Engineered tgfβ receptors repurposed for il-9 signaling and their use in immune cells

Abstract

An engineered TGFβ receptor system is provided that is expressed in an immune cell (e.g., a T cell), upon TGFβ binding, redirects to transduce an immune-supportive interleukin 9 (IL-9) signal, rather than the usual immune-suppressive TGFβ signal mediated by endogenous TGFβ receptors expressed in the immune cell. The engineered TGFβ receptor system includes two engineered chimeric TGFβ receptor chains whose extracellular portions encode the extracellular domains of TGFβR1 and TGFβR2, respectively, and whose intracellular portions encode the intracellular domains of IL9R and IL2γ, respectively. T cells modified with such engineered TGFβ receptor systems exhibit strong JAK-STAT signaling and, upon TGFβ stimulation, especially under repeated antigen stimulation, exhibit high survival and proliferation capacity. Such engineered immune cells are expected to have enhanced persistence and improved control in treating solid tumors that are typically characterized by an immune-suppressive environment rich in TGFβ.

Description

Background Technology

[0001] One obstacle to adoptive cell therapy (ACT) in treating solid tumors is the suppressive tumor microenvironment (TME), and TGFβ has been identified as one of the major influencing factors. As a soluble immunosuppressive cytokine, TGFβ inhibits anti-tumor immune responses through various mechanisms, including, for example, inhibiting T effector cell activation while promoting T regulatory cell differentiation. High levels of TGFβ expression have been reported in various solid tumors such as liver cancer, breast cancer, prostate cancer, glioma, bladder cancer, and thymic epithelial tumors, posing a significant challenge to effective tumor immunotherapy.

[0002] Another obstacle to ACT is the poor persistence of infused cells in the patient's body. Some types of ACT require the simultaneous infusion of high doses of interleukin-2 or IL-2 (i.e., aldesleukin) to maintain cell persistence in order to produce a robust and prolonged antitumor response; however, this often results in systemic toxicity in patients.

[0003] Therefore, these two obstacles need to be addressed in order to develop immunotherapies with enhanced efficacy and reduced toxicity. Summary of the Invention

[0004] To address the two aforementioned obstacles, this disclosure provides an engineered chimeric TGFβ receptor expressed in immune cells (e.g., T lymphocytes or T cells) that, upon TGFβ binding, is repurposed to transduce immune-supporting interleukin-9 (IL-9) signaling, rather than the immunosuppressive TGFβ signaling mediated by endogenous TGFβ receptors. Compared to unmodified immune cells, immune cells engineered to express this engineered chimeric TGFβ receptor exhibit significantly better survival and proliferative capacity after repeated or sustained antigen stimulation, particularly in solid tumors with a TGFβ-rich immunosuppressive environment, while unmodified immune cells typically experience T cell exhaustion or activation-induced cell death upon repeated antigen stimulation. Such engineered immune cells are expected to offer enhanced persistence and better control in solid tumors, thus holding promise for improving current adoptive cell therapies.

[0005] In the first aspect, an engineered transforming growth factor β (TGFβ) receptor chain is provided.

[0006] When expressed in immune cells, this engineered TGFβ receptor chain encodes a transmembrane polypeptide comprising an extracellular portion and an intracellular portion operatively linked by the transmembrane portion. The extracellular portion comprises the extracellular domain (ECD) of either TGFβR1 or TGFβR2, or a functional portion or variant thereof; and the intracellular portion comprises the intracellular domain (ICD) of either IL9R or IL2Rγ, or a functional portion or variant thereof.

[0007] In the engineered TGFβ receptor chain, the transmembrane portion may comprise a transmembrane domain (TM) of any transmembrane protein, such as TGFβR1, TGFβR2, IL2Rγ, IL4R, IL9R, IL7R, IL15R, IL10R, IL21R, CD28, CTLA4, PD1, TIM3, TIGIT, CD4, CD8, or 4-1BB, or a functional portion or variant thereof. In some embodiments, the transmembrane portion comprises a transmembrane domain (TM) of one of TGFβR1, TGFβR2, IL2Rγ, or IL9R, or a functional portion or variant thereof.

[0008] According to some embodiments of the engineered TGFβ receptor chain, the extracellular portion comprises an ECD of TGFβR1 containing a sequence having at least 70% identity with SEQ ID NO: 1; and the intracellular portion comprises an ICD of IL9R containing a sequence having at least 70% identity with SEQ ID NO: 3.

[0009] In some embodiments, the transmembrane portion comprises a transmembrane domain (TM) of TGFβR1, which contains a sequence having at least 70% identity with SEQ ID NO: 5. In some embodiments, the engineered TGFβ receptor chain contains a sequence having at least 70% identity with SEQ ID NO: 9.

[0010] In some further embodiments, the transmembrane portion comprises a transmembrane domain (TM) of IL9R, which contains a sequence having at least 70% identity with SEQ ID NO: 7. In some embodiments, the engineered TGFβ receptor chain contains a sequence having at least 70% identity with SEQ ID NO: 10.

[0011] According to some other embodiments of the engineered TGFβ receptor chain, the extracellular portion comprises an ECD of TGFβR1 containing a sequence having at least 70% identity with SEQ ID NO: 1; and the intracellular portion comprises an ICD of IL2Rγ containing a sequence having at least 70% identity with SEQ ID NO: 4.

[0012] In some embodiments, the transmembrane portion comprises a transmembrane domain (TM) of TGFβR1, which contains a sequence having at least 70% identity with SEQ ID NO: 5. In some embodiments, the engineered TGFβ receptor chain contains a sequence having at least 70% identity with SEQ ID NO: 11.

[0013] In some further embodiments, the transmembrane portion comprises a transmembrane domain (TM) of IL2Rγ, which contains a sequence having at least 70% identity with SEQ ID NO: 8. In some embodiments, the engineered TGFβ receptor chain contains a sequence having at least 70% identity with SEQ ID NO: 12.

[0014] According to some other embodiments of the engineered TGFβ receptor chain, the extracellular portion comprises an ECD of TGFβR2 containing a sequence having at least 70% identity with SEQ ID NO: 2; and the intracellular portion comprises an ICD of IL9R containing a sequence having at least 70% identity with SEQ ID NO: 3.

[0015] In some embodiments, the transmembrane portion comprises a transmembrane domain (TM) of TGFβR2, which contains a sequence having at least 70% identity with SEQ ID NO: 6. In some embodiments, the engineered TGFβ receptor chain contains a sequence having at least 70% identity with SEQ ID NO: 13.

[0016] In some further embodiments, the transmembrane portion comprises a transmembrane domain (TM) of IL9R, which contains a sequence having at least 70% identity with SEQ ID NO: 7. In some embodiments, the engineered TGFβ receptor chain contains a sequence having at least 70% identity with SEQ ID NO: 14.

[0017] According to some other embodiments of the engineered TGFβ receptor chain, the extracellular portion comprises an ECD of TGFβR2 containing a sequence having at least 70% identity with SEQ ID NO: 2; and the intracellular portion comprises an ICD of IL2Rγ containing a sequence having at least 70% identity with SEQ ID NO: 4.

[0018] In some embodiments, the transmembrane portion comprises a transmembrane domain (TM) of TGFβR2, which contains a sequence having at least 70% identity with SEQ ID NO: 6. In some embodiments, the engineered TGFβ receptor chain contains a sequence having at least 70% identity with SEQ ID NO: 15.

[0019] In some further embodiments, the transmembrane portion comprises a transmembrane domain (TM) of IL2Rγ, which contains a sequence having at least 70% identity with SEQ ID NO: 8. In some embodiments, the engineered TGFβ receptor chain contains a sequence having at least 70% identity with SEQ ID NO: 16.

[0020] In a second aspect, an engineered TGFβ receptor system is further provided, which substantially comprises at least one engineered TGFβ receptor chain, each engineered TGFβ receptor chain being based on an engineered TGFβ receptor chain according to any embodiment provided in the first aspect as described above.

[0021] Here, the engineered TGFβ receptor system may comprise a first engineered TGFβ receptor chain and a second engineered TGFβ receptor chain. It is configured such that the extracellular portion of the first engineered TGFβ receptor chain and the extracellular portion of the second engineered TGFβ receptor chain respectively contain an ECD of TGFβR1 or a functional portion or variant thereof, and an ECD of TGFβR2 or a functional portion or variant thereof, or vice versa. Furthermore, it is configured such that the intracellular portion of the first engineered TGFβ receptor chain and the intracellular portion of the second engineered TGFβ receptor chain respectively contain an ICD of IL9R or a functional portion or variant thereof, and an ICD of IL2Rγ or a functional portion or variant thereof, or vice versa.

[0022] According to some embodiments of the engineered TGFβ receptor system, the extracellular and intracellular portions of the first engineered TGFβ receptor chain respectively comprise: an ECD of TGFβR1 or a functional portion or variant thereof, the ECD comprising a sequence having at least 70% identity with SEQ ID NO: 1; and an ICD of IL9R or a functional portion or variant thereof, the ICD comprising a sequence having at least 70% identity with SEQ ID NO: 3. The extracellular and intracellular portions of the second engineered TGFβ receptor chain respectively comprise: an ECD of TGFβR2 or a functional portion or variant thereof, the ECD comprising a sequence having at least 70% identity with SEQ ID NO: 2; and an ICD of IL2Rγ or a functional portion or variant thereof, the ICD comprising a sequence having at least 70% identity with SEQ ID NO: 4.

[0023] Furthermore, in some embodiments of the engineered TGFβ receptor system, the transmembrane portion of the first engineered TGFβ receptor chain comprises a TGFβR1 TM or a functional portion or variant thereof, the TM comprising a sequence having at least 70% identity with SEQ ID NO: 5; and the transmembrane portion of the second engineered TGFβ receptor chain comprises a TGFβR2 TM or a functional portion or variant thereof, the TM comprising a sequence having at least 70% identity with SEQ ID NO: 6. In some embodiments, the first engineered TGFβ receptor chain comprises a sequence having at least 70% identity with SEQ ID NO: 9; and the second engineered TGFβ receptor chain comprises a sequence having at least 70% identity with SEQ ID NO: 15.

