis. Relief may occur before or after the signs or symptoms of the disease or condition appear. Therefore, "treatment" or "management" can include "prevention" or "avoidance" of the disease or adverse condition. Furthermore, "treatment" or "management" does not need to completely reduce the signs or symptoms, does not need to cure the condition, and particularly includes protocols that have only a marginal effect on the patient.

[0031] The terms “treatment benefit” or “treatment effectiveness” as used throughout this application refer to any manner that promotes or enhances the health of a subject in relation to the medical treatment of the condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of the disease. For example, cancer treatment may involve, for instance, a reduction in tumor size, a reduction in tumor invasiveness, a reduction in the rate of cancer growth, or prevention of metastasis. Cancer treatment may also refer to prolonging the survival of a subject with cancer.

[0032] "Subject" and "patient" refer to humans or non-humans, such as primates, mammals, and vertebrates. In some specific implementations, the subject is a human.

[0033] As used in this article, “SSR” or “synapse-stabilizing receptor” refers to a polypeptide that contains at least one extracellular receptor domain, a transmembrane domain, and an intracellular scaffold domain.

[0034] As used herein, "CAR" or "chimeric antigen receptor" refers to an engineered polypeptide that contains at least one extracellular antigen receptor domain, a transmembrane domain, and an intracellular signal transduction domain. Once the target binds to the extracellular receptor domain, the intracellular signal transduction domain triggers effector T cell function, including enhanced proliferation, cytokine release, cytotoxicity, or a combination thereof.

[0035] As used herein, “TCR” or “T cell receptor” refers to an engineered polypeptide containing at least an extracellular receptor domain that targets intracellular antigens presented on MHC molecules.

[0036] As used herein, "Antigen Receptor for Immune Cell Activation (AgRICA)" refers to an engineered peptide containing an antigen-binding domain that targets an antigen on the exterior of a target cell and leads to the activation of immune effector cells. AgRICA may include transmembrane domains and intracellular signaling domains, as seen in CARs and TCRs. AgRICA can also be a BiTE peptide that creates a link between a target cell containing the targeted antigen and an immune effector cell, leading to the activation of the immune effector cell. In other words, the antigen conjugate can be a receptor or an adaptor, as long as binding to the target cell antigen results in the activation of the immune effector cell. CARs, TCRs, and BiTE peptides are all AgRICAs.

[0037] "AgRICA immune cells" refer to immune effector cells, such as T cells, that express AgRICA. For example, AgRICA immune cells may contain AgRICA with extracellular receptors, transmembrane domains, and intracellular signal transduction domains, or AgRICA immune cells may secrete, for example, BiTE.

[0038] As used in this article, "BiTE" refers to a bispecific T-cell connector that simultaneously binds to target antigens on tumor cells and TCRs on T cells, triggering T-cell effector functions. BiTEs can be recombinant or secreted by engineered cells.

[0039] As used herein, “target cell” refers to a cell that contains an antigen on its outer surface, wherein immune effector cells can bind the antigen and trigger cell death in the target cell.

[0040] As used in this article, a "scaffold domain" refers to an intracellular domain that stabilizes the signal transduction function of other polypeptides within the cell but does not contain any signal transduction activity of its own.

[0041] As used herein, "intracellular signal transduction domain" refers to a polypeptide that initiates a signal transduction cascade. In some embodiments, intracellular signal transduction domains induce immune effector cell functions, including enhanced proliferation, cytokine release, cytotoxicity, or combinations thereof.

[0042] As used in this article, “immune effector cells” refers to cells that produce an immune response.

[0043] III. Overview

[0044] The embodiments disclosed herein include adding additional antigen receptors (SSRs) to AgRICA immune effector cells or immune effector cells used in combination with recombinant BiTE. SSRs enhance the efficacy of AgRICA immune effector cells by stabilizing cell-cell interactions and immune synapse formation. Because such synaptic stabilizing receptors (SSRs) are non-cytotoxic, they can target a broad range of antigens that are otherwise difficult to target with conventional cytotoxic AgRICAs (e.g., CD38, EGFR, etc.), thereby enhancing activity against a wider range of targetable tumors.

[0045] Figure 1 Examples of CD38-specific SSRs co-expressed with suboptimal CARs are shown, which promote tumor killing by: (1) physically enhancing intercellular contact; (2) promoting the polarization of the cytoskeleton and supramolecular complexes to form stable immune synapses; (3) inducing CAR-independent CD38 crosslinking and apoptosis in target cells; and (4) shielding CAR-T cells from daratumumab by masking CD38 with SSRs, thereby enabling combination immunotherapy.

[0046] Typically, embodiments of this disclosure can be used to enhance the antitumor activity of engineered immune effector cells against antigenically heterogeneous tumors. Furthermore, SSR co-expression on immune effector cells enables "Boolean AND" gating, where target cells expressing low levels of AgRICA target antigens also express SSR antigens, and are more effectively killed. Embodiments of this disclosure can increase the cytotoxicity of immune effector cells against cells with low or heterogeneous expression of target antigens. In some specific embodiments, co-expression of the AgRICA immune effector cell synaptic stabilizing receptor (SSR) or its combination with recombinant BiTE increases the cytotoxicity of immune effector cells.

[0047] The extracellular receptor domain of the SSR targets the same target cells as AgRICA; however, the SSR receptor and the AgRICA binding domain target different antigens. That is, the target cell presents at least two different antigens externally, with the SSR receptor targeting one and the AgRICA binding domain targeting another. In some embodiments, the antigen bound by the SSR receptor is present on cells other than the target cell. For example, the SSR receptor may target antigens that are also present on healthy cells.

[0048] In some embodiments of this disclosure, SSR enhances the cytotoxicity of immune effector cells and the stability of immune synapse formation compared to AgRICA alone. SSR activity also increases T cell proliferation in response to AgRICA signaling. In some embodiments, SSR works synergistically with AgRICA compared to AgRICA alone. In some embodiments, AgRICA only has target cell death efficacy when used in combination with SSR; that is, no target cell death occurs unless both SSR and AgRICA are used together.

[0049] In some embodiments of this disclosure, SSR enhances the cytotoxicity of T cells activated by soluble BiTE. BiTE targets a peptide derived from the leukemia antigen TdT presented on HLA class I molecules. Adding soluble BiTE to unarmed T cells triggers moderate cell lysis in TdT+ leukemia, which is enhanced by the expression of SSR on the T cells.

[0050] Some embodiments of this disclosure include the use of a CAR targeting CLL1 (CLEC12a) and an SSR targeting CD38 to treat AML. CD38 is an antigen widely expressed in both normal and malignant lymphoid and myeloid cells. As shown in Example 1, co-expression of CD38 on CLL1 CAR T cells with the SSR enhances their cytotoxicity against CLL1-low AML cells in vitro and in vivo. Example 1 shows that the SSR enhances apoptosis in targeted leukemia cells, one way being by cross-linking CD38 on the cell surface, and also by masking CD38 expression on activated T cells, thereby protecting them from the CD38-specific cytotoxic antibody daratumumab. Therefore, this SSR can be combined with various CARs targeting lymphoid and myeloid leukemia to offset the risk of antigen downregulation and can be used in combination therapy with daratumumab without damaging therapeutic T cells. In some embodiments, cytotoxicity against acute myeloid leukemia (AML) cells with low antigen expression is increased by incorporating the CD38 SSR with an AML-targeting CAR.

[0051] IV. Overview of Chimeric Antigen Receptors and T-cell Receptors

[0052] This document discloses SSRs that can be used in combination with chimeric antigen receptors (CARs). CARs typically comprise an extracellular antigen (or ligand) binding (receptor) domain and one or more intracellular signaling components linked thereto, which are in some respects connected by hinges, linkers, and / or transmembrane domains. Such molecules typically mimic or approximate signaling via native antigen receptors, signaling via such receptors in combination with co-stimulatory receptors, and / or signaling via co-stimulatory receptors alone. In embodiments of this disclosure, these CARs are used in combination with SSRs in immune effector cells.

[0053] This document also discloses SSRs that can be used in combination with T cell receptors (TCRs). TCRs typically comprise two variable chains that recognize peptides presented by MHC molecules and complex with CD3 subunits to form a multi-subunit signaling complex. In some embodiments of this disclosure, these TCRs are used in combination with SSRs in immune effector cells.

[0054] In some implementations, the CAR nucleic acid contains sequences encoding additional co-stimulatory receptors, such as transmembrane domains and one or more intracellular signaling domains. In addition to primary T cell activation signals, such as those initiated by CD3ζ and / or FcεRIγ, additional stimulatory signals can be utilized for immune effector cell proliferation and effector function after the chimeric receptor binds to the target antigen. For example, some or all of the human co-stimulatory receptors used to enhance cell activation can be utilized, which can help improve in vivo persistence and increase the therapeutic success of adoptive immunotherapy. Examples include co-stimulatory domains from molecules such as DAP12, DAP10, NKG2D, CD2, CD28, CD27, 4-1BB (CD137), OX-40 (CD134), ICOS (CD278), CD30, HVEM, CD40, LFA-1 (CD11a / CD18), and ICAM-1, although in specific alternative implementations, any of these listed may be excluded from use in the CAR.

[0055] In some specific implementations, this document covers certain CAR and TCR molecules. In some cases, the antigen-binding domain of the CAR and / or TCR is an scFv. When scFv is used in the extracellular domain, the variable heavy chain and variable light chain of the scFv can be in any order in the N-terminal to C-terminal direction. For example, the variable heavy chain can be on the N-terminal side of the variable light chain, or vice versa. The antigen-binding scFv and / or ligand may or may not be codon-optimized.

[0056] In some implementations, CAR can be co-expressed with cytokines to improve persistence, for example, when the amount of tumor-associated antigen is low. For example, CAR can be co-expressed with IL-15, IL-7, L-12, IL-18 and / or IL-21.

[0057] A. CAR antigen-binding domain

[0058] The polypeptide disclosed herein may comprise one or more antigen-binding (receptor) domains. An "antigen-binding domain" describes a region of the polypeptide capable of binding to an antigen under appropriate conditions. In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv) based on one or more antibodies (e.g., a CD20 antibody). In some embodiments, the antigen-binding domain comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH and VL regions are located on the same polypeptide. In some embodiments, the antigen-binding domain comprises a linker between the VH and VL regions. The linker enables the antigen-binding domain to form the desired structure for antigen binding.

[0059] The variable regions of the antigen-binding domains of the disclosed peptides can be modified by mutating amino acid residues within the CDR 1, CDR 2, and / or CDR 3 regions of VH and / or VL to improve one or more binding properties of the antibody (e.g., affinity). The term "CDR" refers to a complementarity-determining region based on a portion of the variable chain in immunoglobulins (antibodies) and T-cell receptors, generated by B cells and T cells, respectively, where these molecules bind to their specific antigens. Since most sequence variations associated with immunoglobulins and T-cell receptors occur in the CDRs, these regions are sometimes referred to as hypervariable regions. Mutations can be introduced by site-directed mutagenesis or PCR-mediated mutagenesis, and their effects on antibody binding or other functional properties of interest can be assessed in appropriate in vitro or in vivo assays. Conserved modifications are preferred, and typically no more than one, two, three, four, or five residues within the CDR region are altered. Mutations can be amino acid substitutions, additions, or deletions.

[0060] It also considers that the antigen-binding domain can be multispecific or multivalent, by polymerizing antigen-binding domains that have VH and VL regions that bind the same antigen (multivalent) or different antigens (multispecific).

[0061] The binding affinity of antigen-binding regions, such as variable regions (heavy chain and / or light chain variable regions) or CDRs, can be at least 10. -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11M, 10 -12 M or 10 -13 M. In some implementations, the antigen-binding region, such as the variable region (heavy chain and / or light chain variable region) or the K of the CDR, D It can be at least 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 - 9 M, 10 -10 M, 10 -11 M, 10 -12 M or 10 -13 M (or any of its derivative ranges).

[0062] Combining affinity, K A or K D It can be determined by methods known in the art, such as by surface plasmon resonance (SRP) based biosensors, by kinetic exclusion assay (KinExA), by optical scanners for detection by microarrays based on polarization-modulated oblique incidence reflectivity difference (OI-RD), or by ELISA.

[0063] In some implementations, peptides containing humanized binding regions have equal, better, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 104%, 106%, 106%, 108%, 109%, 110%, 115%, or 120% binding affinity and / or expression levels in host cells compared to peptides containing non-humanized binding regions.

[0064] B. CAR transmembrane domain

[0065] In some embodiments, the antigen-specific binding or recognition component is associated with one or more transmembrane and intracellular signaling domains. In some embodiments, the CAR includes a transmembrane domain fused to an extracellular domain of the CAR. In some embodiments, a transmembrane domain naturally associated with one of the domains in the CAR is used. In some embodiments, a transmembrane domain not naturally associated with one of the domains of the CAR is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to prevent such domains from binding to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex. In some embodiments, the transmembrane domain is derived from a natural or synthetic source. In some embodiments, when the source is natural, the transmembrane domain is derived from any membrane-binding or transmembrane protein. In some embodiments, the transmembrane region includes transmembrane regions derived from (i.e., transmembrane regions comprising at least the following; also including transmembrane regions “from” the following) the α, β, or ζ chains of the T cell receptor, CD28, DAP12, DAP10, NKG2D, CD3ζ, CD3ε, CD3γ, CD3δ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD30, CD33, CD37, CD64, CD80, CD86, OX-40 (CD134), 4-1BB (CD137), CD154, ICOS / CD278, etc. In some embodiments, the transmembrane domain of this disclosure is a transmembrane domain derived from CD28. In some embodiments, the CD8a hinge appears between the antigen receptor domain and the transmembrane domain.

[0066] The polypeptides disclosed herein may include a transmembrane domain. In some embodiments, the transmembrane domain is a hydrophobic α-helix that spans the membrane. Different transmembrane domains may result in different receptor stability.

[0067] In some embodiments, the transmembrane domain is located between the extracellular septum and the cytoplasm. In some embodiments, the transmembrane domain is located between the extracellular septum and one or more co-stimulatory regions. In some embodiments, the adapter is located between the transmembrane domain and one or more co-stimulatory regions.

[0068] In some embodiments, the transmembrane domain may have a length of at least, at most, or exactly 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids. In some embodiments, the transmembrane domain is about 24 amino acids long.