[0024] In further embodiments of the engineered TGFβ receptor system, the transmembrane portion of the first engineered TGFβ receptor chain comprises a TM of IL9R or a functional portion or variant thereof, the TM comprising a sequence having at least 70% identity with SEQ ID NO: 7; and the transmembrane portion of the second engineered TGFβ receptor chain comprises a TM of IL2Rγ or a functional portion or variant thereof, the TM comprising a sequence having at least 70% identity with SEQ ID NO: 8. In some embodiments, the first engineered TGFβ receptor chain comprises a sequence having at least 70% identity with SEQ ID NO: 10; and the second engineered TGFβ receptor chain comprises a sequence having at least 70% identity with SEQ ID NO: 16.

[0025] According to some other embodiments of the engineered TGFβ receptor system, the extracellular and intracellular portions of the first engineered TGFβ receptor chain respectively comprise: an ECD of TGFβR1 or a functional portion or functional variant thereof, the ECD comprising a sequence having at least 70% identity with SEQ ID NO: 1; and an ICD of IL2Rγ or a functional portion or functional variant thereof, the ICD comprising a sequence having at least 70% identity with SEQ ID NO: 4. The extracellular and intracellular portions of the second engineered TGFβ receptor chain respectively comprise: an ECD of TGFβR2 or a functional portion or functional variant thereof, the ECD comprising a sequence having at least 70% identity with SEQ ID NO: 2; and an ICD of IL9R or a functional portion or functional variant thereof, the ICD comprising a sequence having at least 70% identity with SEQ ID NO: 3.

[0026] Furthermore, in some embodiments of the engineered TGFβ receptor system, the transmembrane portion of the first engineered TGFβ receptor chain comprises a TM of TGFβR1 or a functional portion or variant thereof, the TM comprising a sequence having at least 70% identity with SEQ ID NO: 5; and the transmembrane portion of the second engineered TGFβ receptor chain comprises a TM of TGFβR2 or a functional portion or variant thereof, the TM comprising a sequence having at least 70% identity with SEQ ID NO: 6. In some embodiments, the first engineered TGFβ receptor chain comprises a sequence having at least 70% identity with SEQ ID NO: 11; and the second engineered TGFβ receptor chain comprises a sequence having at least 70% identity with SEQ ID NO: 13.

[0027] In further embodiments of the engineered TGFβ receptor system, the transmembrane portion of the first engineered TGFβ receptor chain comprises a TM of IL2Rγ or a functional portion or variant thereof, the TM comprising a sequence having at least 70% identity with SEQ ID NO: 8; and the transmembrane portion of the second engineered TGFβ receptor chain comprises a TM of IL9R or a functional portion or variant thereof, the TM comprising a sequence having at least 70% identity with SEQ ID NO: 7. In some embodiments, the first engineered TGFβ receptor chain comprises a sequence having at least 70% identity with SEQ ID NO: 12; and the second engineered TGFβ receptor chain comprises a sequence having at least 70% identity with SEQ ID NO: 14.

[0028] In some embodiments of the engineered TGFβ receptor system, when the engineered TGFβ receptor system is expressed in immune cells, the immune cells stimulated with TGFβ exhibit activation of the JAK-STAT signaling pathway compared to the absence of TGFβ stimulation.

[0029] In some embodiments of the engineered TGFβ receptor system, when the engineered TGFβ receptor system is expressed in immune cells, the immune cells stimulated with TGFβ exhibit increased phosphorylation of at least one of STAT1, STAT3, or STAT5 compared to when TGFβ stimulation is absent.

[0030] In some embodiments of the engineered TGFβ receptor system, when the engineered TGFβ receptor system is expressed in immune cells, the phosphorylation levels of STAT3 and / or STAT5 in the immune cells stimulated with TGFβ are at least 50% higher than when TGFβ stimulation is absent.

[0031] In some embodiments of the engineered TGFβ receptor system, the extracellular and transmembrane portions of either the first or second engineered TGFβ receptor chain are derived from the same protein, and when the engineered TGFβ receptor system is expressed in immune cells, the phosphorylation levels of STAT3 and / or STAT5 in the TGFβ-stimulated immune cells are at least 2.5 times higher than in the absence of TGFβ stimulation.

[0032] In some embodiments of the engineered TGFβ receptor system, the extracellular, transmembrane, and intracellular portions of the first engineered TGFβ receptor chain comprise the ECD of TGFβR1 or a functional portion or variant thereof, the TM of TGFβR1 or a functional portion or variant thereof, and the ICD of IL9R or a functional portion or variant thereof; and the extracellular, transmembrane, and intracellular portions of the second engineered TGFβ receptor chain comprise the ECD of TGFβR2 or a functional portion or variant thereof, the TM of TGFβR2 or a functional portion or variant thereof, and the ICD of IL2Rγ or a functional portion or variant thereof. When the engineered TGFβ receptor system is expressed in immune cells, the phosphorylation level of STAT3 in the TGFβ-stimulated immune cells is at least 4.0 times that in the absence of TGFβ stimulation.

[0033] In some embodiments of the engineered TGFβ receptor system, when the engineered TGFβ receptor system is expressed in immune cells cultured in the presence of IL-2, the immune cells stimulated with TGFβ exhibit enhanced proliferative capacity compared to when the engineered TGFβ receptor system is not present in the immune cells.

[0034] In some embodiments of the engineered TGFβ receptor system, when the engineered TGFβ receptor system is expressed in immune cells cultured in the absence of IL-2, the immune cells stimulated with TGFβ exhibit a slower reduction in proliferation in the presence of TGFβ1 compared to when the engineered TGFβ receptor system is not present in the immune cells.

[0035] In some embodiments of the engineered TGFβ receptor system, when the engineered TGFβ receptor system is expressed in immune cells cultured in the absence of IL-2, the immune cells stimulated with TGFβ exhibit better viability in the presence of TGFβ1 compared to when the engineered TGFβ receptor system is not present in the immune cells.

[0036] In some embodiments of the engineered TGFβ receptor system, the extracellular and transmembrane portions of either the first or second engineered TGFβ receptor chain are derived from the same protein, and when the engineered TGFβ receptor system is expressed in a population of immune cells cultured in the absence of IL-2, the population of immune cells stimulated with TGFβ has a viability of at least 50% 12 days after IL-2 withdrawal.

[0037] In some embodiments of the engineered TGFβ receptor system, the extracellular, transmembrane, and intracellular portions of the first engineered TGFβ receptor chain comprise an ECD of TGFβR1 or a functional portion or variant thereof, a TM of TGFβR1 or a functional portion or variant thereof, and an ICD of IL9R or a functional portion or variant thereof; and the extracellular, transmembrane, and intracellular portions of the second engineered TGFβ receptor chain comprise an ECD of TGFβR2 or a functional portion or variant thereof, a TM of TGFβR2 or a functional portion or variant thereof, and an ICD of IL2Rγ or a functional portion or variant thereof. When the engineered TGFβ receptor system is expressed in a population of immune cells cultured in the absence of IL-2, the population of immune cells stimulated with TGFβ exhibits a viability of at least 65% 12 days after IL-2 withdrawal.

[0038] In some embodiments of the engineered TGFβ receptor system, when the engineered TGFβ receptor system is expressed in immune cells cultured in the absence of IL-2 and under repeated antigen stimulation, the immune cells stimulated with TGFβ exhibit enhanced proliferative capacity compared to when the engineered TGFβ receptor system is not present in the immune cells.

[0039] In some embodiments of the engineered TGFβ receptor system, the extracellular and transmembrane portions of either the first or second engineered TGFβ receptor chain are derived from the same protein, and when the engineered TGFβ receptor system is expressed in immune cells cultured in the absence of IL-2 and under repeated antigen stimulation, the immune cells stimulated with TGFβ expand at least 300-fold.

[0040] In some embodiments of the engineered TGFβ receptor system, the extracellular, transmembrane, and intracellular portions of the first engineered TGFβ receptor chain comprise an ECD of TGFβR1 or a functional portion or variant thereof, a TM of TGFβR1 or a functional portion or variant thereof, and an ICD of IL9R or a functional portion or variant thereof; and the extracellular, transmembrane, and intracellular portions of the second engineered TGFβ receptor chain comprise an ECD of TGFβR2 or a functional portion or variant thereof, a TM of TGFβR2 or a functional portion or variant thereof, and an ICD of IL2Rγ or a functional portion or variant thereof. When the engineered TGFβ receptor system is expressed in immune cells cultured in the absence of IL-2 and under repeated antigen stimulation, the immune cells stimulated with TGFβ expand at least 500-fold.

[0041] In some embodiments of the engineered TGFβ receptor system, when the engineered TGFβ receptor system is expressed in a population of immune cells cultured in the absence of IL-2 and under repeated antigen stimulation, the population of immune cells stimulated with TGFβ exhibits enhanced viability compared to the population of immune cells in the absence of the engineered TGFβ receptor system.

[0042] In some embodiments of the engineered TGFβ receptor system, when the engineered TGFβ receptor system is expressed in a population of immune cells cultured in the absence of IL-2 and under repeated antigen stimulation, the population of immune cells stimulated with TGFβ has a viability of at least 70% 9 days after IL-2 is withdrawn.

[0043] In a third aspect, a vector system is further provided comprising at least one recombinant vector (hereinafter referred to as the "vector") encoding an engineered TGFβ receptor system according to any embodiment provided in the second aspect above. It is configured such that when the vector system is transduced in an immune cell, the immune cell is able to activate JAK-STAT signaling upon TGFβ stimulation.

[0044] According to some embodiments of the vector system, the engineered TGFβ receptor system comprises a first engineered TGFβ receptor chain and a second engineered TGFβ receptor chain. The extracellular portion of the first engineered TGFβ receptor chain and the extracellular portion of the second engineered TGFβ receptor chain respectively comprise an ECD of TGFβR1 or a functional portion or variant thereof, and an ECD of TGFβR2 or a functional portion or variant thereof, or vice versa. The intracellular portion of the first engineered TGFβ receptor chain and the intracellular portion of the second engineered TGFβ receptor chain respectively comprise an ICD of IL9R or a functional portion or variant thereof, and an ICD of IL2Rγ or a functional portion or variant thereof, or vice versa.

[0045] In some embodiments of the vector system, a first engineered TGFβ receptor chain and a second engineered TGFβ receptor chain in the engineered TGFβ receptor system are encoded in a single vector. Optionally, the first and second engineered TGFβ receptor chains are encoded within an open reading frame (ORF) in the single vector. Further optionally, the ORF contains a polynucleotide encoding a self-cleaving peptide operatively linked to a polynucleotide encoding the first engineered TGFβ receptor chain and a polynucleotide encoding the second engineered TGFβ receptor chain, wherein the self-cleaving peptide is selected from T2A, P2A, F2A, or E2A.

[0046] In some other embodiments, the vector system comprises two vectors that encode a first engineered TGFβ receptor chain and a second engineered TGFβ receptor chain, respectively.

[0047] In some embodiments of the vector system, a first polynucleotide encoding a first signal peptide is operatively linked to a polynucleotide encoding a first engineered TGFβ receptor chain, and a second polynucleotide encoding a second signal peptide is operatively linked to a polynucleotide encoding a second engineered TGFβ receptor chain. Here, the extracellular portions of the first signal peptide and the first engineered TGFβ receptor chain are optionally derived from the same protein, and the extracellular portions of the second signal peptide and the second engineered TGFβ receptor chain are optionally derived from the same protein.

[0048] Here, the vector system may be based on a viral vector, such as a vector derived from simian virus 40 (SV40), adenovirus, adeno-associated virus (AAV), lentivirus, or retrovirus.

[0049] In a fourth aspect, an immune cell is provided that expresses the engineered TGFβ receptor system according to any embodiment provided in the second aspect.

[0050] According to some implementation schemes of the immune cells, when stimulated with TGFβ, the immune cells exhibit activation of the JAK-STAT signaling pathway compared to when TGFβ stimulation is absent.

[0051] Furthermore, in some embodiments, when stimulated with TGFβ, the immune cells exhibit increased phosphorylation of at least one of STAT1, STAT3, or STAT5 compared to when TGFβ stimulation is absent.

[0052] In some embodiments, when the immune cell is stimulated with TGFβ, the phosphorylation level of STAT3 and / or STAT5 in the immune cell is at least 50% higher than when TGFβ stimulation is absent.

[0053] Here, according to some embodiments of the immune cell, the extracellular and transmembrane portions of either the first engineered TGFβ receptor chain or the second engineered TGFβ receptor chain are derived from the same protein, and when the immune cell is stimulated with TGFβ, the phosphorylation level of STAT3 and / or STAT5 in the immune cell is at least 2.5 times that in the absence of TGFβ stimulation.

[0054] In some embodiments, the extracellular, transmembrane, and intracellular portions of the first engineered TGFβ receptor chain comprise the ECD of TGFβR1 or a functional portion or variant thereof, the TM of TGFβR1 or a functional portion or variant thereof, and the ICD of IL9R or a functional portion or variant thereof; and the extracellular, transmembrane, and intracellular portions of the second engineered TGFβ receptor chain comprise the ECD of TGFβR2 or a functional portion or variant thereof, the TM of TGFβR2 or a functional portion or variant thereof, and the ICD of IL2Rγ or a functional portion or variant thereof. When the immune cell is stimulated with TGFβ, the phosphorylation level of STAT3 in the immune cell is at least 4.0 times that in the absence of TGFβ stimulation.

[0055] According to some embodiments of the immune cells, when cultured in the presence of IL-2, the immune cells stimulated with TGFβ exhibit enhanced proliferative capacity compared to those in the absence of the engineered TGFβ receptor system.

[0056] According to some embodiments of the immune cells, when cultured in the absence of IL-2, the immune cells stimulated with TGFβ showed a slower reduction in proliferation in the presence of TGFβ1 compared to when the engineered TGFβ receptor system was absent from the immune cells.

[0057] According to some embodiments of the immune cells, when cultured in the absence of IL-2, the immune cells stimulated with TGFβ exhibit better viability in the presence of TGFβ1 compared to when the engineered TGFβ receptor system is absent from the immune cells.

[0058] In some embodiments of the immune cells, the extracellular and transmembrane portions of either the first engineered TGFβ receptor chain or the second engineered TGFβ receptor chain are derived from the same protein, wherein, when cultured in the absence of IL-2, a population of the immune cells stimulated with TGFβ has a viability of at least 50% 12 days after IL-2 withdrawal.

[0059] In some embodiments, the extracellular, transmembrane, and intracellular portions of the first engineered TGFβ receptor chain comprise an ECD of TGFβR1 or a functional portion or variant thereof, a TM of TGFβR1 or a functional portion or variant thereof, and an ICD of IL9R or a functional portion or variant thereof; and the extracellular, transmembrane, and intracellular portions of the second engineered TGFβ receptor chain comprise an ECD of TGFβR2 or a functional portion or variant thereof, a TM of TGFβR2 or a functional portion or variant thereof, and an ICD of IL2Rγ or a functional portion or variant thereof. When cultured in the absence of IL-2, the population of these immune cells stimulated with TGFβ exhibits a viability of at least 65% 12 days after IL-2 withdrawal.

[0060] According to some embodiments of the immune cells, when cultured in the absence of IL-2 and under repeated antigen stimulation, the immune cells stimulated with TGFβ exhibit enhanced proliferative capacity compared to the immune cells in the absence of the engineered TGFβ receptor system.

[0061] In some embodiments, the extracellular and transmembrane portions of either the first engineered TGFβ receptor chain or the second engineered TGFβ receptor chain are derived from the same protein, wherein the immune cells stimulated with TGFβ expand at least 300-fold when cultured in the absence of IL-2 and under repeated antigen stimulation.

[0062] In some embodiments, the extracellular, transmembrane, and intracellular portions of the first engineered TGFβ receptor chain comprise an ECD of TGFβR1 or a functional portion or variant thereof, a TM of TGFβR1 or a functional portion or variant thereof, and an ICD of IL9R or a functional portion or variant thereof; and the extracellular, transmembrane, and intracellular portions of the second engineered TGFβ receptor chain comprise an ECD of TGFβR2 or a functional portion or variant thereof, a TM of TGFβR2 or a functional portion or variant thereof, and an ICD of IL2Rγ or a functional portion or variant thereof, wherein, when cultured in the absence of IL-2 and under repeated antigen stimulation, the immune cells stimulated with TGFβ expand at least 500-fold.

[0063] According to some embodiments of the immune cells, when cultured in the absence of IL-2 and under repeated antigen stimulation, the population of immune cells stimulated with TGFβ exhibits enhanced viability compared to the immune cells in the absence of the engineered TGFβ receptor system.

[0064] Furthermore, in some embodiments, when cultured in the absence of IL-2 and under repeated antigen stimulation, the population of immune cells stimulated with TGFβ has a survival rate of at least 70% 9 days after IL-2 withdrawal.

[0065] In any embodiment of the immune cell described above, the immune cell may be a T cell, a natural killer (NK) cell, or a tumor-infiltrating lymphocyte (TIL), and the immune cell may further express a target recognition receptor capable of binding to target molecules on target cells. Optionally, the target recognition receptor is selected from chimeric antigen receptors (CARs), T cell receptors (TCRs), or Aspire-TCRs. Aspire-TCRs are disclosed in International Application PCT / US2023 / 085790, the disclosure of which is incorporated herein by reference in its entirety.

[0066] In a fifth aspect, a method for treating tumors in patients in need is further provided, comprising: administering a population of immune cells as described in any embodiment provided in the fourth aspect above.

[0067] Here, according to some embodiments of the method, the immune cells further express a target recognition receptor capable of binding to target molecules on target cancer cells within the tumor, wherein the target recognition receptor is selected from chimeric antigen receptor (CAR), T cell receptor (TCR), or Aspire-TCR.

[0068] According to some embodiments of the method, the tumor is a solid tumor characterized by high levels of TGFβ expression therein, and may be a tumor selected from liver cancer, breast cancer, prostate cancer, glioma, bladder cancer, or thymic epithelial tumor, etc.

[0069] As used in this article, the term “high level of TGFβ expression” is defined as: TGFB1 expression at the mRNA level is at least 10% higher than that in neighboring non-tumor tissues.

[0070] As used herein and throughout the disclosure, “a” or “an” means “at least one (kind)” or “one (kind) or more (kinds)”.

[0071] As used herein, the term "immune cell" can refer to any cell of the following: T lymphocytes (including αβ T cells or γδ T cells), tumor-infiltrating lymphocytes (TILs), natural killer (NK) cells, or NK T cells, or any of these cells that have been engineered (e.g., cells expressing TCRs or chimeric antigen receptors (CARs)). In the embodiments provided in this disclosure, T lymphocytes or T cells are used as illustrative but non-limiting examples of immune cells.

[0072] As used herein, the term "linker" (L), "linker domain," or "linker region" refers to an oligopeptide or polypeptide region of approximately 1 to 100 amino acids in length that links any domain / region together. Linkers may consist of flexible residues such as glycine and serine, allowing adjacent protein domains to move freely relative to each other. Longer linkers may be used when it is necessary to ensure that two adjacent domains do not spatially interfere with each other. Linkers may be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (e.g., P2A, T2A), 2A-like linkers, or their functional equivalents, and combinations thereof. In some embodiments, linkers include small RNA virus 2A-like linkers, the CHYSEL sequence (P2A) of porcine cyclovir, Thoseea asigna virus (T2A), or combinations thereof, variants, and functional equivalents. Other linkers will be apparent to those skilled in the art and may be used in the methods described herein.