[0069] In some embodiments, providing a polypeptide insertion into any transmembrane domain of the cell membrane of a eukaryotic (e.g., mammalian) cell may be suitable for use. In some embodiments, the transmembrane domain is derived from CD30, CD28, CD8, CD4, CD3ζ, OX-40 (CD134), or CD7. In some embodiments, the transmembrane domain is derived from the α, β, or ζ chain of the T cell receptor, CD28, CD2, CD3ζ, CD3ε, CD3γ, CD3δ, CD45, CD4, CD5, CD8 (including CD8α), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, OX-40 (CD134), 4-1BB (CD137), CD154, ICOS / CD278, GITR / CD357, NKG2D, PD-1, CTLA4, and DAP molecules. In some embodiments, the transmembrane domain is synthetic. In some aspects, the synthesized transmembrane domain primarily comprises hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan, and valine will appear at each end of the synthesized transmembrane domain. In some embodiments, the transmembrane domain comprises, is substantially composed of, or is composed of a sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the transmembrane domains listed above.

[0070] In some embodiments, a hinge domain exists between the antigen-binding domain and the transmembrane domain. In some embodiments, the hinge domain does not dimerize with other hinge domains. In some embodiments, the hinge domain in the CAR will not dimerize with another identical hinge domain. In some embodiments, the hinge domain in the CAR will not dimerize with the hinge domain in the SSR. In some embodiments, the hinge domain in the CAR has been mutated such that it will not dimerize with another identical hinge domain. In some embodiments, the hinge domain in the CAR has been mutated such that it will not dimerize with the hinge domain in the SSR. In some embodiments, the hinge domain is CD8α, and cysteine ​​164 in CD8α is replaced by serine (C164S) (SEQ ID NO: 9).

[0071] C. CAR cytoplasmic region (intracellular region)

[0072] Following antigen recognition, receptors can aggregate, and signals can be transmitted to the cell via the cytoplasmic region. In some embodiments, the co-stimulatory domains described herein are part of the cytoplasmic region. In some embodiments, the cytoplasmic region contains intracellular signal transduction domains. Intracellular signal transduction domains may contain primary signal transduction domains and one or more co-stimulatory domains.

[0073] The cytoplasmic and / or co-stimulatory regions of the CARs applicable to this disclosure include any desired signaling domains that provide a unique and detectable signal in response to activation via antigen-binding domains (e.g., increased production of one or more cytokines; changes in target gene transcription; changes in protein activity; changes in cell behavior, such as cell death; cell proliferation; cell differentiation; cell survival; regulation of cell signaling responses, etc.). In some embodiments, the cytoplasmic region includes at least one (e.g., one, two, three, four, five, six, etc.) ITAM motif as described herein. In some embodiments, the cytoplasmic region includes a DAP10 / CD28 type signaling chain.

[0074] The cytoplasmic region of the polypeptides applicable to this disclosure includes an intracellular signaling polypeptide containing an activating motif based on an immune receptor tyrosine (ITAM). The ITAM motif is YX1X2(L / I), where X1 and X2 are independently any amino acid. In some cases, the cytoplasmic region contains 1, 2, 3, 4, or 5 ITAM motifs. In some cases, the ITAM motif is repeated twice in an intracellular domain, where the first and second instances of the ITAM motif are spaced 6 to 8 amino acids apart, for example, (YX1X2(L / I))(X3)n(YX1X2(L / I)), where n is an integer from 6 to 8, and each of the 6-8 X3 motifs can be any amino acid.

[0075] In some implementations, a suitable cytoplasmic region is a portion containing the ITAM motif, derived from a polypeptide containing the ITAM motif. For example, a suitable cytoplasmic region can be an ITAM motif-containing domain from any protein containing the ITAM motif. Therefore, a suitable intracellular domain does not need to contain the entire sequence of the protein from which it originates. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to: DAP12, DAP10, FCER1G (Fcε receptor Iγ chain); CD3D (CD3δ); CD3E (CD3ε); CD3G (CD3γ); CD3-ζ; and CD79A (antigen receptor complex-associated protein α chain).

[0076] In some embodiments, a suitable cytoplasmic region may comprise a portion of the full-length DAP12 amino acid sequence containing the ITAM motif. In some embodiments, the cytoplasmic region is derived from FCERI1G (also known as FCERG; Fcε receptor Iγ chain; Fc receptor γ chain; fcεRIγ; fcRγ; fceRIγ; high-affinity immunoglobulin ε receptor subunit γ; immunoglobulin E receptor, high affinity, γ chain, etc.). In some embodiments, a suitable cytoplasmic region may comprise a portion of the full-length FCERI1G amino acid sequence containing the ITAM motif.

[0077] In some embodiments, the cytoplasmic region is derived from the T cell surface glycoprotein CD3δ chain (also known as CD3D; CD3-δ; T3D; CD3 antigen, δ subunit; CD3 delta; CD3δ; CD3d antigen, δ polypeptide (TiT3 complex); OKT3, δ chain; T cell receptor T3δ chain; T cell surface glycoprotein CD3δ chain, etc.). In some embodiments, a suitable cytoplasmic region may include a portion of the full-length CD3δ amino acid sequence containing the ITAM motif. In some embodiments, the cytoplasmic region is derived from the T cell surface glycoprotein CD3ε chain (also known as CD3e, CD3ε; T cell surface antigen T3 / Leu-4ε chain, T cell surface glycoprotein CD3ε chain, AI504783, CD3, CD3-ε, T3e, etc.). In some embodiments, a suitable cytoplasmic region may include a portion of the full-length CD3ε amino acid sequence containing the ITAM motif. In some embodiments, the cytoplasmic region is derived from the T cell surface glycoprotein CD3γ chain (also known as CD3G, CD3γ, T cell receptor T3γ chain, CD3-γ, T3G, γ polypeptide (TiT3 complex), etc.). In some embodiments, a suitable cytoplasmic region may include a portion of the full-length CD3γ amino acid sequence containing the ITAM motif. In some embodiments, the cytoplasmic region is derived from the T cell surface glycoprotein CD3ζ chain (also known as CD3Z, CD3ζ, T cell receptor T3ζ chain, CD247, CD3-ζ, CD3H, CD3Q, T3Z, TCRZ, etc.). In some embodiments, a suitable cytoplasmic region may include a portion of the full-length CD3ζ amino acid sequence containing the ITAM motif.

[0078] In specific embodiments of a particular CAR molecule, the CAR may utilize CD28, DAP10, DAP12, 4-1BB, NKG2D, or other co-stimulatory domains (which may be referred to herein as intracytoplasmic domains). In some cases, CD3ζ may be utilized without any co-stimulatory domains. In specific embodiments of a particular CAR molecule, the CAR may utilize any suitable transmembrane domain, such as transmembrane domains derived from CD30, DAP12, DAP10, 4-1BB, 2B4, OX40, CD27, NKG2D, CD8, CD28, IL12Rβ1, or IL12Rβ2.

[0079] In some embodiments, the cytoplasmic region is derived from CD79A (also known as the B-cell antigen receptor complex-associated protein α chain; CD79a antigen (immunoglobulin-associated α); MB-1 membrane glycoprotein; ig-α; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein, etc.). In some embodiments, a suitable cytoplasmic region may contain a portion of the full-length CD79A amino acid sequence containing the ITAM motif.

[0080] Non-limiting examples of suitable co-stimulatory regions (such as those included in the cytoplasm) include, but are not limited to, peptides derived from 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM. In some embodiments, the co-stimulatory region is derived from CD8, 4-1BB (CD137), CD27, CD28, CD30, OX-40 (CD134), CD3ε, CD3ζ, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, or CD154. In some embodiments, the co-stimulatory domain includes, but is not limited to, one or more of CD28, CD27, OX-40 (CD134), ICOS, HVEM, GITR, LIGHT, CD40L, DR3, CD30, SLAM, CD2, CD226 (DNAM-1), MyD88, CD244, TMIGD2, BTNL3, NKG2D, DAP10, DAP12, 4-1BB (CD137), or synthetic molecules. In some embodiments, in addition to the primary signal initiated by CD3ζ, additional signaling provided by the co-stimulatory receptor inserted into the CAR is important for the complete activation of immune cells and may help improve in vivo persistence and the therapeutic success of cell therapy.

[0081] The co-stimulatory region may have a length of at least, at most, or exactly 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300 amino acids, or any range thereof. In some embodiments, the co-stimulatory region is derived from the transmembrane protein 4-1BB (also known as TNFRSF9; CD137; CDwl37; ILA, etc.). In some embodiments, the co-stimulatory region is derived from the intracellular portion of the transmembrane protein CD28 (also known as Tp44). In some embodiments, the co-stimulatory region is derived from the intracellular portion of the transmembrane protein ICOS (also known as AILIM, CD278, and CVID1). In some embodiments, the co-stimulatory region is derived from the intracellular portion of the transmembrane protein OX-40 (also known as TNFRSF4, RP5-902P8.3, ACT35, CD134, OX40, TXGP1L). In some embodiments, the co-stimulatory region is derived from the intracellular portion of the transmembrane protein BTLA (also known as BTLA1 and CD272). In some embodiments, the co-stimulatory region is derived from the intracellular portion of the transmembrane protein CD27 (also known as S152, T14, TNFRSF7, and Tp55). In some embodiments, the co-stimulatory region is derived from the intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, D1S166E, and Ki-1). In some embodiments, the co-stimulatory region is derived from the intracellular portion of the transmembrane protein GITR (also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D). In some embodiments, the co-stimulatory region is derived from the intracellular portion of the transmembrane protein HVEM (also known as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR, and TR2).

[0082] In some embodiments, the polypeptides described herein may further comprise a detection peptide. Suitable detection peptides include hemagglutinin, FLAG, c-myc, etc. Other suitable detection peptides are known in the art.

[0083] V. Synaptic Stabilizing Receptors (SSRs)

[0084] Some embodiments of this disclosure are SSRs. In some embodiments, SSRs are used in combination with agRICA in immune effector cells to target and kill disease cells. An SSR includes at least an extracellular receptor domain, a transmembrane domain, and an intracellular scaffold domain. In some embodiments, an SSR may additionally include one or more spacer regions, hinge domains, cytoplasmic domains, motifs with co-stimulatory signaling, or combinations thereof.

[0085] A. SSR antigen receptor domain

[0086] In some embodiments, the extracellular receptor domain targets the antigen. In some embodiments, the antigen is present on diseased or healthy cells. In some embodiments, the antigen is disease-specific. In some embodiments, the antigen is cell-type specific.

[0087] The binding affinity of antigen-binding regions, such as variable regions (heavy chain and / or light chain variable regions) or CDRs, can be at least 10. -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or 10 -13 M. In some implementations, the antigen-binding region, such as the variable region (heavy chain and / or light chain variable region) or the K of the CDR, D It can be at least 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 - 9 M, 10 -10 M, 10 -11 M, 10 -12 M or 10 -13 M (or any of its derivative ranges).

[0088] Combining affinity, K A or K D It can be determined by methods known in the art, such as by surface plasmon resonance (SRP) based biosensors, by kinetic exclusion assay (KinExA), by optical scanners for detection by microarrays based on polarization-modulated oblique incidence reflectivity difference (OI-RD), or by ELISA.

[0089] In some implementations, peptides containing humanized binding regions have equal, better, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 104%, 106%, 106%, 108%, 109%, 110%, 115%, or 120% binding affinity and / or expression levels in host cells compared to peptides containing non-humanized binding regions.

[0090] In some embodiments, the antigen receptor domain is an antibody targeting a known receptor on cancer cells. Antibodies targeting known receptors on cancer cells are known in the art. In some embodiments, the antigen receptor domain is derived from an antibody targeting the CD38 antigen or the EGFR antigen. In some embodiments, the antigen receptor is an anti-CD38 or anti-EGFR molecule or peptide.

[0091] B. SSR transmembrane domain

[0092] The polypeptides disclosed herein may include a transmembrane domain. In some embodiments, the transmembrane domain is a hydrophobic α-helix that spans the membrane. Different transmembrane domains may result in different receptor stability.

[0093] In some embodiments, the transmembrane domain is situated between the extracellular septum and the intracellular region. In some embodiments, the transmembrane domain is situated between the hinge domain and the intracellular region. In some embodiments, the connector is located between the transmembrane domain and the scaffold domain.

[0094] In some implementations, the transmembrane domain comprises, is substantially composed of, or consists of sequences derived from the transmembrane regions of the CD28, DAP12, DAP10, NKG2D, CD3ζ, CD3ε, CD3γ, CD3δ, CD45, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD30, CD33, CD37, CD64, CD80, CD86, OX-40 (CD134), 4-1BB (CD137), CD154, or ICOS / CD278 genes. In some embodiments, the transmembrane domain may have a length of at least, at most, or exactly 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids. In some embodiments, the transmembrane domain is about 24 amino acids long. In some embodiments, the transmembrane domain comprises, is substantially composed of, or is composed of a sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similar to the transmembrane region of CD28, DAP12, DAP10, NKG2D, CD3ζ, CD3ε, CD3γ, CD3δ, CD45, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD30, CD33, CD37, CD64, CD80, CD86, OX-40 (CD134), 4-1BB (CD137), CD154, or ICOS / CD278.

[0095] In some embodiments, the transmembrane domain and peptide spacer region comprise, substantially comprise, or comprise a sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similar to the transmembrane region of CD28, DAP12, DAP10, NKG2D, CD3ζ, CD3ε, CD3γ, CD3δ, CD45, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD30, CD33, CD37, CD64, CD80, CD86, OX-40 (CD134), 4-1BB (CD137), CD154, or ICOS / CD278.

[0096] In some embodiments, the antigen-specific receptor component is associated with a transmembrane domain and one or more intracellular signaling domains. In some embodiments, the SSR includes a transmembrane domain fused to an extracellular domain of the SSR. In some embodiments, a transmembrane domain naturally associated with one of the domains in the SSR is used. In some embodiments, a transmembrane domain not naturally associated with one of the domains of the SSR is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to prevent such domains from binding to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other transmembrane proteins. In some embodiments, the transmembrane domain is derived from a natural or synthetic source. In some embodiments, when the source is natural, the transmembrane domain is derived from any membrane-binding or transmembrane protein. In some implementations, the transmembrane region includes transmembrane regions derived from the α, β or ζ chain of the T cell receptor, CD28, DAP12, DAP10, NKG2D, CD3ζ, CD3ε, CD3γ, CD3δ, CD45, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD30, CD33, CD37, CD64, CD80, CD86, OX-40 (CD134), 4-1BB (CD137), CD154, ICOS / CD278, etc. (i.e., transmembrane regions containing at least the above items; i.e., transmembrane regions "from" at least the above items).