[0073] As used herein, the term "functional variant" refers to a sequence variant of a specific functional domain of interest (e.g., TGFβR1 ECD, TGFβR2 ECD, IL9R ICD, IL2Rγ ICD, TGFβR1 TM, or TGFβR2 TM, etc.) with a predefined sequence, containing one or more sequence alterations such as substitution, deletion, insertion, transposition, etc., but still retaining all or part of the functionality of the specific functional domain with the predefined sequence. Similarly, the term "functional portion" refers to a part, not all, of a specific functional domain of interest (e.g., TGFβR1 ECD, TGFβR2 ECD, IL9R ICD, IL2Rγ ICD, TGFβR1 TM, or TGFβR2 TM, etc.) with a predefined sequence, which substantially retains all or part of the functionality of the specific functional domain with the complete sequence.

[0074] As used herein, the terms “increase,” “increase,” “enhance,” “enhanced,” “elevated,” “decrease,” “change,” “altered,” “variable,” “higher,” “lower,” or similar terms refer to a change of at least 10% (e.g., pSTAT3 level) compared to a reference level.

[0075] As used in this paper, the phrase “substantially unchanged” means that the level of the tested variable (e.g., the pSTAT3 level) changes by less than 5% when comparing a later time point to an earlier reference time point. If the change is greater than or equal to 5%, such a change can be considered as an “increase” or “decrease” as referred to in this paper.

[0076] As used in this article, the phrase “target cell” refers to a cell that can be specifically targeted or killed by immune cells.

[0077] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, these sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. The two molecules are identical at that position when the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence. The percentage of identity between two sequences is a function of the number of common positions shared by the two sequences, taking into account the number of vacancies introduced for optimal alignment and the length of each vacancy. For example, the Blossum 62 scoring matrix can be used to perform sequence comparisons and determine the percentage of identity between two sequences with the following parameters: vacancy penalty of 12, vacancy extension penalty of 4, and frameshift vacancy penalty of 5.

[0078] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. This document describes the methods and materials used in this invention; other suitable methods and materials known in the art may also be used. These materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the specification including the definitions shall prevail.

[0079] Other features and advantages of the invention will become apparent from the following detailed description, the accompanying drawings, and the claims. Attached Figure Description

[0080] Figures 1A-1D Block diagrams of two types of engineered transforming growth factor β (TGFβ) receptor systems provided in certain embodiments of this disclosure are shown. Figure 1A and Figure 1C ), and the corresponding TGFβ-responsive signaling complex formed therefrom ( Figure 1B and Figure 1D , respectively corresponding to Figure 1A and Figure 1C ).

[0081] Figures 2A-2D A block diagram is shown that encodes a construct of an engineered TGFβ receptor system according to four embodiments of the present disclosure.

[0082] Figures 3A-3C The transduction efficiency of two TGFβR1R2-IL9R receptors and their effect on T cell proliferation were shown.

[0083] Figures 4A-4C The results show the comparison of pSTAT1 / 3 / 5 signal transduction during TGFβ stimulation.

[0084] Figures 5A-5D The results show a comparison of cell proliferation and survival of T cells cultured with or without IL-2 and with or without TGFβ1.

[0085] Figures 6A-6D The results show a comparison of cell proliferation and survival after repeated antigen stimulation in the presence or absence of IL-2 and in the presence or absence of TGFβ1.

[0086] Figures 7A-7C The transduction efficiency of the TGFβR1R2-IL9R receptor in tumor-infiltrating lymphocytes (TILs) and its effect on cell proliferation or survival were demonstrated.

[0087] Figure 8This study demonstrates the activation of TGFβ-stimulated pSTAT signaling in TILs expressing the TGFβR1R2-IL9R chimeric receptor.

[0088] Figures 9A-9B The effects of TGFβR1R2-IL9R receptor modification on cell expansion were shown in the presence or absence of IL-2 and in the presence or absence of TGFβ1.

[0089] Figures 10A-10B The effects of TGFβR1R2-IL9R receptor modification on T cell proliferation were demonstrated in the presence or absence of IL-2 and in the presence or absence of TGFβ1 following repeated antigen stimulation.

[0090] Figure 11 A sequence list is provided in this disclosure. Detailed Implementation

[0091] This disclosure provides an engineered chimeric transforming growth factor β (TGFβ) receptor system, a recombinant vector system, and engineered immune cells expressing the engineered chimeric TGFβ receptor system, as well as a method for treating patients in need using the engineered immune cells. The engineered chimeric TGFβ receptor system is configured such that, when expressed in immune cells, the immune cells are repurposed to stimulate the IL-9 signaling pathway after TGFβ binding. Further details are provided below.

[0092] Engineered TGFβ receptor system

[0093] Figures 1A-1D Two types of engineered TGFβ receptor systems, and the corresponding TGFβ-responsive signaling complexes formed therefrom, are illustrated according to certain embodiments of this disclosure. These two types of engineered TGFβ receptor systems (i.e., type I and type II, respectively corresponding to…) Figure 1A and Figure 1C Each of the first and second engineered TGFβ receptor chains comprises a first engineered TGFβ receptor chain and a second engineered TGFβ receptor chain. Each of the first and second engineered TGFβ receptor chains comprises an extracellular portion, a transmembrane portion, and an intracellular portion, which are operatively interconnected.

[0094] Specifically, in such Figure 1AIn the class I engineered TGFβ receptor system shown, the extracellular and intracellular portions of the first engineered TGFβ receptor chain respectively contain the extracellular domain (ECD) of mammalian TGFβR1 or its functional portion or functional variant, and the ICD of mammalian IL9R or its functional portion or functional variant; and the extracellular and intracellular portions of the second engineered TGFβ receptor chain respectively contain the extracellular domain (ECD) of mammalian TGFβR2 or its functional portion or functional variant, and the ICD of mammalian IL2Rγ or its functional portion or functional variant.

[0095] In such Figure 1C In the class II engineered TGFβ receptor system shown, the extracellular and intracellular portions of the first engineered TGFβ receptor chain respectively contain the extracellular domain (ECD) of mammalian TGFβR1 or its functional portion or functional variant, and the ICD of mammalian IL2Rγ or its functional portion or functional variant; and the extracellular and intracellular portions of the second engineered TGFβ receptor chain respectively contain the extracellular domain (ECD) of mammalian TGFβR2 or its functional portion or functional variant, and the ICD of mammalian IL9R or its functional portion or functional variant.

[0096] Here, mammalian TGFβR1, mammalian TGFβR2, mammalian IL9, or mammalian IL2Rγ can be derived from any mammal, such as humans, apes, rats, mice, dogs, cats, etc. According to some embodiments, each ECD and each ICD in the first and second chains of each type of engineered TGFβ receptor system is derived from humans; therefore, the ECD of human TGFβR1, the ECD of human TGFβR2, the ICD of human IL9, or the ICD of human IL2Rγ contained in the first and second engineered TGFβ receptor chains respectively contain sequences as shown in SEQ ID NO: 1, 2, 3, and 4.

[0097] In some implementations, class I and class II engineered TGFβ receptor systems ( Figure 1A and Figure 1C The extracellular portion of the first engineered TGFβ receptor chain in each of the following can contain the ECD of TGFβR1 or its functional portion or functional variant, which contains a sequence having at least 70% identity with SEQ ID NO: 1; Class I and Class II engineered TGFβ receptor systems ( Figure 1A and Figure 1C The extracellular portion of the second engineered TGFβ receptor chain in the system may contain the ECD of TGFβR2 or a functional portion or functional variant thereof, which contains a sequence having at least 70% identity with SEQ ID NO: 2. Class I engineered TGFβ receptor systems ( Figure 1A The intracellular portion of the first engineered TGFβ receptor chain or the class II engineered TGFβ receptor system ( Figure 1C The intracellular portion of the second engineered TGFβ receptor chain in the SEQ ID NO: 3 may each contain an ICD of IL9R or a functional portion or functional variant thereof, which contains a sequence having at least 70% identity with SEQ ID NO: 3; and the class I engineered TGFβ receptor system ( Figure 1A The intracellular portion of the second engineered TGFβ receptor chain or the class II engineered TGFβ receptor system ( Figure 1C The intracellular portion of the first engineered TGFβ receptor chain in SEQ ID NO: 4 may contain an ICD of IL2Rγ or a functional portion or functional variant thereof, which contains a sequence having at least 70% identity with SEQ ID NO: 4.

[0098] In such Figure 1A and Figure 1C In each engineered TGFβ receptor chain of each engineered TGFβ receptor system shown, the transmembrane portion (i.e., TM1, TM2, TM3, or TM4) operatively connecting the extracellular and intracellular portions can optionally be derived from the transmembrane domain of any transmembrane protein (e.g., TGFβR1, TGFβR2, IL2Rγ, IL4T, IL9R, IL7R, IL15R, IL10R, IL21R, CD28, CTLA4, PD1, TIM3, TIGIT, CD4, CD8, or 4-1BB, etc.). In some embodiments, the transmembrane portion can be derived from the same protein as the extracellular portion, and may contain the transmembrane domain (TM) of TGFβR1 (SEQ ID NO: 5) or the transmembrane domain (TM) of TGFβR2 (SEQ ID NO: 6), or a functional portion or functional variant thereof. In some other embodiments, the transmembrane portion may be derived from the same protein as the intracellular portion, and may contain the transmembrane domain (TM) of IL9R (SEQ ID NO: 7) or the transmembrane domain (TM) of IL2Rγ (SEQ ID NO: 8), or a functional portion or functional variant thereof. Table 1 summarizes the sequences of each domain of several embodiments of the engineered TGFβ receptor chain.

[0099] Table 1: Domain structures and amino acid sequences of several embodiments of the engineered TGFβ receptor chain used in the engineered TGFβ receptor system provided in this disclosure.

[0100] TGFβR1(ECD): SEQ ID NO: 1 TGFβR1(TM): SEQ ID NO: 5 IL9R(ICD): SEQ ID NO: 3 TGFβR1(ECD)-TGFβR1(TM)-IL9R(ICD): SEQ ID NO: 9 TGFβR1(ECD): SEQ ID NO: 1 IL9R(TM): SEQ ID NO: 7 IL9R(ICD): SEQ ID NO: 3 TGFβR1(ECD)-IL9R(TM)-IL9R(ICD):SEQ ID NO: 10 TGFβR1(ECD): SEQ ID NO: 1 TGFβR1(TM): SEQ ID NO: 5 IL2Rγ(ICD): SEQ ID NO: 4 TGFβR1(ECD)-TGFβR1(TM)-IL2Rγ(ICD): SEQ ID NO: 11 TGFβR1(ECD): SEQ ID NO: 1 IL2Rγ(TM): SEQ ID NO: 8 IL2Rγ(ICD): SEQ ID NO: 4 TGFβR1(ECD)-IL2Rγ(TM)-IL2Rγ(ICD): SEQ ID NO: 12 TGFβR2(ECD): SEQ ID NO: 2 TGFβR2(TM): SEQ ID NO: 6 IL9R(ICD): SEQ ID NO: 3 TGFβR2(ECD)-TGFβR2(TM)-IL9R(ICD): SEQ ID NO: 13 TGFβR2(ECD): SEQ ID NO: 2 IL9R(TM): SEQ ID NO: 7 IL9R(ICD): SEQ ID NO: 3 TGFβR2(ECD)-IL9R(TM)-IL9R(ICD):SEQ ID NO: 14 TGFβR2(ECD): SEQ ID NO: 2 TGFβR2(TM): SEQ ID NO: 6 IL2Rγ(ICD): SEQ ID NO: 4 TGFβR2(ECD)-TGFβR2(TM)-IL2Rγ(ICD): SEQ ID NO: 15 TGFβR1(ECD): SEQ ID NO: 2 IL2Rγ(TM): SEQ ID NO: 8 IL2Rγ(ICD): SEQ ID NO: 4 TGFβR2(ECD)-IL2Rγ(TM)-IL2Rγ(ICD): SEQ ID NO: 16

[0101] Recombinant vector

[0102] In another aspect, this disclosure further provides a recombinant vector system designed to express the engineered chimeric TGFβ receptor system as described above in immune cells. Such a recombinant vector system may comprise at least one recombinant vector (e.g., an expression vector).

[0103] Herein, each recombinant vector may include isolated polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein), a host cell into which the recombinant vector is introduced (i.e., such that the host cell contains the polynucleotide and / or the vector containing the polynucleotide), and the production of a recombinant polypeptide or a fragment thereof by recombinant technology.

[0104] As used herein, a “vector” is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into that host cell. An “expression vector” is capable of delivering and expressing one or more polynucleotides of interest in the form of a polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned within the vector for expression by being operatively linked to regulatory elements such as promoters, enhancers, and / or poly-A tails located within the vector or near or flanking the integration site of the polynucleotide of interest in the host cell genome, such that the polynucleotide of interest will be translated in the host cell into which the expression vector has been introduced.

[0105] Vectors can be introduced into host cells by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-glucan), transformation, transfection, and infection and / or transduction (e.g., using recombinant viruses). Therefore, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, granules, phage vectors, and DNA or RNA expression vectors associated with cationic condensers.

[0106] This disclosure provides a recombinant vector containing a nucleic acid construct suitable for genetic modification of cells, which can be used to treat pathological diseases or conditions.

[0107] Any vector or vector type can be used to deliver genetic material into cells. These vectors include, but are not limited to, plasmid vectors, viral vectors, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and human artificial chromosomes (HACs). Viral vectors may include, but are not limited to, recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenovirus vectors, foam virus vectors, recombinant adeno-associated virus (AAV) vectors, hybridization vectors, and plasmid transposons (e.g., the Sleeping Beauty transposon system and the PiggyBac transposon system) or integrase-based vector systems. Other vectors known in the art may also be used in the methods described herein.

[0108] In some embodiments, the vector is a viral vector. The viral vector can be grown in a culture medium specifically designed for preparing viral vectors. According to the embodiments described herein, any suitable growth medium and / or supplements can be used to culture the viral vector.

[0109] In some implementations, the vector used is a recombinant retroviral vector. Retroviral vectors are capable of directing the expression of nucleic acid molecules of interest. Retroviruses exist in their viral capsid as RNA and form a double-stranded DNA intermediate when replicating in a host cell. Similarly, retroviral vectors exist in both RNA and double-stranded DNA forms. Retroviral vectors also include DNA forms containing recombinant DNA fragments and RNA forms containing recombinant RNA fragments. Vectors may include at least one transcription promoter / enhancer or other element controlling gene expression. Such vectors may also include packaging signals, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites suitable for the retrovirus used. Long terminal repeats (LTRs) are identical DNA sequences repeated multiple times (e.g., hundreds or thousands of times) at either end of a retrotransposon or proviral DNA formed by reverse transcription of retroviral RNA. Viruses use them to insert genetic material into the host genome. Optionally, the vector may also include a signal to guide polyadenylation, a selective marker such as ampicillin resistance, neomycin resistance, TK, hygromycin resistance, fosetyl-aluminum resistance, histamine resistance, or DHFR, and one or more restriction sites and translation termination sequences. For example, such vectors may include a 5' LTR, a leader sequence, an tRNA binding site, a packaging signal, a second-strand DNA synthesis origin, and a 3' LTR or a portion thereof. Additionally, the retroviral vectors used herein may also refer to recombinant vectors generated by removing the retroviral gag, pol, and env genes and replacing them with the gene of interest.

[0110] In some embodiments, the vector may include additional nucleic acid encoding a repressor protein (e.g., a checkpoint inhibitor). In different embodiments, the cell expresses a genetically engineered antigen receptor and a repressor protein. In different embodiments, the repressor protein is constitutively expressed.

[0111] In some embodiments, the vector or construct may contain a single promoter driving the expression of one or more nucleic acid molecules. In some embodiments, such a promoter may be polycistronic (bicistronic or tricistronic). For example, in some embodiments, a transcription unit may be engineered to be a bicistronic unit containing an IRES (internal ribosome entry site), which allows for co-expression of gene products from a single promoter. Alternatively, in some cases, a single promoter may direct the expression of RNA containing two or three genes in a single open reading frame (ORF), the sequences of which encode self-cleaving peptides (e.g., P2A or T2A) or protease recognition sites (e.g., furin protease) separated from each other. Thus, the ORF encodes a single polyprotein that is cleaved into individual proteins during translation (in the case of 2A, such as T2A) or post-translational. In some cases, a peptide such as T2A may cause ribosome skipping (ribosome jumping) of the synthesis of a peptide bond at the C-terminus of a 2A element, resulting in separation between the end of the 2A sequence and the next downstream peptide.

[0112] Various cell lines can be used in conjunction with the vectors described herein. Exemplary eukaryotic cells that can be used to express peptides include, but are not limited to: COS cells, including COS7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44. Lec13 CHO cells, and FUT8 CHO cells; PER.C6® cells; and NSO cells. In some embodiments, specific eukaryotic host cells are selected based on their ability to perform the desired post-translational modifications to the bound molecules. For example, in some embodiments, peptides produced by CHO cells have a higher level of sialylation than the same peptides produced by 293 cells.

[0113] According to some implementation schemes, the vector system may comprise two vectors designed to express a first engineered TGFβ receptor chain and a second engineered TGFβ receptor chain from an engineered TGFβ receptor system, respectively.

[0114] According to some other embodiments, the vector system may comprise a vector expressing both a first engineered TGFβ receptor chain and a second engineered TGFβ receptor chain from the engineered TGFβ receptor system. Thus, the first and second engineered TGFβ receptor chains are substantially within a single vector. Further, in some embodiments, the two engineered TGFβ receptor chains may be encoded within an open reading frame (ORF) of the single vector. Additionally, the ORF may contain a polynucleotide encoding a self-cleaving peptide (e.g., T2A, P2A, F2A, or E2A), which operatively links the polynucleotide encoding the first engineered TGFβ receptor chain and the polynucleotide encoding the second engineered TGFβ receptor chain. In some other embodiments, the two engineered TGFβ receptor chains may each reside in a different ORF within the same vector.

[0115] In any embodiment of the above-described vector system, the polynucleotide encoding the first engineered TGFβ receptor chain and the polynucleotide encoding the second engineered TGFβ receptor chain are each further operably linked to a polynucleotide encoding a signal peptide (SP). Thus, the polypeptide encoded by the polynucleotide in the vector of this vector system substantially contains a first signal peptide at the N-terminus of the first engineered TGFβ receptor chain of the engineered TGFβ receptor system and a second signal peptide at the N-terminus of the second engineered TGFβ receptor chain.

[0116] In some embodiments, the extracellular portion of the first signal peptide and the first engineered TGFβ receptor chain originates from the same protein, and the extracellular portion of the second signal peptide and the second engineered TGFβ receptor chain originates from the same protein. Therefore, in class I as described above ( Figure 1A ) or Class II ( Figure 1C In the engineered TGFβ receptor system, according to some specific embodiments, the first signal peptide of the first engineered TGFβ receptor chain may contain the signal peptide of TGFβR1 (SEQ ID NO: 17) or a functional portion or functional variant thereof, and the second signal peptide of the second engineered TGFβ receptor chain may contain the signal peptide of TGFβR2 (SEQ ID NO: 18) or a functional portion or functional variant thereof. Table 2 summarizes the amino acid sequences of several embodiments of the engineered TGFβ receptor chains encoded by the vector system provided in this disclosure.