[0097] In some embodiments, providing a polypeptide insertion into any transmembrane domain of the cell membrane of a eukaryotic (e.g., mammalian) cell may be suitable for use. In some embodiments, the transmembrane domain is derived from CD30, CD28, CD8, CD4, CD3ζ, OX-40 (CD134), or CD7. In some embodiments, the transmembrane domain is derived from the α, β, or ζ chain of the T cell receptor, CD28, CD2, CD3ζ, CD3ε, CD3γ, CD3δ, CD45, CD4, CD5, CD8 (including CD8α), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, OX-40 (CD134), 4-1BB (CD137), CD154, ICOS / CD278, GITR / CD357, NKG2D, PD-1, CTLA4, and DAP molecules. In some embodiments, the transmembrane domain is synthetic. In some cases, the synthetic transmembrane domain primarily contains hydrophobic residues such as leucine and valine. In others, a triplet of phenylalanine, tryptophan, and valine will appear at each end of the synthetic transmembrane domain.

[0098] In some embodiments, a hinge domain exists between the antigen-binding domain and the transmembrane domain. In some embodiments, the hinge domain does not dimerize with other transmembrane domains it will encounter in the cell membrane. In some embodiments, the hinge domain in the SSR will not dimerize with another identical hinge domain. In some embodiments, the hinge domain in the SSR will not dimerize with the hinge domain in the CAR. In some embodiments, the hinge domain in the SSR has been mutated such that it will not dimerize with another identical hinge domain. In some embodiments, the hinge domain in the SSR has been mutated such that it will not dimerize with the hinge domain in the CAR. In some embodiments, the hinge domain is CD8α, and cysteine ​​164 in CD8α is replaced by serine (C164S) (SEQ ID NO: 9).

[0099] C. SSR intracellular domain

[0100] Embodiments of this disclosure include SSRs having a scaffold protein in the intracellular region of the SSR. In specific embodiments, the scaffold protein has at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence similarity to LAT (SEQ ID NO: 7). In some embodiments, the scaffold protein has at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence similarity to LAT177 (SEQ ID NO: 8). LAT amplifies signal transduction from existing antigen receptors, rather than triggering additional signal transduction.

[0101] VI. Other structural domains

[0102] In embodiments of this disclosure, the SSR, AgRICA, or both may include additional elements. In embodiments, the SSR, AgRICA, or both may include spacers, hinges, markers, or combinations thereof.

[0103] Peptide spacer regions (e.g., spacer regions), such as extracellular spacer regions, can connect antigen-binding domains to transmembrane domains. In some embodiments, the peptide spacer region is flexible enough to allow the antigen-binding domains to orient in different directions to facilitate antigen binding. In some embodiments herein, the peptide spacer region is a “hinge,” for example, a flexible polypeptide linker region that links one or more domains of AgRICA and / or SSR to one or more other domains of AgRICA or SSR. As used herein, the term “hinge” refers to a flexible polypeptide linker region (also referred to herein as a “hinge region”) that provides structural flexibility and spacer between flanking polypeptide regions and can be composed of native or synthetic polypeptides. In an embodiment, the peptide spacer region of the SSR is derived from CD8a and carries a C164S mutation.

[0104] In some embodiments, the extracellular peptide spacer region containing the hinge may have a length of at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids. In some embodiments, the length of the peptide spacer region is 12 amino acids.

[0105] The length of the extracellular spacer region can influence AgRICA signaling activity (if present), AgRICA expression levels (transcription and / or translation), cytotoxic and / or cancer cell killing efficacy, and / or the amplification characteristics of AgRICA signaling in response to antigen stimulation by AgRICA immune effector cells. In some embodiments, the sequence of the AgRICA extracellular spacer region depends on the location of the target antigen. In some embodiments, a longer extracellular spacer region is used when the target antigen is close to the cell membrane. In some embodiments, a shorter extracellular spacer region is used when the target antigen is far from the cell membrane. In some embodiments, a longer extracellular spacer region is used when a more flexible AgRICA is required. In some embodiments, a shorter extracellular spacer region is used when a more rigid AgRICA is required.

[0106] In some implementations, shorter spacer regions are used, such as less than or no more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids. In these implementations, shorter spacer regions may have advantages in terms of AgRICA-mediated signaling activity, AgRICA expression levels (transcription and / or translation), cytotoxicity and / or cancer cell killing efficacy, and / or the amplification properties of AgRICA signaling in response to antigen stimulation.

[0107] The length of the extracellular spacer region can influence the scaffold activity of the SSR, the expression level of the SSR (transcription and / or translation), antigen binding, or combinations thereof. In some embodiments, the sequence of the extracellular spacer region of the SSR depends on the location of the target antigen. In some embodiments, a longer extracellular spacer region is used when the target antigen is close to the cell membrane. In some embodiments, a shorter extracellular spacer region is used when the target antigen is far from the cell membrane. In some embodiments, a longer extracellular spacer region is used when a more flexible SSR is required. In some embodiments, a shorter extracellular spacer region is used when a more rigid SSR is required.

[0108] In some implementations, shorter spacer regions are used, such as less than or no more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids. In some implementations, shorter spacer regions may have advantages in terms of SSR expression levels (transcription and / or translation), scaffolding, optimal antigen binding, or combinations thereof.

[0109] VII. Production of polypeptides in immune effector cells

[0110] Engineered constructs (such as AgRICA or SSR) can be introduced into immune cells as naked DNA or RNA, transposons, or in suitable vectors. Methods for stable transfection of cells using naked DNA via electroporation are known in the art. See, for example, U.S. Patent No. 6,410,319. Naked DNA generally refers to DNA encoding a chimeric receptor contained in a plasmid or viral expression vector in a direction suitable for expression.

[0111] In some embodiments, CAR constructs can be introduced into any type of cell, including at least immune cells, using viral vectors (e.g., retroviral vectors, adenovirus vectors, adeno-associated virus vectors, or lentiviral vectors). Suitable vectors used according to the methods of this disclosure are non-replicating in immune cells. A large number of virus-based vectors are known, in which the viral copy number maintained in the cell is low enough to maintain cell viability, such as vectors based on HIV, MoMLV, MSCV, SV40, EBV, HSV, or BPV.

[0112] Certain embodiments of this disclosure involve the use of nucleic acids, including nucleic acids encoding cancer antigen-specific AgRICA peptides, and in some cases, CARs (hCARs) that have been humanized to reduce immunogenicity, comprising at least one intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising one or more signaling motifs. In some embodiments, the binding region may comprise a complementarity-determining region of a monoclonal antibody, a variable region of the monoclonal antibody, and / or an antigen-binding fragment thereof. In another embodiment, the specificity is derived from a peptide that binds to a receptor (e.g., a cytokine).

[0113] The CAR nucleic acid may or may not contain at least a portion of a human gene for enhancing cellular immunotherapy in human patients. In specific embodiments, this disclosure includes a full-length CAR cDNA or coding region. The antigen-binding region or domain may comprise fragments of the VH and VL chains of a single-stranded variable fragment (scFv) derived from a specific human monoclonal antibody (e.g., an anti-CD19 antibody, such as FMC63.3). In some embodiments, the fragment may also be any number of different antigen-binding domains of a human antigen-specific antibody. In a more specific embodiment, the fragment is a cancer antigen-specific scFv encoded by a sequence optimized for expression in human cells using human codons. In some embodiments, the antigen-binding region comprises a protein or polypeptide that acts as a ligand and / or receptor for another protein and / or polypeptide.

[0114] In some embodiments, the arrangement can be multimeric, such as a bivalent antibody or a multimer. Multimers are most likely formed by cross-pairing variable portions of the light and heavy chains to create a bivalent antibody. The hinge portion of the construct can have various options, from complete deletion to retaining the first cysteine ​​residue, to proline substitution instead of serine, to being truncated to the first cysteine ​​residue. In some embodiments, the Fc portion can be deleted. In some embodiments, any stable and / or dimerized protein can be used for this purpose. In some embodiments, only one of the Fc domains is used, for example, the CH2 or CH3 domain from human immunoglobulins. In some embodiments, the hinge, CH2, and CH3 regions of human immunoglobulins that have been modified to improve dimerization can be used. In some embodiments, only the hinge portion of the immunoglobulin can be used.

[0115] The sequence encoding the open reading frame of the chimeric receptor can be obtained from genomic DNA, cDNA, or can be synthesized (e.g., by PCR), or a combination thereof. Depending on the size of the genomic DNA and the number of introns, cDNA or a combination thereof may be necessary, as introns have been found to stabilize mRNA. Furthermore, using endogenous or exogenous non-coding regions to stabilize mRNA may be further advantageous.

[0116] VIII. SSR and AgRICA combination

[0117] The embodiments disclosed herein are SSRs that can be used in combination with AgRICA. Embodiments of this disclosure include a combination of an antigen targeted by AgRICA and an antigen targeted by an SSR, which work together to treat a disease. In some embodiments, neither the SSR nor AgRICA promotes cytotoxicity when present alone on immune effector cells, and cytotoxicity is promoted only when used in combination. In some embodiments, both the SSR and AgRICA are contained within the same immune effector cell.

[0118] In one implementation, the AgRICA / SSR combination can target blood cancers such as leukemia, lymphoma, or myeloma. In one implementation, the disease type is AML, the SSR receptor is anti-CD38 and the antigen targeted by the SSR receptor is CD38, and the CAR receptor is anti-CLL1 and the antigen targeted by the CAR receptor is CLL1. In another example, the disease is B-cell leukemia, acute myeloid leukemia, or T-cell leukemia (i.e., BV173, THP1, or TALL1), and the SSR targets CD38 and the TCR targets survivin. In yet another example, the disease is acute lymphoblastic leukemia, and the SSR targets CD38 and BiTE targets the TdT peptide-HLA complex.

[0119] In some embodiments, the AgRICA-SSR combination targets solid tumors. In some embodiments, AgRICA or SSR targets tumor markers overexpressed in solid tumors. In some specific instances, AgRICA targets GRP78 or B7-H3. In one specific embodiment, the SSR targets EGFR and the TCR targets the survivin peptide-HLA complex on the solid tumor.

[0120] Table 1: Examples of combinations of SSR and CAR / TCL targeted antigens that can be used to treat specific diseases.

[0121]

[0122] IX. Therapy

[0123] Certain aspects of this implementation can be used to prevent or treat diseases or conditions. In one implementation, the disease is a blood cancer, such as leukemia, lymphoma, or myeloma. In a specific implementation, the disease is acute myeloid leukemia (AML). In some implementations, the disease is a solid tumor. In another implementation, AgRICA immune effector cells target pathogenic lymphocytes, for example, to treat autoimmune or alloimmune diseases.

[0124] Some embodiments of this disclosure involve obtaining and administering cells to a subject to target cancer cells. The cells may deliver antibody compositions covered herein, but they may or may not be immune cells themselves. In specific embodiments, the cells are immune effector cells. Examples of cells include T cells (including αβ T cells or γδ T cells), natural killer (NK) cells, invariant NKT (iNKT) cells, B cells, macrophages, any type of stem cell (including MSCs or induced pluripotent stem cells), or dendritic cells.

[0125] In some embodiments, the method further includes administering a cancer therapy to the patient. The cancer therapy may be selected solely based on expression level measurements or in combination with a clinical risk score calculated for the patient. In some embodiments, the cancer therapy includes local cancer therapy. In some embodiments, the cancer therapy does not include systemic cancer therapy. In some embodiments, the cancer therapy does not include local therapy. In some embodiments, the cancer therapy includes local cancer therapy without the administration of systemic cancer therapy. In some embodiments, the cancer therapy includes immunotherapy, which may be an immune checkpoint therapy. Any of these cancer therapies may also be excluded. A combination of these therapies may also be administered.

[0126] As used herein, the term "cancer" can be used to describe solid tumors, metastatic cancer, or non-metastatic cancer. In some embodiments, cancer can originate in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, rectum, anus, gums, head, kidneys, liver, lungs, nasopharynx, neck, ovaries, pancreas, prostate, skin, stomach, testes, tongue, or uterus. In some embodiments, cancer is recurrent cancer. In some embodiments, cancer is stage I cancer. In some embodiments, cancer is stage II cancer. In some embodiments, cancer is stage III cancer. In some embodiments, cancer is stage IV cancer.

[0127] This treatment method is useful for tumors including any malignant cell type, such as those found in solid tumors or hematologic malignancies. Exemplary solid tumors may include, but are not limited to, tumors of organs selected from the group consisting of: pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematologic malignancies include bone marrow tumors, T-cell or B-cell malignancies, leukemia, lymphoma, blastoma, myeloma, etc. Other examples of cancers that can be treated using the methods described in this article include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, gastric cancer or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal carcinoma), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, ductal carcinoma, vulvar cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0128] Cancer can specifically be, but is not limited to, the following histological types: neoplasm (malignant); carcinoma; undifferentiated carcinoma; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; and malignant gastrinoma. malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma (familial polyposis coli); solid carcinoma; malignant carcinoid tumor; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophilic carcinoma. carcinoma; oxyphilicadenocarcinoma; basophilic carcinoma;Clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenalcortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma. carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease (mammary); acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; malignant thymoma. malignant ovarian stromal tumor; malignant theca cell tumor.Malignant granulosa cell tumor; malignant androblastoma; Sertoli cell carcinoma; malignant leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; lentigo malignant melanoma; acrallentiginous melanomas; nodular melanoma. melanomas; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor; Müllerian mixed tumor Tumors; Nephroblastoma; Hepatoblastoma;Carcinosarcoma; Malignant mesenchymoma; Malignant Brenner tumor; Malignant phyllodes tumor; Synovial sarcoma; Malignant mesothelioma; Dysgerminoma; Embryonal carcinoma; Malignant teratoma; Malignant struma ovarii; Choriocarcinoma; Malignant mesonephroma; Hemangiosarcoma; Malignant hemangioendothelioma; Kaposi's sarcoma; Malignant hemangiopericytoma. malignant); lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma. malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma;Astroblastoma; Glioblastoma; Oligodendroglioma; Primitive neuroectodermal tumor; Cerebellar sarcoma; Ganglioneuroblastoma; Neuroblastoma; Retinoblastoma; Olfactory neurogenic tumor; Malignant meningioma; Neurofibrosarcoma; Malignant schwannoma; Malignant granular cell tumor; Malignant lymphoma; Hodgkin's disease. Disease); Hodgkin's lymphoma; paragranuloma; malignant lymphoma (small lymphocytic); malignant lymphoma (large cell, diffuse); malignant lymphoma (follicular); mycosis fungoids; other specified non-Hodgkin's lymphomas; B-cell lymphoma; low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic non-Hodgkin's lymphoma (SLNHL); intermediate-grade / follicular non-Hodgkin's lymphoma (SLNHL). NHL); intermediate grade diffuse non-Hodgkin lymphoma; high grade immunoblastic non-Hodgkin lymphoma;High-grade lymphoblastic non-Hodgkin lymphoma; high-grade small non-cleaved cell non-Hodgkin lymphoma; bulky disease non-Hodgkin lymphoma; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; malignanthistiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia. Cellleukemia; Erythroleukemia; Lymphosarcoma cellleukemia; Myeloid leukemia; Basophilic leukemia; Eosinophilic leukemia; Monocyticle leukemia; Mast cell leukemia; Megakaryoblastic leukemia; Myeloid sarcoma; Hairy cell leukemia; Chronic lymphocytic leukemia (CLL); Acute lymphoblastic leukemia (ALL); Acute myeloid leukemia. AML; and chronic myeloblastic leukemia.

[0129] Some implementation schemes involve treatments for leukemia. Leukemia is a cancer of the blood or bone marrow characterized by the abnormal proliferation (through amplification) of blood cells (usually white blood cells, leukocytes). It is part of a broad group of diseases known as hematologic malignancies. Leukemia is a broad term encompassing a range of diseases. Leukemia is clinically and pathologically classified into acute and chronic forms.

[0130] In some embodiments, this disclosure provides methods for immunotherapy, the method comprising administering an effective amount of the composition of this disclosure comprising AgRICA and SSR. In one embodiment, a medical disease or condition is treated by administering a population of cells expressing AgRICA and SSR that elicits an immune response. In some embodiments of this disclosure, cancer is treated by administering a population of immune cells expressing AgRICA and SSR that elicits an immune response. This document also provides methods for treating or delaying the progression of cancer in an individual, the method comprising administering an effective amount of antigen-specific cell therapy to the individual. For example, the methods of the present invention can be used to treat immune disorders, solid tumors, and hematologic malignancies. Specifically, cancer can be B-cell malignancies, such as diffuse large B-cell lymphoma, high-grade B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, mantle cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, and chronic lymphocytic leukemia.

[0131] In some implementations, non-myeloablative lymphoablative chemotherapy may be administered to the subject prior to cell therapy. Non-myeloablative lymphoablative chemotherapy can be any suitable such therapy, which can be administered via any suitable route. Non-myeloablative lymphoablative chemotherapy may include, for example, the administration of cyclophosphamide and fludarabine, particularly if the cancer is melanoma, which may be metastatic. An exemplary route of administration for cyclophosphamide and fludarabine is intravenous. Similarly, any suitable dose of cyclophosphamide and fludarabine can be administered. In a particular aspect, approximately 60 mg / kg of cyclophosphamide is administered over two days, followed by approximately 25 mg / kg of cyclophosphamide. 2 Fludarabine for 5 days.

[0132] In some embodiments of T-cell therapy, a T-cell growth factor that promotes the growth and activation of autologous T cells is administered to the subject concurrently with or after autologous T cells. The T-cell growth factor can be any suitable growth factor that promotes the growth and activation of autologous T cells. Examples of suitable T-cell growth factors include interleukin (IL)-2, IL-7, IL-15, and / or IL-12, which can be used alone or in various combinations, such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL2.

[0133] Therapeutic amounts of immune cells can be administered via a variety of routes, including parenteral administration, such as intravenous, intraperitoneal, intramuscular, intrasternal, or intra-articular injections, or infusions.

[0134] For discrete, solid, palpable tumors, intratumoral injection or injection into the tumor vascular system is specifically considered. Local, regional, or systemic administration may also be suitable. In some embodiments, for tumors >4 cm, the volume to be administered will be about 4-10 ml (especially 10 ml), while for tumors <4 cm, a volume of about 1-3 ml (especially 3 ml) will be used. In some embodiments, multiple injections as a single-dose delivery may contain a volume of about 0.1 ml to about 0.5 ml.

[0135] In some implementations, cell populations may be administered in a treatment regimen consistent with the disease, such as a single or multiple doses administered over one to several days to improve the disease state, or periodic administration over a longer period to inhibit disease progression and prevent recurrence. The precise dosage used in the formulation will also depend on the route of administration and the severity of the disease or condition, and should be determined based on the practitioner's judgment and the individual patient's situation. The therapeutically effective dose of cells will depend on the subject being treated, the severity and type of the ailment, and the method of administration. In some implementations, the dose range that can be used to treat human subjects is at least 3.8 × 10⁻⁶. 4 At least 3.8 × 10 5 At least 3.8 × 10 6 At least 3.8 × 10 7 At least 3.8 × 10 8 At least 3.8 × 10 9 Or at least 3.8 × 10 10 cells / m 2 In one implementation, the dose range for treating human subjects is approximately 3.8 × 10⁻⁶. 9 Approximately 3.8 × 10 10 cells / m2 In another implementation, the therapeutically effective dose of cells can be from approximately 5 × 10⁻⁶. 6 Cells / kg body weight to approximately 7.5 × 10⁻⁶ 8 Variation between cells / kg body weight, such as approximately 2×10 7 One cell to approximately 5 × 10 8 Cells / kg body weight, or approximately 5 × 10⁻⁶ 7 One cell to approximately 2 × 10 8 Cells / kg body weight. The exact number of cells can be easily determined by a person skilled in the art based on the subject's age, weight, sex, and physiological condition. The effective dose can be obtained by extrapolation from dose-response curves derived from in vitro or animal model testing systems.

[0136] In some embodiments of this disclosure, an effective amount of immune cells expressing AgRICA and SSR are delivered to an individual in need, such as an individual with cancer. In some embodiments, the cells can then enhance the individual's immune system to attack cancer cells. In some cases, one or more doses of immune cells are provided to the individual (e.g., those described herein). In some embodiments, where two or more doses of immune cells are provided to the individual, the duration between administrations should be sufficient to allow for proliferation in the individual, and in some specific embodiments, the duration between doses is 1, 2, 3, 4, 5, 6, 7, or more days.

[0137] In some specific implementations, cells that have been engineered to express AgRICA and SSR are used to treat effective doses (ranging from 10). 3 Up to 10 10 This cell is provided to an individual, and it improves at least one symptom associated with cancer cells in that individual. The therapeutically effective dose can be 10... 3 Up to 10 10 10 3 Up to 10 9 10 3 Up to 10 8 10 3 Up to 10 7 10 3 Up to 10 6 10 3 Up to 10 5 10 3 Up to 10 4 10 4 Up to 10 10 10 4 Up to 10 9 10 4 Up to 10 8 10 4 Up to 10 710 4 Up to 10 6 10 4 Up to 10 5 10 5 Up to 10 10 10 5 Up to 10 9 10 5 Up to 10 8 10 5 Up to 10 7 10 5 Up to 10 6 10 6 Up to 10 10 10 6 Up to 10 9 10 6 Up to 10 8 10 6 Up to 10 7 10 7 Up to 10 10 10 7 Up to 10 9 10 7 Up to 10 8 10 8 Up to 10 10 10 8 Up to 10 9 , or 10 9 Up to 10 10 Individual cells. Therefore, in a particular implementation, an individual suffering from a certain cancer is provided with a therapeutically effective amount of cells expressing one or more AgRICA / SSRs, either once or multiple times.

[0138] X. Cell therapy

[0139] Some embodiments involve cells containing the AgRICA and SSR peptides or nucleic acids disclosed herein. In some embodiments, the cells are immune effector cells. In some T cell embodiments, as used herein, "T cell" includes all types of immune cells expressing CD3, including T helper cells, invariant natural killer T (iNKT) cells, cytotoxic T cells, T regulatory cells (Treg), γδ T cells, natural killer (NK) cells, and neutrophils. T cells may refer to CD4+ or CD8+ T cells.

[0140] Suitable mammalian cell lines include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, and rodent (e.g., mouse, rat) cell lines. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) CCL-2), CHO cells (e.g., ATCC CRL9618, CCL61, CRL9096), human embryonic kidney (HEK) 293 cells (e.g., ATCC CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC CCL10), PC12 cells (ATCC CRL1721), COS cells, COS-7 cells (ATCC CRL1651), RATI cells, mouse L cells (ATCC CCL1.3), HLHepG2 cells, Hut-78, Jurkat, HL-60, and NK cell lines (e.g., NKL, NK92, and YTS).

[0141] In some cases, the cells are not immortalized cell lines, but cells obtained from an individual (e.g., primary cells). For example, in some cases, the cells are immune cells obtained from an individual. As one example, the cells are T lymphocytes obtained from an individual. As another example, the cells are cytotoxic cells obtained from an individual. As yet another example, the cells are stem cells (e.g., peripheral blood stem cells) or progenitor cells obtained from an individual.

[0142] Immune cells can be any type of immune cell, including T cells (such as regulatory T cells, CD4+). + T cells, CD8 + Immune cells can be T cells, αβ T cells, γδ T cells, or mixtures thereof; NK cells, invariant NKT cells, NKT cells, innate lymphocytes, or mixtures thereof. Immune cells can be virus-specific, expressing CAR, expressing TCR, expressing BiTE, or a combination thereof. In some embodiments, the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells (DCs), mast cells, eosinophils, and / or basophils. This document also provides methods for generating and engineering immune cells, as well as methods for using and administering cells for adoptive cell therapy, in which case the cells can be autologous or allogeneic. Therefore, immune cells can be used as immunotherapies, such as targeting cancer cells. These immune cells can be used as a single cell type or as a combination of multiple immune cell types for therapy. In specific embodiments, immune cells are CD3+, CD4+, CD8+, CD16+, or mixtures thereof.

[0143] Immune cells can be isolated from subjects, particularly human subjects. Immune cells can be obtained from subjects of interest, such as subjects suspected of having a specific disease or condition, subjects suspected of having a susceptibility to a specific disease or condition, or subjects undergoing treatment for a specific disease or condition. Immune cells can be collected from any location in the subject where they are present, including but not limited to blood, umbilical cord blood, spleen, thymus, lymph nodes, and bone marrow. Isolated immune cells can be used directly, or they can be stored for a period of time, such as through freezing.

[0144] Immune cells can be enriched / purified from any tissue in which they are present, including but not limited to blood (including blood collected from blood banks or cord blood banks), spleen, bone marrow, tissue removed and / or exposed during surgical procedures, and tissue obtained through biopsy procedures. The tissue / organ from which immune cells are enriched, isolated, and / or purified can be isolated from living and non-living subjects, where the non-living subject is an organ donor. In certain embodiments, immune cells are isolated from blood, such as peripheral blood or cord blood. In some aspects, immune cells isolated from cord blood have enhanced immunomodulatory capabilities, such as those measured by suppression of CD4-positive or CD8-positive T cells. In specific aspects, immune cells are isolated from pooled blood, particularly pooled cord blood, to enhance immunomodulatory capabilities. The pooled blood may come from two or more sources, such as three, four, five, six, seven, eight, nine, ten, or more sources (e.g., donor subjects).

[0145] Immune cell populations can be obtained from subjects requiring therapy or suffering from diseases associated with reduced immune cell activity. Therefore, the cells are autologous for the subjects requiring therapy. Alternatively, immune cell populations can be obtained from donors, such as partially or completely tissue-compatible donors or completely tissue-incompatible donors. Immune cell populations can be collected from the peripheral blood, umbilical cord blood, bone marrow, spleen, or any other organ / tissue in which immune cells are present, of the subject or donor. Immune cells can be isolated from a bank of the subject and / or donor, such as from pooled umbilical cord blood.

[0146] When a population of immune cells is obtained from a donor different from the subject, the donor can be allogeneic, provided that the obtained cells are compatible with the subject, i.e., they can be introduced into the subject. Allogeneic donor cells may or may not be compatible with human leukocyte antigens (HLA).

[0147] A. T cells

[0148] In some implementations, the immune cells are T cells. Over the past two decades, several fundamental approaches for the derivation, activation, and expansion of functional anti-tumor effector cells have been described. These include: autologous cells, such as tumor-infiltrating lymphocytes (TILs); T cells activated in vitro using autologous dendritic cells or PBMCs, lymphocytes, artificial antigen-presenting cells (APCs), soluble or plastic-coated antibodies, or beads coated with T cell ligands and activating antibodies, or cells separated by capturing target cell membranes; allogeneic cells naturally expressing anti-host tumor T cell receptors (TCRs); and non-tumor-specific autologous or allogeneic donor-derived or iPSC-derived cells that have been genetically reprogrammed or “redirected” to express tumor-responsive TCRs, CARs, or BiTEs. These approaches have yielded numerous protocols for T cell preparation and immunization that can be used in the methods described herein.

[0149] In some implementations, the T cells are derived from blood, bone marrow, lymph, umbilical cord, or lymphatic organs. In some aspects, the cells are human cells. The cells are typically primary cells, such as cells isolated directly from the subject and / or cells isolated from and frozen from the subject. In some implementations, the cells include one or more subsets of T cells or other cell types, such as the entire T cell population, CD4+, etc. + Cells, CD8 + Cells and their subpopulations, such as those defined by: function, activation state, maturity, differentiation potential, expansion, recycling capacity, localization and / or persistence, antigen specificity, antigen receptor type, presence in a specific organ or compartment, biomarker or cytokine secretion profile, and / or degree of differentiation. For the subject to be treated, cells may be allogeneic and / or autologous. In some aspects, such as with off-the-shelf technologies, cells are pluripotent and / or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the method includes isolating cells from the subject, preparing, processing, culturing and / or engineering these cells as described herein, and reintroducing them into the same patient before or after cryopreservation.

[0150] In T cell subtypes and subsets (e.g., CD4) + and / or CD8 + T cells are either naïve or naïve T cells. N ) cells, effector T cells (T cells) EFF ), memory T cells and their subtypes, such as stem cell memory T cells (TSCs). M Central Memory T (TC) M ), effect memory T (T EMThis includes terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, and helper T cells such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α / β T cells, and γ / δ T cells. In some implementations, the T cells are γ / δ T cells.