[0117] Table 2: Amino acid sequences of several embodiments of engineered TGFβ receptor chains encoded by the vector system provided in this disclosure.

[0118] TGFβR1(SP): SEQ ID NO: 17 TGFβR1(ECD)-TGFβR1(TM)-IL9R(ICD): SEQ ID NO: 9 TGFβR1(SP)-TGFβR1(ECD)-TGFβR1(TM)-IL9R(ICD): SEQ ID NO: 19 TGFβR1(SP): SEQ ID NO: 17 TGFβR1(ECD)-IL9R(TM)-IL9R(ICD): SEQ ID NO: 10 TGFβR1(SP)-TGFβR1(ECD)-IL9R(TM)-IL9R(ICD): SEQ ID NO: 20 TGFβR1(SP): SEQ ID NO: 17 TGFβR1(ECD)-TGFβR1(TM)-IL2Rγ(ICD): SEQ ID NO: 11 TGFβR1(SP)-TGFβR1(ECD)-TGFβR1(TM)-IL2Rγ(ICD): SEQ ID NO: 21 TGFβR1(SP): SEQ ID NO: 17 TGFβR1(ECD)-IL2Rγ(TM)-IL2Rγ(ICD): SEQ ID NO: 12 TGFβR1(SP)-TGFβR1(ECD)-IL2Rγ(TM)-IL2Rγ(ICD): SEQ ID NO: 22 TGFβR2(SP): SEQ ID NO: 18 TGFβR2(ECD)-TGFβR2(TM)-IL9R(ICD): SEQ ID NO: 13 TGFβR2(SP)-TGFβR2(ECD)-TGFβR2(TM)-IL9R(ICD): SEQ ID NO: 23 TGFβR2(SP): SEQ ID NO: 18 TGFβR2(ECD)-IL9R(TM)-IL9R(ICD): SEQ ID NO: 14 TGFβR2(SP)-TGFβR2(ECD)-IL9R(TM)-IL9R(ICD): SEQ ID NO: 24 TGFβR2(SP): SEQ ID NO: 18 TGFβR2(ECD)-TGFβR2(TM)-IL2Rγ(ICD): SEQ ID NO: 15 TGFβR2(SP)-TGFβR2(ECD)-TGFβR2(TM)-IL2Rγ(ICD): SEQ ID NO: 25 TGFβR2(SP): SEQ ID NO: 18 TGFβR2(ECD)-IL2Rγ(TM)-IL2Rγ(ICD): SEQ ID NO: 16 TGFβR2(SP)-TGFβR2(ECD)-IL2Rγ(TM)-IL2Rγ(ICD): SEQ ID NO: 26

[0119] Here, as used herein, the term "linker" (L), "linker domain," or "linker region" refers to an oligopeptide or polypeptide region of approximately 1 to 100 amino acids in length that links any domain / region together. Linkers may consist of flexible residues such as glycine and serine, allowing adjacent protein domains to move freely relative to each other. Longer linkers can be used when it is necessary to ensure that two adjacent domains do not spatially interfere with each other. Linkers can be cleavable or non-cleavable.

[0120] As used herein, “nucleic acid” may include “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally refers to a polymer of DNA or RNA that may be single-stranded or double-stranded, synthetic or derived from a natural source, and may contain natural, non-natural, or modified nucleotides. Furthermore, nucleic acids may also contain complementary DNA (cDNA). Typically, preferably, nucleic acids do not contain any insertions, deletions, inversions, and / or substitutions. However, as discussed herein, in certain circumstances, it may be appropriate for nucleic acids to contain one or more insertions, deletions, inversions, and / or substitutions.

[0121] Nucleic acids as described herein can be constructed using procedures known in the art based on chemical synthesis and / or enzymatic ligation reactions. For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or nucleotides with different modifications. In any of these embodiments, the nucleotide sequence is codon-optimized.

[0122] This disclosure also provides nucleic acids comprising nucleotide sequences complementary to the nucleotide sequences of any nucleic acid described herein, or nucleotide sequences that hybridize under stringent conditions to the nucleotide sequences of any nucleic acid described herein.

[0123] In some embodiments, the nucleotide sequences encoding two functional polypeptides (e.g., a first engineered TGFβ receptor chain and a second engineered TGFβ receptor chain) are separated by a peptide sequence that causes ribosome jumping. In some embodiments, the peptide causing ribosome jumping is a P2A, F2A, or T2A peptide. In some embodiments, the nucleic acid is synthetic. In some embodiments, the nucleic acid is cDNA.

[0124] In some embodiments, the first and second engineered TGFβ receptor chains described herein are each encoded by a codon-optimized nucleotide sequence. In some embodiments, the first and second engineered TGFβ receptor chains each further comprise a signal peptide (SP). In specific embodiments, the first and second engineered TGFβ receptor chains described herein are each isolated, purified, or recombinant.

[0125] This disclosure also provides nucleic acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any nucleotide sequence described herein, and amino acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any amino acid sequence described herein. In some embodiments, this disclosure relates to a nucleotide sequence encoding any peptide described herein, or any amino acid sequence encoded by any nucleotide sequence described herein.

[0126] In some embodiments, the nucleic acid sequence is at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is at least or about 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acid residues. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acid residues.

[0127] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, these sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. The two molecules are identical at that position when the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence. The percentage of identity between two sequences is a function of the number of common positions shared by the two sequences, taking into account the number of vacancies introduced for optimal alignment and the length of each vacancy.

[0128] Figures 2A-2D Block diagrams are shown of four constructs that respectively encode four specific embodiments of the engineered TGFβ receptor system according to the present disclosure (i.e., embodiments 1-4, sequences summarized in Table 3). As shown, each embodiment essentially employs a single ORF configuration that encodes both the first and second engineered TGFβ receptor chains separated by the self-cleaving peptide “F2A” (SEQ ID NO: 27).

[0129] Table 3: Amino acid sequences of four embodiments encoding the engineered TGFβ receptor system provided in this disclosure.

[0130] 1 TGFβR1(SP)-TGFβR1(ECD)-TGFβR1(TM)-IL9R(ICD)-F2A-TGFβR2(SP)-TGFβR2(ECD)-TGFβR2(TM)-IL2Rγ(ICD): SEQ ID NO: 28 (Nucleotide sequence: SEQ ID NO: 32) "TGFβR1-IL9R-TGFβR2-IL2Rγ(TGFβR1 / R2 TM)"; or "TR1 / 2-IL9R(TR TM)" 2 TGFβR1(SP)-TGFβR1(ECD)-IL9R(TM)-IL9R(ICD)-F2A-TGFβR2(SP)-TGFβR2(ECD)-IL2Rγ(TM)-IL2Rγ (ICD): SEQ ID NO: 29 "TGFβR1-IL9R-TGFβR2-IL2Rγ(IL9R / IL2Rγ TM)"; or "TR1 / 2-IL9R (9R TM)" 3 TGFβR1(SP)-TGFβR1(ECD)-TGFβR1(TM)-IL2Rγ(ICD)-F2A-TGFβR2(SP)-TGFβR2(ECD)-TGFβR2(TM)-IL9R (ICD): SEQ ID NO: 30 "TGFβR1-IL2Rγ-TGFβR2-IL9R(TGFβR1 / R2 TM)" 4 TGFβR1(SP)-TGFβR1(ECD)-IL2Rγ(TM)-IL2Rγ(ICD)-F2A-TGFβR2(SP)-TGFβR2(ECD)-IL9R(TM)-IL9R(ICD): SEQ ID NO: 31 "TGFβR1-IL2Rγ-TGFβR2-IL9R(IL9R / IL2Rγ TM)"

[0131] engineered cells

[0132] This disclosure further provides engineered cells, particularly immune cells (e.g., T cells) comprising the engineered TGFβ receptor system described herein. These engineered cells can be used to treat various conditions or diseases described herein (e.g., viral infections, cancer, virus-induced conditions).

[0133] In various embodiments, the engineered cells may be obtained from, for example, human and non-human animals. In various embodiments, the engineered cells may be obtained from bacteria, fungi, humans, rats, mice, rabbits, monkeys, pigs, or any other species. Preferably, the cells are derived from humans, rats, or mice. More preferably, the cells are derived from humans. In various embodiments, the engineered cells are blood cells. Preferably, the cells are white blood cells (e.g., T cells), lymphocytes, or any other suitable blood cell type. In some embodiments, the cells are peripheral blood cells. In some embodiments, the cells are T cells, tumor-infiltrating lymphocytes (TILs), B cells, or NK cells.

[0134] In some embodiments, the cell is a T cell. In some embodiments, the T cell expresses a cell surface receptor that recognizes a specific antigenic moiety on the surface of a target cell. This cell surface receptor may be a wild-type or recombinant T cell receptor (TCR), a chimeric antigen receptor (CAR), or any other surface receptor capable of recognizing an antigenic moiety associated with the target cell. The T cell can be obtained by various methods known in the art, such as in vitro culture of T cells isolated from a patient (e.g., tumor-infiltrating lymphocytes). TCR-modified T cells can be obtained by transducing T cells with a viral vector (e.g., isolated from a patient's peripheral blood). In some embodiments, the T cell is a TCR-modified T cell. In some embodiments, the T cell is a CD4+ T cell, a CD8+ T cell, or a regulatory T cell. In some embodiments, the T cell is a type 1 helper T cell and a type 2 helper T cell. In some embodiments, the T cell expressing this receptor is an αβ-T cell. In alternative embodiments, the T cell expressing this receptor is a γδ-T cell.

[0135] In some embodiments, the cells are NK cells. In some embodiments, the preparation of engineered cells includes one or more culture and / or preparation steps. Cells used to introduce binding molecules such as TCRs may be isolated from a sample, such as a biological sample, for example, a sample obtained from or derived from a subject. In some embodiments, the subject from whom the cells are isolated is a subject suffering from a disease or symptom or requiring cell therapy or to whom cell therapy will be administered. In some embodiments, the subject is a person requiring a specific therapeutic intervention, such as adoptive cell therapy for whom cells have been isolated, processed, and / or engineered.