[0151] In some implementations, cells that are positive for or negative for a specific biomarker (such as a surface biomarker) are enriched or removed from one or more T cell populations. In some cases, such biomarkers are those that are absent or expressed at relatively low levels in some T cell populations (e.g., non-memory cells), but present or expressed at relatively high levels in some other T cell populations (e.g., memory cells).

[0152] In some implementations, T cells are isolated from PBMC samples by negative selection of markers (such as CD14) expressed on non-T cells (such as B cells, monocytes, or other leukocytes). In some aspects, CD4... + or CD8 + Select the step for separating CD4 + Support and CD8 + Cytotoxic T cells. These are CD4 cells. + and CD8 + The population can be further sorted into subpopulations by positive or negative selection of biomarkers that are expressed or expressed at relatively high levels in one or more naive, memory, and / or effector T cell subpopulations.

[0153] In some implementations, CD8 + T cells are further enriched or depleted of primary, central memory, effector memory, and / or central memory stem cells through methods such as positive or negative selection based on surface antigens associated with the corresponding subsets. In some embodiments, central memory T cells (T0) are further enriched or depleted. CM Enrichment of memory cells or stem cells to enhance efficacy, such as improving long-term survival, expansion and / or engraftment after administration, is particularly robust in some respects in this subpopulation.

[0154] In some implementations, the T cells are autologous T cells. In this method, a tumor sample is obtained from the patient, and a single-cell suspension is obtained. The single-cell suspension can be obtained in any suitable manner, for example, mechanically (e.g., using gentleMACS). TMDissociators (such as Miltenyi Biotec, Auburn, and Calif) or enzymatic digests (e.g., collagenases or DNases) are used to digest tumor cells. Single-cell suspensions of tumor enzyme digests are cultured in interleukin-2 (IL-2) or other growth factors.

[0155] The cultured T cells can pool and rapidly expand. Rapid expansion provides at least a 50-fold (e.g., 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more) increase in the number of antigen-specific T cells over a period of about 10 to about 14 days. More preferably, rapid expansion provides at least a 200-fold (e.g., 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold or more) increase over a period of about 10 to about 14 days.

[0156] Expansion can be accomplished by any of a variety of methods known in the art. For example, T cells can be rapidly expanded using nonspecific T cell receptor stimulation in the presence of fed lymphocytes and interleukin-2 (IL-2) or interleukin-15 (IL-15) (preferably IL-2). This nonspecific T cell receptor stimulation may include about 30 ng / ml of OKT3, a mouse monoclonal anti-CD3 antibody (available from Ortho-McNeil®, Raritan, NJ). Alternatively, T cells can be rapidly expanded in vitro by stimulating peripheral blood mononuclear cells (PBMCs) with one or more antigens of cancer (including their antigenic portions, such as epitopes, or cells) in the presence of T cell growth factors (such as 300 IU / ml IL-2 or IL-15, preferably IL-2), said antigens optionally expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide or a peptide bound to other MHC class I or II molecules. In vitro induced T cells can be rapidly expanded by repeated stimulation with the same cancer antigen, which is pulsed onto antigen-presenting cells expressing HLA-A2 or other HLA molecules. In vitro induced T cells can also be expanded in the absence of antigen-presenting cells.

[0157] Autologous T cells can be modified to express T cell growth or differentiation factors that promote the growth, differentiation, and activation of autologous T cells. Suitable T cell growth factors include, for example, interleukin (IL)-2, IL-7, IL-15, IL-18, IL-21, and IL-12. Suitable modification methods are known in the art. See, for example, Sambrook et al. Molecular Cloning: A Laboratory ManualThird edition, edited by [author's name], Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al. Current Protocols in Molecular Biology , Greene Publishing Associates and John Wiley & Sons, NY, 1994. In a particular aspect, the modified autologous T cells express T cell growth factors at high levels. T cell growth factor coding sequences, such as those for IL-12, and promoters that can be operably linked thereto are readily available in the art, and operable linkage of these promoters to T cell growth factor coding sequences promotes high-level expression.

[0158] B. NK cells

[0159] In some implementations, the immune cells are natural killer (NK) cells. NK cells are a subset of lymphocytes that exhibit spontaneous cytotoxicity against a variety of tumor cells, virus-infected cells, and some normal cells in the bone marrow and thymus. NK cells differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus. NK cells can be detected by specific surface markers such as CD16, CD56, and / or CD8 in humans. NK cells do not express T-cell antigen receptors, the pan-T marker CD3, or surface immunoglobulin B-cell receptors.

[0160] In some implementations, NK cells are derived from human peripheral blood mononuclear cells (PBMCs), unstimulated leukocyte removal products (PBSCs), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, tissues, or umbilical cord blood using methods known in the art.

[0161] C. NKT cells

[0162] Natural killer T (NKT) cells are a heterogeneous population of T cells, sharing characteristics of both T cells and natural killer cells. Many of these cells recognize non-polymorphic CD1d molecules, antigen-presenting molecules that bind to both self- and exogenous lipids and glycolipids. They constitute only about 0.1% of all peripheral blood T cells. NKT cells are a subset of T cells that co-express αβ T cell receptors and also express several molecular markers typically associated with NK cells, such as NK1.1. Invariant natural killer T (iNKT) cells highly express the transcriptional regulator promyelocytic leukemia zinc finger protein and depend on this regulator for their development. Currently, there are five major distinct iNKT cell subsets. These subsets produce different groups of cytokines upon activation. Isotypes iNKT1, iNKT2, and iNKT17 are similar to Th cell subsets in cytokine production. In addition, there are subtypes specifically for T follicle helper-like functions and IL-10-dependent regulatory functions.

[0163] D. Congenital lymphocytes

[0164] Innate lymphoid cells (ILCs) are a group of innate immune cells derived from common lymphoid progenitors (CLPs) and belonging to the lymphoid lineage. These cells are defined as lacking antigen-specific B or T cell receptors due to the absence of the recombination activating gene (RAG). ILCs do not express myeloid or dendritic cell markers. They play a role in protective immunity and the regulation of homeostasis and inflammation in the body; therefore, their dysregulation can lead to immunopathologies such as allergies, bronchial asthma, and autoimmune diseases. ILCs can be classified according to the cytokines they can produce and the transcription factors that regulate their development and function.

[0165] E. Cell Culture

[0166] In some embodiments, the engineered cells can be cultured for at least about 10 days to about 40 days, at least about 15 days to about 35 days, at least about 15 days to 21 days, such as at least about 15, 16, 17, 18, 19, or 21 days. In some embodiments, the cells of this disclosure can be cultured for no more than 60 days, or no more than 50 days, or no more than 45 days. Cells can be cultured for 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days. Cells can be cultured in the presence of a liquid culture medium. Typically, the culture medium may contain a basal culture medium formulation known in the art. Many basal culture medium formulations can be used to culture the cells described herein, including but not limited to Eagle Minimum Essential Medium (MEM), Dulbecco Modified Eagle Medium (DMEM), α Modified Minimum Essential Medium (α-MEM), Basal Essential Medium (BME), Iscove Modified Dulbecco Medium (IMDM), BGJb Medium, F-12 Nutrient Mixture (Ham), Liebovitz L-15, DMEM / F-12, Essential Modified Eagle Medium (EMEM), RPMI-1640, and modifications and / or combinations thereof. The composition of the aforementioned basal media is well known in the art, and altering or adjusting the concentration of the medium and / or supplements according to the needs of the cultured cells is within the skill of those skilled in the art. In some embodiments, the culture medium formulation may be explant medium (CEM) consisting of IMDM supplemented with 10% fetal bovine serum (FBS), 100 U / ml penicillin G, 100 μg / ml streptomycin, and 2 mmol / L L-glutamine. Other embodiments may employ further basal culture medium formulations, such as those selected from the formulations described above.

[0167] Any culture medium capable of supporting cells in vitro can be used for cell culture. Culture medium formulations that can support cell growth include, but are not limited to, Dulbecco Modified Eagle Medium (DMEM), α Modified Minimal Essential Medium (αMEM), and Roswell Park Memorial Institute Medium 1640 (RPMI Media 1640). Typically, up to 20% fetal bovine serum (FBS) or 1-20% horse serum is added to the aforementioned media to support cell growth. However, specific media may also be used if the growth factors, cytokines, and hormones required for cell culture are provided in the medium at appropriate concentrations. Culture media used in the methods of this disclosure may contain one or more compounds of interest, including but not limited to antibiotics, mitotic compounds, or differentiation compounds suitable for cell culture. Cells can be grown at temperatures from 27°C to 40°C (e.g., 31°C to 37°C) and can be grown in a humidified incubator. Carbon dioxide levels can be maintained at 2% to 10%, and oxygen levels can be maintained at 1% to 22%. However, this disclosure should not be construed as limiting oneself to any particular method of isolating and culturing cells. Conversely, any method of isolating and culturing cells should be interpreted as being included in this disclosure.

[0168] For cell culture, the culture medium may be provided with one or more further components. For example, additional supplements may be used to provide cells with essential trace elements and substances for optimal growth and expansion. Such supplements include insulin, transferrin, selenium salts, and combinations thereof. These components may be contained in salt solutions, such as, but not limited to, Hanks' Balanced Salt Solution (HBSS) and Earle's Salt Solution. Further antioxidant supplements, such as β-mercaptoethanol, may be added. While many culture media already contain amino acids, some amino acids may be added later, such as L-glutamine, which is known to be poorly stable in solution. The culture medium may further provide antibiotics and / or antifungal compounds, such as, typically, a mixture of penicillin and streptomycin, and / or other compounds, examples including, but not limited to, amphotericin B, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampin, spectinomycin, tetracycline, tylosin, and zeocin. Supplementing the cell culture medium with mammalian plasma or serum was also considered. Plasma or serum typically contains cytokines and components essential for cell viability and expansion. The use of suitable serum alternatives was also considered.

[0169] References to specific buffers, culture media, reagents, cells, culture conditions, etc., or subclasses thereof, are not intended to be limiting, but should be interpreted to include all such relevant materials that a person skilled in the art would consider interesting or valuable in the specific context in which the discussion is presented. For example, one buffer system or culture medium can often be substituted for another, thereby using different but known methods to achieve the same objective as that oriented towards the use of the proposed methods, materials, or compositions. In a particular embodiment, cells are cultured in a cell culture system containing cell culture medium, preferably in a culture vessel, particularly in a cell culture medium supplemented with suitable and defined substances for protecting cells from in vitro aging and / or inducing nonspecific or specific reprogramming.

[0170] F. Cell generation

[0171] Some methods of this disclosure involve culturing cells obtained from human tissue samples. In specific embodiments of this disclosure, cells are plated onto a matrix that allows cells to adhere thereto. This can be done, for example, by plated cells in a culture plate that displays one or more matrix surfaces compatible with cell adhesion. Cell adhesion between the cells and the matrix surface may occur when said one or more matrix surfaces come into contact with a cell suspension (e.g., a suspension in a culture medium) introduced into the culture system. Thus, in some embodiments, cells are introduced into a culture system characterized by at least one matrix surface that is generally compatible with cell adhesion thereto, such that plated cells can contact said matrix surface; such embodiments include plated onto a matrix that allows cells to adhere thereto.

[0172] Cells disclosed herein can be identified and characterized by expressing specific biomarker proteins, such as cell surface markers. Detection and isolation of these cells can be achieved, for example, by flow cytometry, ELISA, and / or magnetic beads. Reverse transcription polymerase chain reaction (RT-PCR) can be used to quantify cell-specific genes and / or monitor changes in gene expression in response to differentiation. In some embodiments, the biomarker proteins used for cell identification and characterization are selected from a list of CD3, CD4, CD8, CD5, CD7, CD45RA, CD45RO, CD45, and any combinations thereof.

[0173] XI. Pharmaceutical Compositions

[0174] This document also provides pharmaceutical compositions and formulations comprising immunocellular therapies and pharmaceutically acceptable carriers. The cells disclosed herein can be cultured and infused in common culture medium formulations known in the art. Examples of such formulations are described below.

[0175] Pharmaceutically acceptable carriers are generally non-toxic to recipients at the doses and concentrations used, and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl parabens (such as methylparaben or propylparaben); catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10). The following are pharmaceutically acceptable carriers: polypeptides (residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers described herein further include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX). ® (Baxter International, Inc.). In one aspect, sHASEGP is combined with one or more other glycosaminoglycans, such as chondroitinase.

[0176] A. Combination therapy

[0177] In some embodiments, the compositions and methods of this embodiment involve the use of immunocellular therapy in combination with at least one additional therapy. The additional therapy may be radiation therapy, surgery (e.g., tumor resection and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, antibody-drug conjugates, siRNA therapy, vaccination, or a combination of the above therapies. The additional therapy may be in the form of adjuvant or neoadjuvant therapy.

[0178] In some embodiments, the additional therapy is the administration of a small molecule enzyme inhibitor or an anti-transfer agent. In some embodiments, the additional therapy is the administration of a side effect limiter (e.g., an agent designed to reduce the occurrence and / or severity of treatment side effects, such as an antinausea agent). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a therapy targeting the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. In some embodiments, the additional therapy is monoclonal antibody therapy or an antibody-based therapy. In some embodiments, the additional therapy is an immunomodulator, such as an antibody against PD-1, PDL-1, CTLA-4, or other checkpoint therapies, or an immune activator, such as an IL2, IL7, IL15, IL-12, IL-18, or a STING agonist. The additional therapy may be one or more chemotherapeutic agents known in the art.

[0179] Cell therapy can be administered before, during, after, or in various combinations with other cancer therapies, such as immune checkpoint therapy. The intervals between administrations can range from simultaneous to minutes to days to weeks. In implementations that separately administer immunocellular therapy and other therapeutic agents to a patient, it is generally ensured that the time between each administration is not too long so that the two compounds can still exert a beneficial combined effect on the patient. In such cases, it may be considered to administer antibody therapy and anticancer therapy to the patient within approximately 12 to 24 hours or 72 hours of each other, more specifically within approximately 6 to 12 hours of each other. In some cases, it may be desirable to significantly extend the duration of treatment, in which case the intervals between corresponding administrations may be several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks).

[0180] Multiple combinations can be used. In the following example, immunotherapy is designated "A," and anticancer therapy is designated "B":

[0181]

[0182] Administering any compound or therapy according to this embodiment to a patient will follow the general protocol for administering such compounds, taking into account the toxicity of the agent (if any). Therefore, in some embodiments, there are steps for monitoring toxicities attributable to the combination therapy.