[0136] Different cell types can be obtained through appropriate isolation methods. Isolation methods include separating different cell types based on the expression or presence of one or more specific molecules (such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids) in the cells. In some embodiments, any known method for isolation based on such markers can be used. In some embodiments, the isolation is based on affinity or immunoaffinity. For example, in some aspects, the isolation includes separating cells and cell populations based on the cellular expression or expression level of one or more markers (typically cell surface markers), for example, by incubating with an antibody or binding coupler that specifically binds to such a marker, followed typically by a washing step, and separating cells that have bound the antibody or binding coupler from those that have not.

[0137] Such separation steps can be based on positive and / or negative selection. In positive selection, cells that have bound to the reagent are retained for further use, while in negative selection, cells that have not yet bound to the antibody or binding coupler are retained. In some examples, both fractions are retained for further use. In some respects, negative selection can be particularly useful when antibodies that specifically identify a certain cell type in a heterogeneous population are unavailable, and therefore separation is best based on markers expressed by cells outside the desired population.

[0138] Methods, nucleic acids, compositions, and kits for expressing binding molecules and generating genetically engineered cells expressing such binding molecules are also provided. Genetic engineering typically involves introducing nucleic acids encoding therapeutic molecules such as TCRs, CARs (e.g., TCR-like CARs), peptides, or fusion proteins into cells, for example, through retroviral transduction, transfection, or transformation. In some embodiments, gene transfer is accomplished by the following steps: first, stimulating cells, for example by combining cells with stimuli that induce responses such as proliferation, survival, and / or activation, for example, as measured by the expression of cytokines or activation markers; subsequently, transducing the activated cells; and expanding them in culture to a quantity sufficient for clinical application.

[0139] In some embodiments, recombinant infectious viral particles, such as vectors derived from simian virus 40 (SV40), adenovirus, or adeno-associated virus (AAV), are used to transfer recombinant nucleic acids into cells. In some embodiments, recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors, are used to transfer recombinant nucleic acids into T cells. In some embodiments, the retroviral vector has a long terminal repeat (LTR) sequence, such as a retroviral vector derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), or spleen lesion-forming virus (SFFV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retrovirus includes retroviruses from any avian or mammalian cell source. Retroviruses are generally amphiphilic, meaning they can infect host cells of several species, including humans. In some embodiments, the vector is a lentiviral vector. In some embodiments, the recombinant nucleic acid is transferred into T cells via electroporation. In some implementations, recombinant nucleic acids are transferred to T cells via transposition. Other methods for introducing and expressing genetic material in immune cells include calcium phosphate transfection, protoplast fusion, cationic liposome-mediated transfection, tungsten particle-promoted microparticle bombardment, and strontium phosphate DNA coprecipitation. Many such methods are described, for example, in WO2019195486, which is incorporated herein by reference in its entirety.

[0140] Also provided are engineered cell populations, compositions containing such cells and / or enriched with such cells, for example, wherein cells expressing the binding molecule constitute a percentage of the total cells in the composition or specific cell types such as T cells, CD8 cells, etc. + or CD4 + At least 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher percentage of cells.

[0141] Methods for preparing engineered cells

[0142] This disclosure further provides a method or process for preparing and using engineered cells to treat pathological diseases or conditions.

[0143] Cells used to introduce an engineered TGFβ receptor system can be isolated from a sample, such as a biological sample, for example, a sample obtained from or derived from a subject. In some embodiments, the subject from whom the cells are isolated is a subject suffering from a disease or symptom, requiring cell therapy, or to whom a cell therapy will be administered. In some embodiments, the subject is a person requiring a specific therapeutic intervention, such as adoptive cell therapy for whom cells have been isolated, processed, and / or engineered.

[0144] Therefore, in some embodiments, the cells are primary cells, such as primary human cells. Samples include tissues, fluids, and other samples taken directly from the subject, as well as samples obtained through one or more processing steps such as separation, centrifugation, genetic engineering (e.g., transduction with a viral vector), washing, and / or incubation. Biological samples can be samples obtained directly from biological sources or processed samples. Biological samples include, but are not limited to, bodily fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, and tissue and organ samples, including processed samples derived therefrom.

[0145] In some respects, the sample from which cells are derived or isolated is blood or a blood-derived sample, or is derived from apheresis or leukapheresis products. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy material, tumors, leukemia, lymphoma, lymph nodes, intestinal-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lungs, stomach, intestines, colon, kidneys, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsils, or other organs, and / or cells derived therefrom. In the context of cell therapy, such as adoptive cell therapy, samples include those from autologous and allogeneic sources.

[0146] In some embodiments, the cells are derived from a cell line, such as a T cell line. In some embodiments, the cells are obtained from a xenogeneic source, such as from mice, rats, or non-human primates.

[0147] In some embodiments, blood cells collected from a subject are washed, for example, to remove plasma components and the cells are placed in a suitable buffer or medium for subsequent processing steps. In some embodiments, cells are washed with phosphate-buffered saline (PBS). In some embodiments, the wash solution is deficient in calcium and / or magnesium and / or many or all divalent cations. In some aspects, the washing step is performed by a semi-automatic "flow-through" centrifuge. In some aspects, the washing step is performed by tangential flow filtration (TFF). In some embodiments, after washing, the cells are resuspended in various biocompatible buffers, such as those free of calcium. 2+ / Mg 2+ In PBS. In some embodiments, components of the blood cell sample are removed and the cells are directly resuspended in the culture medium. In some embodiments, the method includes density-based cell separation methods, such as preparing leukocytes from peripheral blood by lysing red blood cells and centrifuging via Percoll or Ficoll gradient.

[0148] In some embodiments, the method includes one or more of the following steps: for example, isolating T cells from a patient's blood; transducing a population of T cells with a viral vector comprising a nucleic acid construct encoding a genetically engineered antigen receptor; expanding the transduced cells in vitro; and / or infusing the expanded cells into a patient, wherein the engineered cells (e.g., engineered T cells) will seek out and destroy antigen-positive tumor cells. In some embodiments, the nucleic acid construct further comprises a sequence encoding a repressor protein. In some embodiments, these engineered cells (e.g., engineered T cells) can block PD-1 / PD-L1 immunosuppression and enhance anti-tumor immune responses. In some embodiments, the method further includes: transfecting T cells with a viral vector containing the nucleic acid construct.

[0149] In some embodiments, the method involves introducing any of the vectors described herein into cells, either in vitro or ex vivo. In some embodiments, the vector is a viral vector, and the introduction is performed via transduction. In some embodiments, one or more agents are repressive nucleic acids (e.g., siRNA). In some embodiments, one or more agents are fusion proteins comprising a DNA-targeting protein and a nuclease or RNA-directed nuclease (e.g., clustered regularly spaced short palindromic nucleic acid (CRISPR)-associated nuclease).

[0150] T cell transfection can be achieved using any standard method, such as calcium phosphate, electroporation, liposome-mediated transfer, microinjection, biological projectile particle delivery systems, or any other method known to the technician. In some implementations, T cell transfection is performed using the calcium phosphate method.

[0151] According to the various embodiments described herein, this disclosure provides an immunotherapy for tumors. In some embodiments, engineered cells (e.g., engineered T cells) recognize tumor-associated antigens. In some embodiments, these engineered cells (e.g., engineered T cells) exhibit a stronger anti-tumor response and reduced T cell exhaustion.

[0152] This disclosure provides a method for creating personalized anti-tumor immunotherapy. In some embodiments, engineered cells can be generated from a patient's blood cells. These engineered cells are then infused into the patient as a cell therapy product. This product can be used in any patient carrying a tumor.

[0153] Treatment

[0154] In another aspect, treatment methods are also provided, which can be used for various therapeutic purposes. In one aspect, this disclosure provides methods for treating a tumor in a subject, methods for reducing the rate of increase in tumor volume over time in a subject, methods for reducing the risk of metastasis, or methods for reducing the risk of additional metastasis in a subject. In some embodiments, the treatment can prevent, delay, slow, or inhibit the progression of cancer. In some embodiments, the treatment can result in a reduction in the number, severity, and / or duration of one or more symptoms of cancer in the subject. Here, the subject may be a patient requiring the treatment (i.e., a patient with a tumor or a patient identified or diagnosed with a tumor).

[0155] According to some embodiments, a method for treating tumors in patients in need is provided, the method comprising: administering a population of immune cells as described in any embodiment provided in the fourth aspect above. Here, the population of immune cells may be a therapeutically effective amount.

[0156] Here, according to some embodiments of the method, the immune cells further express a target recognition receptor capable of binding to target molecules on target cancer cells within the tumor, wherein the target recognition receptor is selected from chimeric antigen receptor (CAR), T cell receptor (TCR), or Aspire-TCR.

[0157] According to some embodiments of the method, the tumor is a solid tumor, such as breast cancer (e.g., triple-negative breast cancer), carcinoid tumor, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, stomach cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematologic malignancies. In some embodiments, the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-resistant prostate cancer.

[0158] According to some embodiments of the method, the solid tumor is characterized by having a high level of TGFβ expression therein, and may be a tumor selected from liver cancer, breast cancer, prostate cancer, glioma, bladder cancer, or thymic epithelial tumor, etc.

[0159] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk of developing cancer. Cancer patients can be identified using various methods known in the art.

[0160] Furthermore, this disclosure provides methods for treating an infection or infection-related symptom in a subject. In some embodiments, the treatment can prevent, delay, slow, or inhibit the progression of a disease. These methods typically involve administering a therapeutically effective amount of the genetically engineered cells disclosed herein to a subject in need. In some embodiments, the disease or symptom being treated is an infectious disease or symptom, such as, but not limited to, viral, retroviral, bacterial, and protozoan infections, immunodeficiency, human papillomavirus (HPV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, and BK polyomavirus.