[0183] 1. Chemotherapy

[0184] Multiple chemotherapy agents can be used according to this implementation plan. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; polyacetic acids (especially bulbatacin and bulbatacinone); camptothecins (including the synthetic analog topotecan); and lichenin... Yostatin; Callystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin, and bizelesin); Nostocin (especially Nostocin 1 and Nostocin 8); Dolastatin; Duocarmycin (including synthetic analogues KW-2189 and CB1-TM1); Elutherobin; Pancratistatin; Sarcodictyin; Spongistatin; Nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, and mechlorethamine hydrochloride oxide. oxidehydrochloride, melphalan, novombhichin, phenesterine, prednimustine, trofosfamide, and uracil mustard;Nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamycin, especially calicheamycin γ1I and calicheamycin ωI1); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; and neocarcinostatin chromophores. Chromophores and related chromophores of alkenyne antibiotics, including aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin C, carabicin, carminomycin, carzinophilin, chromomycinis, and dactinomycin. Daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholine doxorubicin, cyanomorpholine doxorubicin, 2-pyrrolinoline doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, and mitomycins, such as mitomycin C. C) Mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin;Antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine (a Fiber lysine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and fluxuridine; androgens, such as calusterone and dromostanolone. Propionate, epitiostanol, mepitiostane, and testolactone; antiadrenergic drugs such as mitotane and trilostane; folic acid supplements such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate. acetate); epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansine, such as maytansine and ansamitocins; mitoguazone; mitoxantrone;Mopidanmol; Nitraerine; Pentostatin; Phenamet; Pirarubicin; Losoxantrone; Podophyllinic acid; 2-ethylhydrazide; Procarbazine; PSK polysaccharide complex; Razoxane; Rhizoxin; Sizofiran; Spirogermanium; Tenuazonic acid; Triaziquone; 2,2',2''-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, Verracurin A) A), roridin A and anguidine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactalol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide Taxanes, such as paclitaxel and docetaxel; gemcitabine; 6-thioguanine; mercaptopurine; platinum complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (xeloda);Ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid derivatives, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicamycin, gemcitabine, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, and any pharmaceutically acceptable salts, acids, or derivatives thereof.

[0185] 2. Radiotherapy

[0186] Other factors that cause DNA damage and have been widely used include the targeted delivery of what are commonly referred to as gamma rays, X-rays, and / or radioactive isotopes to tumor cells. Other forms of DNA damage factors, such as microwaves, proton beam irradiation, and UV irradiation, have also been considered. All of these factors are most likely to cause extensive damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray dose ranges from 50 to 200 roentgens per day for extended periods (3 to 4 weeks) to 2000 to 6000 roentgens per single dose. Radioactive isotope dose ranges vary considerably and depend on the isotope's half-life, the intensity and type of emitted radiation, and the uptake by tumor cells.

[0187] 3. Immunotherapy

[0188] Technicians should understand that immunotherapy can be used in combination with or in conjunction with the methods employed in the treatment plan. In the context of cancer treatment, immunotherapy typically relies on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN) ® This is one such example. For instance, an immune effector can be an antibody that is specific to certain markers on the surface of tumor cells. An antibody alone can act as an effector for a therapy, or it can recruit other cells to actually achieve cell killing. Antibodies can also be conjugated to drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc.) and used as a targeting agent. Alternatively, an effector can be a lymphocyte carrying a surface molecule that interacts directly or indirectly with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0189] Antibody-drug conjugates (ADCs) comprise monoclonal antibodies (Mabs) covalently linked to cytotoxic drugs and can be used in combination therapies. This approach combines the high specificity of a Mab against its antigenic target with a highly potent cytotoxic drug to create an "armed" MAb that delivers the payload (drug) to tumor cells with enriched levels of said antigen. Targeted delivery of the drug also minimizes its exposure to normal tissues, thereby reducing toxicity and improving the therapeutic index. Exemplary ADC drugs include ADCETRIS. ® (brentuximab vedotin) and KADCYLA ® (Trastuzumab emtansine or T-DM1).

[0190] In one aspect of immunotherapy, tumor cells must carry certain markers suitable for targeting, i.e., markers that are absent in most other cells. Many tumor markers exist, and any one of them can be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialic acid Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, erb B2, and p155. An alternative aspect of immunotherapy is combining anticancer effects with immunostimulatory effects. Immunostimulatory molecules also exist, including: cytokines such as IL-2, IL-4, IL-12, GM-CSF, and γ-IFN; chemokines such as MIP-1, MCP-1, and IL-8; and growth factors such as FLT3 ligand.

[0191] Examples of immunotherapy include immune adjuvants such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds; cytokine therapies such as interferon α, β, and γ, IL-1, GM-CSF, and TNF; gene therapies such as TNF, IL-1, IL-2, and p53; and monoclonal antibodies such as anti-CD20, anti-ganglioside GM2, and anti-p185. One or more anticancer therapies may be used in conjunction with the antibody therapies described herein.

[0192] In some implementations, immunotherapy can be an immune checkpoint inhibitor. Immune checkpoints either upregulate (e.g., co-stimulatory molecules) or downregulate signaling. Inhibitory immune checkpoints that can be targeted by immune checkpoint blockade include: adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene 3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain Ig suppressor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA 4.

[0193] Immune checkpoint inhibitors can be drugs such as small molecules, recombinant forms of ligands or receptors, or, in particular, antibodies such as human antibodies. Inhibitors of known immune checkpoint proteins or analogues thereof can be used, especially chimeric, humanized, or human-form antibodies. Those skilled in the art will appreciate that alternative and / or equivalent names may be used for certain antibodies mentioned in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of this disclosure. For example, lambrolizumab is also known by the alternative and equivalent names MK 3475 and pembrolizumab.

[0194] In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In one specific aspect, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In one specific aspect, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, a PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In one specific aspect, the PDL2 binding partner is PD-1. The antagonist may be an antibody, its antigen-binding fragment, an immunoadhesin, a fusion protein, or an oligopeptide.

[0195] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular portion of PDL1 or PDL2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence) or a PD-1 binding portion). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab is also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO. ® It is a usable anti-PD-1 antibody. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, and KEYTRUDA. ® SCH-900475 is an exemplary anti-PD-1 antibody. CT-011, also known as hBAT or hBAT-1, is also an anti-PD-1 antibody. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor.

[0196] Another immune checkpoint that can be targeted using the methods presented in this article is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is present on the surface of T cells and acts as a “switch” when it binds to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily and is expressed on the surface of helper T cells, transmitting inhibitory signals to them. CTLA4 is similar to the T cell costimulatory protein CD28; both molecules bind to CD80 and CD86 (also known as B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA4 is also present in regulatory T cells and is likely important for their function. T cell activation via T cell receptors and CD28 leads to increased expression of CTLA-4, which is an inhibitory receptor for the B7 molecule.

[0197] In some implementations, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, an immunoadhesin, a fusion protein, or an oligopeptide.

[0198] Anti-human CTLA-4 antibodies (or their VH and / or VL domains) suitable for this method can be generated using methods known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. Exemplary anti-CTLA-4 antibodies are ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments thereof and variants thereof. In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes with the aforementioned antibody for binding to and / or binding to the same epitopes on CTLA-4. In another embodiment, the antibody has at least about 90% amino acid sequence identity in the variable region with the aforementioned antibody (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).

[0199] 4. Surgery

[0200] Approximately 60% of people diagnosed with cancer will undergo some type of surgery, including preventative, diagnostic or staging, curative, and palliative surgeries. Curative surgeries include resection, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed. This can be combined with other therapies, such as treatments in this protocol, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or replacement therapy. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery).

[0201] After partial or complete removal of cancer cells, tissue, or tumors, cavities may form in the body. Treatment can be performed by perfusion, direct injection, or local application of additional anticancer therapies to the area. This treatment can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may also be administered at different doses.

[0202] 5. Other pharmaceuticals

[0203] Other agents may be used in combination with certain aspects of this embodiment to enhance the therapeutic efficacy. These additional agents include agents that affect the upregulation of cell surface receptors and gap (GAP) junctions, cell inhibitors and differentiation agents, cell adhesion inhibitors, agents that increase the sensitivity of overproliferating cells to apoptosis inducers, or other biological agents. Increasing intercellular signaling by increasing the number of gap junctions will increase the anti-overproliferative effect against neighboring overproliferating cell populations. In other embodiments, cell inhibitors or differentiation agents may be used in combination with certain aspects of this embodiment to enhance the anti-overproliferative efficacy of the treatment. Cell adhesion inhibitors are considered to enhance the efficacy of this embodiment. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin.

[0204] XII. Products or reagent kits

[0205] This document also provides articles or kits comprising polynucleotides, peptides, immune cells, antibodies, reagents, buffers, or combinations thereof. The articles or kits may further include a packaging insert containing instructions for using immune cells to treat or delay the progression of cancer in an individual or to enhance the immune function of an individual with cancer. The articles or kits may include any antigen-specific immune cells described herein. Suitable containers include, for example, bottles, vials, bags, and syringes. Containers may be formed from a variety of materials, such as glass, plastics (such as polyvinyl chloride or polyolefins), or metal alloys (such as stainless steel or Hastelloy). In some embodiments, the container contains the formulation, and a label on or associated with the container may indicate usage instructions. The articles or kits may further include other materials required from a commercial and user perspective, including additional buffers, diluents, filters, needles, syringes, and packaging inserts with instructions for use. In some embodiments, the articles further include one or more additional reagents (e.g., chemotherapeutic agents and antitumor agents). Suitable containers for such one or more reagents include, for example, bottles, vials, bags, and syringes.

[0206] XIII. Sequences used in certain implementations

[0207] Table 2: Polypeptide sequences disclosed herein.

[0208]

[0209]

[0210]

[0211]

[0212] As used herein, "protein" or "peptide" refers to a molecule containing at least five amino acid residues. As used herein, the term "wild-type" refers to an endogenous version of a molecule that is naturally present in an organism. In some embodiments, a wild-type version of a protein or peptide is employed; however, in many embodiments of this disclosure, a modified protein or peptide is employed to generate an immune response. The above terms are used interchangeably. "Modified protein" or "modified peptide" or "variant" refers to a protein or peptide whose chemical structure (particularly its amino acid sequence) is altered relative to a wild-type protein or peptide. In some embodiments, the modified / variant protein or peptide has at least one modified activity or function (recognizing that a protein or peptide can have multiple activities or functions). It is particularly considered that a modified / variant protein or peptide may be altered for one activity or function but retain wild-type activity or function in other respects, such as immunogenicity.

[0213] When a protein is specifically referred to herein, it generally means a natural (wild-type) or recombinant (modified) protein, or optionally a protein in which any signal sequence has been removed. The protein can be isolated directly from an organism in which it is naturally present, produced by a recombinant DNA / exogenous expression method, or produced by solid-phase peptide synthesis (SPPS) or other in vitro methods. In a particular embodiment, a recombinant vector comprises an isolated nucleic acid fragment and a nucleic acid sequence incorporating a polypeptide (e.g., an antibody or a fragment thereof). The term “recombinant” may be used in conjunction with the name of a polypeptide or a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has already been manipulated in vitro or a replication product of such a molecule.

[0214] In some embodiments, the size of the protein or polypeptide (wild-type or modified) may include, but is not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or 525 amino acid residues or more, and any range from which they may be derived, or derivatives of the corresponding amino sequences described or cited herein. It is anticipated that peptides can be mutated through truncation, making them shorter than their corresponding wild-type forms. Furthermore, they can be altered by fusing or conjugating heterologous protein or peptide sequences with specific functions (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.). As used herein, the term "domain" refers to any unique functional or structural unit of a protein or peptide, and generally refers to an amino acid sequence having a structure or function recognizable to those skilled in the art.

[0215] The polypeptides, proteins, or polynucleotides encoding such polypeptides or proteins disclosed herein may include 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any derivable range thereof) or more variant amino acid or nucleic acid substitutions.Or, respectively, with at least or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, of SEQ ID NO: 1-12. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more consecutive amino acids or nucleotides,Or any range derived therefrom that is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range derived therefrom) similar, identical, or homologous.

[0216] In some embodiments, the protein or polypeptide may contain SEQ ID NO: Amino acids 1-12: 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81. 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 20 7, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267,268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 32 6, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 44 3, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500 or 501.

[0217] In some embodiments, the polypeptide, protein, or nucleic acid may each contain SEQ ID NO: At least, at most, or exactly 1, 2, 10, or 11: 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77. 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524、525、526、527、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、567、568、569、570、571、572、573、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616、617、618、619、620、621、622、623、624、625、626、627、628、629、630、631、632、633、634、635、636、637、638、639、640、641、642、643、644、645、646、647、648、649、650、651、652、653、654、655、656、657、658、659、660、661、662、663、664、665、666、667、668、669、670、671、672、673、674、675、676、677、678、679、680、681、682、683、684、685、686、687、688、689、690、691、692、693、694、695、696、697、698、699、700、701、702、703、704、705、706、707、708、709、710、711、712、713、714、715、716、717、718、719、720、721、722、723、724、725、726、727、728、729、730、731、732、733、734、735、736、737、738、739、740、741、742、743、744、745、746、747、748、749、750、751、752、753、754、755、756、757、758、759、760、761、762、763、764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 85 5, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 8 86, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 94 7, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 (or any derivable range thereof) consecutive amino acids or nucleotides, each being at least one of SEQ ID NO: 1-12.At most or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any of the derivative ranges thereof) similar, identical, or homologous.

[0218] Variant peptides

[0219] The following is a discussion of altering amino acid subunits of proteins to produce equivalent, or even improved, second-generation variant polypeptides or peptides. For example, certain amino acids can replace other amino acids in a protein or polypeptide sequence, potentially resulting in a significant loss of binding ability to interact with structures such as antigen-binding regions of antibodies or binding sites on substrate molecules. Since the interacting abilities and properties of a protein define its functional activity, certain amino acid substitutions can be made in the protein sequence and its corresponding DNA-coding sequence, still producing proteins with similar or desired properties. Therefore, the inventors anticipate that various alterations can be made in the DNA sequence of the gene encoding the protein without a significant loss of its biological utility or activity.

[0220] The term "functionally equivalent codon" is used in this paper to refer to a codon that encodes the same amino acid, such as the six different codons for arginine. "Neutral substitution" or "neutral mutation" is also considered, which refers to a change in one or more codons that encodes a biologically equivalent amino acid.

[0221] The amino acid sequence variants disclosed herein may be substitution, insertion, or deletion variants. Compared to SEQ ID NO: 1 and / or SEQ ID NO: 2, variations of the polypeptides disclosed herein may affect 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more non-continuous or continuous amino acids. Variants may contain at least 50%, 60%, 70%, 80%, or 90% (inclusive) of the same amino acid sequence as any sequence provided or cited herein. Variants may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more substituted amino acids.

[0222] It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as correspondingly additional N-terminal or C-terminal amino acids, or 5' or 3' sequences, but remain substantially identical to those shown in one of the sequences disclosed herein, provided that the sequence meets the criteria set forth above, including maintaining biological protein activity in the context of protein expression. The addition of terminal sequences is particularly applicable to nucleic acid sequences, which may, for example, include various non-coding sequences flanking the 5' or 3' portions of a coding region.

[0223] Deletion variants typically lack one or more residues from the native or wild-type protein. This can involve the deletion of a single residue or multiple consecutive amino acids. Stop codons can be introduced (through substitution or insertion) to encode nucleic acid sequences to produce truncated proteins.

[0224] Insertion mutants typically involve adding amino acid residues at a non-terminal point of a peptide. This can include the insertion of one or more amino acid residues. Terminal additions can also be produced, and fusion proteins can be included, which are multimers or concatenators of one or more peptides or polypeptides described or cited herein.

[0225] Substitution variants typically involve exchanging one amino acid for another at one or more sites within a protein or polypeptide, and can be programmed to modulate one or more properties of the polypeptide with or without the loss of other functions or properties. Substitution can be conserved, meaning that one amino acid is replaced by another amino acid with similar chemical properties. "Conservative amino acid substitution" can involve replacing a member of one amino acid class with another member of the same class. Conservative substitutions are well known in the art and include, for example, the following variations: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamate; cysteine ​​to serine; glutamine to asparagine; glutamate to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Conservative amino acid substitutions can encompass amino acid residues that are not naturally occurring and are typically incorporated through chemical peptide synthesis rather than synthesis in biological systems. These include other reversed or inverted forms of peptides or amino acid moieties.

[0226] Alternatively, substitution can be "non-conservative," affecting the function or activity of the polypeptide. Non-conservative changes typically involve replacing an amino acid residue with a chemically dissimilar amino acid residue, such as replacing a non-polar or uncharged amino acid with a polar or charged amino acid, or vice versa. Non-conservative substitution can involve replacing a member of one amino acid class with a member of another class.

[0227] Those skilled in the art can use known techniques to identify suitable variants of the peptides as described herein. Those skilled in the art can identify suitable intramolecular regions that can be altered without destroying activity by targeting regions considered unimportant to activity. Skilled artisans will also be able to identify conserved amino acid residues and molecular motifs among similar proteins or peptides. In a further embodiment, conserved amino acid substitutions can be made to regions that are biologically important or structurally important without significantly altering biological activity or adversely affecting the protein or peptide structure.

[0228] When making such changes, the hydrophobicity index of amino acids can be considered. A protein's hydrophobicity map is calculated by assigning a numerical value ("hydrophilicity index") to each amino acid and then averaging these values ​​repeatedly along the peptide chain. Each amino acid is assigned a value based on its hydrophobic and charge properties. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The importance of hydrophilicity / hydrophobicity indexes in conferring interacting biological functions to proteins is widely recognized in the art (Kyte et al., J. Mol. Biol. 157:105-131 (1982)). It is generally accepted that the relative hydrophilicity / hydrophobicity of amino acids contributes to the secondary structure of the resulting protein or polypeptide, which in turn defines the interaction of the protein or polypeptide with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. It is also known that certain amino acids can be substituted with other amino acids having similar hydrophilicity / hydrophobicity indices or scores and still retain similar biological activities. In some embodiments, substitutions based on hydrophilicity / hydrophobicity indices include amino acid substitutions within ±2. In some aspects of this disclosure, substitutions of amino acids with hydrophilicity / hydrophobicity indices within ±1 are included, and in other aspects, substitutions of amino acids with hydrophilicity / hydrophobicity indices within ±0.5 are included.

[0229] It should also be understood in the art that similar amino acid substitutions can be efficiently performed based on hydrophilicity. As noted by reference in U.S. Patent 4,554,101, which is incorporated herein by reference, the maximum local average hydrophilicity of a protein (controlled by the hydrophilicity of its adjacent amino acids) is related to the protein's biological properties. In some embodiments, the maximum local average hydrophilicity of a protein (controlled by the hydrophilicity of its adjacent amino acids) is related to its immunogenicity and antigen binding, i.e., related to the protein's biological properties. The following hydrophilicity values ​​have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). When modifications are made based on similar hydrophilicity values, some embodiments include substitution of amino acids with hydrophilicity values ​​within ±2, others include substitution of amino acids with hydrophilicity values ​​within ±1, and still others include substitution of amino acids with hydrophilicity values ​​within ±0.5. In some cases, epitopes can also be identified from the primary amino acid sequence based on hydrophilicity. These regions are also referred to as “epitope core regions.” It should be understood that an amino acid can be substituted with another amino acid having a similar hydrophilicity value and still produce a biologically and immunologically equivalent protein.

[0230] Furthermore, those skilled in the art can review structure-function studies identifying activity- or structurally important residues in similar peptides or proteins. Based on such comparisons, one can predict the importance of amino acid residues in the protein corresponding to activity- or structurally important amino acid residues in similar proteins. Those skilled in the art can choose chemically similar amino acids to replace the important amino acid residues used for this prediction.

[0231] Those skilled in the art can also analyze the three-dimensional structure and amino acid sequence associated with this structure in similar proteins or peptides. Given such information, those skilled in the art can predict the arrangement of amino acid residues of an antibody relative to its three-dimensional structure. Those skilled in the art may choose not to alter the predicted amino acid residues on the protein surface, as such residues may be involved in important interactions with other molecules. Furthermore, those skilled in the art can generate test variants containing a single amino acid substitution at each desired amino acid residue. These variants can then be screened for binding and / or activity using standard assays, generating information gathered from such routine experiments that allows those skilled in the art to determine the amino acid positions where further substitution should be avoided, alone or in combination with other mutations. Various tools available for determining secondary structure can be found at expasy.org / proteomics / protein_structure.

[0232] In some embodiments of this disclosure, amino acid substitutions are made that: (1) reduce sensitivity to proteolysis, (2) reduce sensitivity to oxidation, (3) alter the binding affinity of the protein complex, (4) alter the binding affinity of the ligand or antigen, and / or (5) impart or modify other physicochemical or functional properties to such peptides. For example, single or multiple amino acid substitutions (in some embodiments, conserved amino acid substitutions) can be made in naturally occurring sequences. Substitutions can be made in portions of the antibody located outside the domain(s) forming intermolecular contacts. In such embodiments, conserved amino acid substitutions that substantially do not alter the structural characterization of the protein or peptide can be used (e.g., one or more substituted amino acids that do not disrupt the secondary structure characterizing the native antibody).

[0233] XIV. Examples

[0234] The following embodiments are included to illustrate specific implementations of this disclosure. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques that the inventors have discovered to perform well in the practice of this invention, and therefore can be considered as preferred modes of practice. However, based on this disclosure, those skilled in the art will understand that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the invention, and similar or related results can still be obtained. It should be understood that the methods used in the following embodiments can be used to test the efficacy of other combinations of SSR and AgRICA.

[0235] Example 1

[0236] This embodiment describes a strategy to enhance the cytotoxicity of immune effector cells against cells with low or heterogeneous expression of target antigens. Here, we show that co-expression of the synaptic stabilizing receptor (SSR) on chimeric antigen receptor (CAR) T cells increases their cytotoxicity against acute myeloid leukemia (AML) cells with low antigen expression and prolongs survival in a human AML xenograft mouse model. The CD38-targeting model SSR also enhanced leukemia cell apoptosis in a CAR-independent manner and protected therapeutic T cells from the CD38-specific cytotoxic antibody daratumumab, enabling combination immunotherapy. In general, this can be used to enhance the antitumor activity of engineered immune effector cells against antigenically heterogeneous tumors. Furthermore, the co-expression of SSRs on immune effector cells enables "Boolean AND" gating, where target cells expressing low levels of the AgRICA target antigen are more effectively killed if they also express the SSR antigen.

[0237] The limitations of current treatments can be overcome by engineering alternative antigen receptors that do not trigger T cell cytotoxicity themselves. Instead, these receptors enhance T cell activation from the primary CAR or TCR receptor by stabilizing cell-cell interactions and immune synapse formation. Because such synaptic stabilizing receptors (SSRs) are non-cytotoxic, they can target a broad range of antigens that are otherwise difficult to target with conventional cytotoxic CARs (e.g., CD38, EGFR, etc.), thereby enhancing activity against a wider range of targetable tumors.

[0238] In this embodiment, we used AML as a model disease and CLL1 (CLEC12) as a model antigen to develop and characterize SSRs targeting CD38 (an antigen widely expressed in both normal and malignant lymphocytes and myeloid cells). We showed that co-expression of CD38 on CLL1 CAR T cells enhanced their targeting of CLL1 in vitro and in vivo. 低 SSR enhances apoptosis in leukemia cells by cross-linking CD38 on the cell surface and masks CD38 expression on activated T cells, thereby protecting them from the CD38-specific cytotoxic antibody daratumumab. Figure 1 Therefore, this SSR can be combined with various CARs targeting lymphoid and myeloid leukemia to offset the risk of antigen downregulation, and can be used in combination therapy with daratumumab without damaging therapeutic T cells.

[0239] The generation of CD38 SSR-armed CAR T cells.

[0240] To generate CD38-specific SSRs, we selected a CD38-specific binder derived from the monoclonal antibody clone HB7 in a model publication (WO / 1999 / 062526). This binder is fused to both the CD8 spacer region and the transmembrane region, with the cysteine ​​164 mutation of CD8α to a serine residue to prevent heterodimerization with CARs 8. In some designs, the SSR contains an intracellular signaling domain at the 3' end of the CD8 transmembrane region. Most vectors have an alternative marker, NGFR, separated from the SSR by an internal ribosome entry site (IRES). Figure 2A In addition to conventional co-stimulatory signaling, we also included the motif of the T cell activation adaptor (LAT), a key linker that provides a central scaffold for the binding of the central supramolecular cluster of the TCR, initiating a signaling cascade of cytoskeleton remodeling, degranulation, and T cell activation. By co-transducing vectors encoding CD38 SSR and CAR in a retroviral system, stable co-expression of CLL1 CAR with truncated (TC) CD38 SSR lacking an intracellular domain or CD27-co-stimulated SSR, or CD28-co-stimulated SSR, and with an SSR possessing a cytoplasmic domain from amino acids 28 to 177 (LAT177) derived from LAT, was detected 7 days post-transduction. Figure 2B This process aims to generate CD38 SSR-armed CAR-T cells. SSR-TC (SEQ ID NO: 1), SSR-CD28 (SEQ ID NO: 2), and SSR-LAT177 (SEQ ID NO: 5) do not affect CAR T cell proliferation, while co-transduction of CD38 CARs may reduce T cell viability due to CD38-specific cannibalism. Figure 2C CD27 co-stimulated SSR expression impaired CAR T cell expansion, possibly due to the toxicity derived from tetanic TNFR-TRAF2 signaling previously characterized in our laboratory. Figure 2D and 2E ).

[0241] CD38 SSR-LAT177 improved the cytolytic activity of CAR-T cells against leukemia cells with low antigen density.

[0242] The activity of CD38 SSR-armed CLL1 CAR T cells was evaluated by co-culturing them with the CD38+ AML cell line Molm-13, which expresses low surface levels of CLL1. Figure 3A and 3BAfter 3 days of co-culture, co-expression of truncated CD38 SSRs slightly reduced tumor counts. CLL1 CAR T cells armed with CD38 SSR-CD28 and CD38 SSR-LAT177 showed enhanced Molm-13 cell killing comparable to CLL1 / CD38 dual CAR-T cells and promoted significant T cell proliferation during co-culture, reflecting increased T cell activation. Figure 3C and Figure 3D However, co-expression of SSR-CD28 on CLL1 CAR T cells also increased the killing of CLL1-negative CD38-positive T-ALL cells (CCRF-CEM), which may reflect non-specific killing of CD38+ targets after heterodimerization of CARs co-stimulated with CD28. Figure 3E and 3F Therefore, the SSR-CD28 construct was excluded from subsequent analyses. To further validate the efficacy of SSR in CAR sensitivity, we co-cultured CLL1.CAR.SSR T cells with CLL1-low THP1 cell derivatives obtained by sorting and purifying THP1 cells expressing dim levels of CLL1 (CLL1.CAR.SSR T cells). Figure 3G Consistent with results obtained using Molm13, expression of CD38.SSR significantly enhanced the cytotoxicity of CLL1.CAR T cells against THP1-low CLL1 cells, while unarmed CAR T cells showed only limited activity. Figure 3H and 3I ).

[0243] When co-cultured with Molm-13, co-expression of CD38 SSR-LAT177 significantly enhanced cytokine production and degranulation (CD107α / granzyme B) in CLL1 CAR T cells. Figure 4E -F), reflecting the improved immune synapse formation of SSR-armed CLL1 CAR T cells with CLL1-low leukemia cells.

[0244] CD38 SSR-LAT177 inhibits the progression of systemic leukemia in an AML xenograft mouse model.

[0245] To evaluate the role of SSR in CAR T cell-mediated tumor control in vivo, we utilized two mouse xenograft models of human AML. In the first model, we transplanted Molm13 cells into NSG mice lacking mouse MHC class I and II genes to reduce nonspecific xenograft reactivity of human T cells (NSG-MHCKO). Three days after AML injection, we injected a single dose of control untransduced T cells or T cells expressing only CLL1.CAR or a combination of CLL1.CAR and CD38.SSR. Figure 5 A). Quantitative analysis of peripheral blood AML cells via continuous tail vein bleeding showed that control and unarmed CLL1.CAR T cells provided only minimal disease control, while SSR-armed T cells suppressed leukemia proliferation in peripheral blood and significantly prolonged animal survival. Figure 5 B and 5C). SSR co-expression also increased the extent of CAR T cell expansion in peripheral blood during the first 10 days after infusion (B and 5C). Figure 5 D), consistent with our in vitro observations. Compared to unarmed CLL1.CAR T cells, SSR-armed CLL1.CAR T cells had a higher frequency of CD62L+CD45RA+ naïve T cells at 19 days post-infusion. Figure 5 E). To validate these findings in another model of AML with suboptimal target antigen expression, we modified CLL1-low THP-1 cells to express firefly luciferase and transplanted them into NSG-MHCKO mice, followed by a single injection of CAR T cells (E). Figure 5 F). Similarly, CLL1.CAR T cells showed poor activity against low-antigen leukemia, and untransduced or SSR-only T cells had minimal effect on tumor growth (if any). Figure 5 G and 5H). In contrast, SSR-armed CLL1.CAR T cells controlled systemic leukemia in most animals, leading to their long-term survival (G and 5H). Figure 5 I).

[0246] To validate these findings in another model of AML with suboptimal target antigen expression, we modified CLL1-low THP-1 cells to express firefly luciferase and transplanted them into NSG-MHCKO mice, followed by a single injection of CAR T cells (… Figure 5 F). Similarly, CLL1.CAR T cells showed poor activity against low-antigen leukemia, and untransduced or SSR-only T cells had minimal effect on tumor growth (if any). Figure 5 G and 5H). In contrast, SSR-armed CLL1.CAR T cells controlled systemic leukemia in most animals, leading to their long-term survival (G and 5H). Figure 5 I).

[0247] Co-targeting CD38 via SSR does not enhance detumescent toxicity and does not increase the risk of bone marrow ablation.

[0248] Conventional approaches to combination antigen targeting involve the co-expression of two cytotoxic receptors, enabling targeting of single antigen deletion mutants while amplifying detumescent cytotoxicity. We compared the activity of CLL1 CAR T cells armed with cytotoxic CD38 CARs or non-cytotoxic CD38 SSRs against normal CD38+ cells. In mature circulating cells, CD38 was expressed at the highest levels on CLL1+ monocytes and CLL1-NK cells (…). Figure 6 A). Co-expression of CD38.SSR does not enhance the cytotoxicity of CLL1.CAR T cells against autologous monocytes, nor does it produce cytotoxicity against CLL1-negative autologous T cells, B cells, and NK cells. Figure 6 B) or allogeneic NK cells ( Figure 6 C) activity. In contrast, co-expression of cytotoxic CD38.CAR predictably produces cell lysis of CLL1-negative, CD38+ NK cells ( Figure 6 B and 6C). In addition to mature blood cells, CD38 is expressed in primitive hematopoietic progenitor cells. To evaluate the activity of CD38.SSR-armed CLL1.CAR T cells against hematopoietic progenitor cells, we purified CD34+ cells from umbilical cord blood (B and 6C). Figure 6 D), and co-cultured them with control untransduced or armed and unarmed CLL1.CAR T cells at an effector-target ratio of 10:1 for 5 hours, and then cultured on semi-solid methylcellulose medium supporting myeloid differentiation for 12 days. We did not observe any activity of CLL1.CAR T cells with or without SSR against CLL1-negative erythroid progenitors (BFU-E, Figure 6 D). Although CLL1.CAR T cells are expected to target granulocyte / monocyte progenitor cells (CLL1+), the addition of SSR does not increase this cytotoxicity (CFU-GM, Figure 6 D). In contrast, co-expression of cytotoxic CD38.CAR led to the ablation of both types of CD38+ progenitor cells (D). Figure 6 D). Therefore, CD38.SSR does not exacerbate the toxicity of CAR T cells against normal CD38+ tissues.

[0249] CAR-independent tumor cell killing via CD38 SSR

[0250] CD38 has been clinically targeted by monoclonal antibodies (including the FDA-approved daratumumab), which mediate the killing of CD38-high leukemia tumor cells at least partially by cross-linking CD38 on the cell surface and activating apoptosis signaling, without impairing the transplantation of hematopoietic stem cells and progenitor cells that also express low levels of CD38. 7. We hypothesize that SSRs can specifically mediate similar activity by cross-linking CD38 on the surface of CD38-high tumor cells upon contact. Although SSR expression alone does not significantly inhibit leukemia growth at a low E:T ratio ( Figure 3H We co-cultured SSR-transduced T cells with CD38-high monocyte AML line THP1 and T-ALL line CCRF-CEM at a high E:T ratio to potentially enhance CD38 crosslinking activity. Figure 7 A). Although untransduced T cells produced only marginal nonspecific killing at an E:T ratio of 4:1, T cells transduced with truncated CD38 SSRs further promoted tumor apoptosis in both high CD38 parental lines at E:T ratios of 2:1 to 4:1, but not in CD38-KO derivatives. Figure 7 B and 7C). As mentioned, even at an E:T ratio of 10:1, SSR expression does not exert any toxicity on CD38+HSPC ( Figure 6 This indicates that SSR has the ability to induce CAR-independent, CD38-specific apoptosis in leukemia cells without adverse effects on primary tissues. This CAR-independent killing potentially provides the ability to eliminate minimal residual leukemia clones in patients that have completely lost their CAR target antigens.

[0251] SSR masks CD38 expression on T cells and protects them from being targeted by daratumumab.

[0252] In the context of adoptive cell therapy, the combination of cell and antibody immunotherapy has been explored. One example of this combination is the co-administration of a CD20 antibody (rituximab) with engineered NK cells capable of antibody-mediated cytotoxicity (ADCC). However, extending this approach to CD38 / daratumumab is more challenging because upregulation of CD38 on activated immune effector cells makes them targets for antibodies. We sought to determine whether CD38-targeting SSRs bind to CD38 on the cell surface, thereby masking it from external antibodies. Indeed, we observed complete masking of surface CD38 in CAR T cells expressing SSRs compared to unmodified controls. Figure 8 A and 8B). In the presence of daratumumab, SSR masking of CD38 prevents NK cell-mediated ADCC, thereby protecting CD38 SSR T cells ( Figure 8C). Therefore, this activity enables combination therapy of CAR T cells and daratumumab without the risk of damaging adoptive T cells.

[0253] Example 2

[0254] CD38 SSR improves the anti-tumor function of T cells expressing TCR.

[0255] To evaluate the utility of SSRs beyond CARs, we tested their activity in T cells expressing tumor-specific T-cell receptors (TCRs). TCRs target intracellular antigens presented on surface MHC molecules and share major signaling pathways with CARs. To assess whether SSRs could enhance TCR-mediated tumor cell lysis, we co-expressed CD38 SSR-TC and CD38 SSR-LAT177 in T cells transduced with a survivin-specific TCR. This TCR recognizes the survivin peptide presented in HLA-A2, allowing the elimination of HLA-high B-cell leukemia without damaging normal proliferating cells that also express survivin. Figure 9 AB). SSR expression on survivin TCR+ T cells derived from HLA-A2+ donors did not affect their proliferation, indicating a lack of T cell cannibalism (AB). Figure 9 C). Although survivin-TCR T cells showed limited effect against HLA-A2+ leukemia cells, expression of CD38SSR-LAT177 improved the killing of HLA-A2-high B-cell leukemia BV173 at a low E:T ratio and extended the cytotoxicity of survivin-TCR to HLA-A2-low AML cells THP1 (C). Figure 9 DE). In short-term co-culture with THP1, CD38 SSR-LAT177 induced the production of IFN-γ and TNF-α in both CD8 and CD4 TCR+ populations, as well as the release of CD107a+ / granzyme B+ lysates from CD8 cells, further supporting the enhancement of TCR activation by CD38 SSR-LAT177. Figure 9 F and 9G).

[0256] Example 3

[0257] CD38 SSR promotes T-cell killing of TdT+ leukemia cells by TdT / HLA-A2-specific BiTE redirection.

[0258] Next, we evaluated whether CD38 LAT177.SSR could enhance the killing of low-antigen targets recognized by TCR or BiTE. We co-cultured T cells expressing a chimeric TCR (cTCR) that was specific for the TdT peptide presented on HLA-A2 by leukemia cells. We found that TdT.cTCR T cells exhibited significantly higher cytotoxicity against TdT+ TALL-1 leukemia cells. Figure 10 A). Furthermore, in the presence of soluble BiTE that recognizes the same TdT-HLA-A2 antigen, SSR-expressing T cells recognize TdT+ leukemia cells TALL-1 and P12 ICHIKAWA (…). Figure 10 B). These results indicate that SSR enhances T cell cytotoxicity against low-antigen target cells mediated by soluble BiTE or chimeric TCR.

[0259] Example 4

[0260] Truncation of LAT at amino acid 177 improves SSR function and T cell phenotype.

[0261] We compared the activity of full-length (FL) and truncated (LAT177) SSRs in CLL1 CAR-armed T cells. The truncated LAT177 retained tyrosine residues Y132 and Y171, essential for docking with PLCg, GADS, and Grb2, but lacked tyrosine residues Y191 and Y226, which are the primary docking sites for Grb2. Figure 11A Co-expression of SSR-LATFL and CLL1.CAR significantly reduced the expansion of dual-transduced T cells, possibly reflecting excessively tetanic CAR signaling amplified by full-length LAT, since T cells expressing only SSR-LATFL exhibited normal expansion. Figure 11B LAT177 truncation attenuated this effect and improved the expansion of SSR-armed CLL1.CAR T cells. Figure 11B Unlike LATFL, CD38.SSR carrying LAT177 has minimal impact on the differentiation status and subset composition of expanded T cells. Figure 11C Furthermore, T cells expressing SSR-LATFL alone exhibited significant cytotoxicity against CD38+ target cell lines Molm-13 and CCRF-CEM, indicating spontaneous activation of cytotoxic signaling after conjugation of the full-length LAT intracellular domain. Figure 11D In contrast, T cells expressing SSR-LAT177 exhibit minimal activity against the same target cells. Figure 11D This indicates that LAT truncation is required to minimize the background cytotoxicity of SSRs.

[0262] According to this disclosure, all methods disclosed and claimed herein can be performed and carried out without improper experimentation. While the compositions and methods of the invention have been described according to preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the methods and steps or order of steps described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically relevant reagents may replace those described herein while yielding the same or similar results. All such similar substitutions and modifications that will be apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the invention as defined in the appended claims.

[0263] References

[0264] The following references are specifically incorporated herein by reference to the extent that they provide exemplary procedures or other details that supplement the content described herein.

[0265]

[0266]

Claims

1. A composition comprising a polypeptide, said polypeptide comprising an antigen receptor domain, a transmembrane domain, and a modified LAT domain or an unmodified LAT domain.

2. The composition of claim 1, further comprising a second polypeptide, the second polypeptide comprising an antigen receptor (AgRICA) for immune cell activation having a second antigen receptor domain, wherein the second antigen receptor domain and the antigen receptor domain bind to different antigens or epitopes.

3. A composition comprising a polynucleotide encoding a polypeptide, said polypeptide comprising an antigen receptor domain, a transmembrane domain, and a modified LAT domain or an unmodified LAT domain.

4. The composition of claim 3, further comprising a second polynucleotide encoding a second polypeptide comprising AgRICA having a second antigen receptor domain, wherein the second antigen receptor domain and the antigen receptor domain bind to different antigens, optionally wherein the polynucleotide and the second polynucleotide are contained on one or more carriers.

5. The composition according to any one of claims 1-4, wherein the modified LAT domain has at least 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or greater sequence similarity to SEQ ID NO:

8.

6. The composition according to any one of claims 1-5, wherein the binding domain is anti-CD38 or anti-EGFR.

7. The composition according to any one of claims 2 and 4-6, wherein the second antigen receptor domain binds to 5T4, 8H9, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EphA3, EpCAM, folate receptor-α FAP, FBP, Fetal AchR, FR, GD2, G250 / CAIX, GD3, Phosphatidylinositol Proteoglycan-3 (GPC3), Her2, IL-13Rα2, Lambda, Lewis-Y, Kappa, KDR, MAGE, MCSP, Mesothelin, Muc1, Muc16, NCAM, NKG2D Ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, Survivin, TAG72, TEMs, Carcinoembryonic Antigen, HMW-MAA, AFP, CA-125, ETA, GRP78 Tyrosinase, MAGE, Laminin Receptor, HPV E6, E7, BING-4, calcium-activated chloride channel 2, cyclin B1, 9D7, EphA3, telomerase, SAP-1, B melanoma antigen, cancer / testis antigen, melanoma-associated antigen, sarcoma antigen, CT antigen, NY-ESO-1 / LAGE-1, SSX-2, Melan-A / MART-1, GP100 / pmel17, TRP-1 / -2, P. polypeptide, MC1R, prostate-specific antigen, β-catenin, BRCA1 / 2, CML66, fibronectin, tendinin C, MART-2, TGF-βRII, CLL1, TdT, or MICA, MICB, NKG2D ligand, Claudin18.2, Claudin 6, cadherin 17.

8. The composition according to any one of claims 1-6, wherein the transmembrane domains or extracellular domains of AgRICA and SSR do not undergo heterodimerization.

9. The composition according to any one of claims 2 and 4-8, wherein the AgRICA is CAR, TCR or BiTE.

10. The composition according to claim 9, wherein the transmembrane domain of the SSR is CD8a.

11. The composition according to any one of claims 1-9, wherein the polypeptide further comprises a hinge.

12. A cell comprising the composition according to any one of claims 1-11.

13. The cell of claim 12, wherein the cell is an immune effector cell.

14. The cell according to claim 13, wherein the immune effector cell is a regulatory T cell, CD4+, or other similar cell. + T cells, CD8 + T cells, αβ T cells, γ-δ T cells, NK cells, invariant NKT cells, NKT cells, innate lymphocytes, B cells, dendritic cells, macrophages, cytotoxic T cells, MAIT cells, virus-specific T cells, iPSC-derived T cells, or mixtures thereof.

15. The cell of claim 13, wherein the immune effector cell is a T cell.

16. A plurality of cells according to any one of claims 12-15.

17. The plurality of cells according to claim 16, wherein the plurality of cells are frozen.

18. A method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of a plurality of cells according to any one of claims 12-17 and / or a composition according to any one of claims 1-11.

19. The method of claim 18, wherein the cancer is a blood cancer.

20. The method of claim 18, wherein the cancer is a solid tumor.

21. The method according to any one of claims 18-20, further comprising administering at least a second therapeutic agent to the subject.

22. The method of claim 21, wherein the at least second therapeutic agent comprises chemotherapy, immunotherapy, surgery, radiotherapy, drug therapy, targeted therapy, hormone therapy, biological therapy, or combinations thereof.

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