[0161] As used herein, "effective amount" or "therapeutic effective amount" means an amount or dose sufficient to achieve a beneficial or desired outcome, including preventing, delaying, slowing, or inhibiting the progression of a disease (e.g., cancer). Effective amounts vary depending on factors such as the age and weight of the subject to be administered the therapeutic agent and / or therapeutic composition, the severity of symptoms, and the route of administration, and therefore the dosage can be determined on an individual basis.

[0162] An effective amount can be administered by a single or multiple doses. For example, an effective amount of a composition is an amount sufficient to improve, terminate, stabilize, reverse, inhibit, slow, and / or delay the progression of cancer in a patient, or an amount sufficient to improve, terminate, stabilize, reverse, slow, and / or delay the proliferation of cells (e.g., biopsy cells, any cancer cells described herein, or cell lines (e.g., cancer cell lines)) in vitro. As will be understood in the art, an effective amount may vary, particularly based on the patient’s medical history and other factors such as the type (and / or dosage) of the composition used.

[0163] Effective dosage and schedule can be determined empirically, and making such a determination is within the scope of the art. Those skilled in the art will understand that the necessary dosage will vary depending on, for example, the mammal to be treated, the route of administration, the specific type of therapeutic agent, and other drugs administered to the mammal. Guidelines for selecting appropriate dosages can be found in the literature. Furthermore, treatment does not necessarily result in 100% or complete cure or prevention of disease or symptom. Various treatment / prevention methods exist with varying degrees of therapeutic effect, which are considered potentially beneficial by those skilled in the art.

[0164] In any of the methods described herein, engineered cells and / or at least one additional therapeutic agent may be administered to a subject at least once weekly (e.g., once weekly, twice weekly, three times weekly, four times weekly, once daily, twice daily, or three times daily). In some embodiments, at least two different engineered cells (e.g., cells expressing different binding molecules) are administered in the same composition (e.g., a liquid composition). In some embodiments, engineered cells and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition). In some embodiments, engineered cells and at least one additional therapeutic agent are administered in two different compositions. In some embodiments, the at least one additional therapeutic agent is administered in the form of a pill, tablet, or capsule. In some embodiments, the at least one additional therapeutic agent is administered in the form of a sustained-release oral formulation.

[0165] In some implementations, one or more additional therapeutic agents may be administered to the subject before, during, or after the administration of engineered cells.

[0166] In some embodiments, one or more adjunctive therapeutic agents may be administered to the subject. The adjunctive therapeutic agent may be a checkpoint inhibitor (CPI). In some embodiments, the checkpoint inhibitor is an inhibitory protein, such as an antibody or its antigen-binding fragment. The checkpoint inhibitor may inhibit or block one or more immune checkpoints, including, for example, PD-1, PD-L1, PD-L2, 2B4 (CD244), 4-1BB, A2aR, B7.1, B7.2, B7-H2, B7-H3, B7-H4, B7-H6, BTLA, prolactin, CD160, CD48, CTLA4, GITR, gp49B, HHLA2, HVEM, ICOS, ILT-2, ILT-4, KIR family receptors, LAG-3, OX-40, PIR-B, SIRPalpha (CD47), TFM-4, TIGIT, TIM-1, TIM-3, TIM-4, VISTA, and combinations thereof. In some embodiments, the inhibitory protein blocks PD-1 or PD-L1. In different embodiments, the inhibitory protein comprises an anti-PD-1 scFv. The inhibitory protein is capable of causing a decrease in the expression of PD-1 or PD-L1, and / or inhibiting the upregulation of PD-1 or PD-L1 in T cells in a population, and / or physically inhibiting the formation of the PD-1 / PD-L1 complex and subsequent signal transduction. In some embodiments, the inhibitory protein blocks PD-1. In some embodiments, the adjunctive therapeutic agent is an anti-OX40 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, or an anti-GITR antibody. In some implementations, the adjunctive therapeutic agent is an anti-CTLA4 antibody (e.g., ipilimumab), an anti-CD20 antibody (e.g., rituximab), an anti-EGFR antibody (e.g., cetuximab), an anti-CD319 antibody (e.g., elotuzumab), or an anti-PD1 antibody (e.g., nivolumab).

[0167] In some embodiments, the adjunctive therapeutic agent may include one or more inhibitors selected from the group consisting of: B-Raf inhibitors, EGFR inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, PI3K / mTOR dual inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2) inhibitors. In some embodiments, the adjunctive therapeutic agent is an inhibitor of indoleamine 2,3-dioxygenase-1 (IDO1) (e.g., epacadostat). In some embodiments, the adjunctive therapeutic agent may include one or more inhibitors selected from the group consisting of: HER3 inhibitors, LSD1 inhibitors, MDM2 inhibitors, BCL2 inhibitors, CHK1 inhibitors, inhibitors activating the hedgehog signaling pathway, and agents that selectively degrade estrogen receptors.

[0168] In some implementations, the adjunctive treatment may include one or more treatments selected from the group consisting of: trabectedin, albumin-bound paclitaxel, Trebananib, Pazopanib, sildenafil, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, and vo... Trient, Pazopanib, IMA-901, AGS-003, Cabozantinib, Vinflunine, Hsp90 inhibitors, Ad-GM-CSF, Temozolomide, IL-2, IFNa, Vincristine, Thalomid, Dacarbazine, Cyclophosphamide, Lenalidomide, Azacytidine, Lenalidomide, Bortezomid, Amrubicin, Carfilzomib, Pralatrexate, and Enzatoline.

[0169] In some implementations, the additional therapeutic agent may include one or more therapeutic agents selected from the group consisting of: adjuvants, TLR agonists, tumor necrosis factor (TNF) α, IL-1, HMGB1, IL-10 antagonists, IL-4 antagonists, IL-13 antagonists, IL-17 antagonists, HVEM antagonists, ICOS agonists, therapies targeting CX3CL1, therapies targeting CXCL9, therapies targeting CXCL10, therapies targeting CCL5, LFA-1 agonists, ICAM1 agonists, and selectin agonists.

[0170] In some implementations, the subject is administered carboplatin, albumin-bound paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI. In some implementations, the adjunctive treatment agent is selected from asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vincristine, vinblastine, and / or combinations thereof.

[0171] Compositions and formulations

[0172] This disclosure provides compositions (including pharmaceutical compositions and therapeutic compositions) containing engineered cells produced by the methods disclosed herein. Methods for administering the engineered cells and compositions thereof to a subject (e.g., a patient) are also provided, such as therapeutic methods.

[0173] Compositions comprising engineered cells for administration are provided, including pharmaceutical compositions and formulations, such as compositions comprising a unit dose form of a number of cells for administration at a given dose or partial dose. The pharmaceutical composition and formulation may comprise one or more optional pharmaceutically acceptable carriers or excipients. In some embodiments, the composition comprises at least one additional therapeutic agent.

[0174] A pharmaceutically acceptable carrier is a component in a pharmaceutical composition other than the active ingredient. A pharmaceutically acceptable carrier does not interfere with the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers may include, but are not limited to, buffers, excipients, stabilizers, or preservatives. Drug formulation refers to the process of combining different substances and / or reagents to produce a final pharmaceutical product. Formulation studies involve developing pharmaceutical articles that are acceptable to patients. Furthermore, the form of the article allows the biological activity of the active ingredient contained therein to be effective and does not contain other components that would have unacceptable toxicity to the subject who will administer the formulation.

[0175] In some embodiments, the choice of carrier depends in part on the specific cells (e.g., T cells or NK cells) and / or the method of administration. A variety of suitable formulations are available. For example, the pharmaceutical composition may contain a preservative. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is used. Preservatives or mixtures thereof are typically present in an amount from about 0.0001% to about 2% (by weight) of the total composition. For example, a carrier is described in Remington's Pharmaceutical Sciences, 16th edition, edited by Osol, A. (1980). 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 esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 1... (0 residues) polypeptides; 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).

[0176] Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and a variety of other acids and salts. In some embodiments, a mixture of two or more buffers is used. Buffers or mixtures thereof are typically present in an amount of about 0.001% to about 4% (by weight) of the total composition. Methods for preparing administerable pharmaceutical compositions are known. Exemplary methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st edition (May 1, 2005).

[0177] The formulation may include an aqueous solution. The formulation or composition may also contain more than one active ingredient that is useful for a specific indication, disease, or symptom treated with engineered cells, preferably those active ingredients whose activity is complementary to that of the engineered cells, wherein the respective activities do not adversely affect each other. Such active ingredients are suitably present in a combination of amounts effective for the intended purpose. Therefore, in some embodiments, the pharmaceutical composition may further comprise other pharmaceutically active agents or drugs, such as checkpoint inhibitors, fusion proteins, chemotherapeutic agents, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vincristine, and / or vinblastine.

[0178] In some embodiments, the pharmaceutical composition contains an amount of engineered cells effective in treating or preventing a disease or symptom, such as a therapeutically effective amount or a preventatively effective amount of engineered cells. In some embodiments, the therapeutic or preventative effect is monitored by periodically evaluating the treated subject. The desired dose can be delivered by a single bolus injection, by multiple bolus injections, or by continuous infusion of engineered cells.

[0179] Engineered cells and compositions can be administered using standard application techniques, formulations, and / or devices. The administration of engineered cells can be autologous or xenogeneic. For example, immunoreactive T cells or progenitor cells can be obtained from a subject and, after genetic modification according to the various embodiments described herein, administered to the same subject or different compatible subjects. Peripheral blood-derived immunoreactive T cells or their progeny (e.g., in vivo, ex vivo, or in vitro derived) can be administered via local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. Typically, when administering therapeutic compositions (e.g., pharmaceutical compositions containing genetically modified immunoreactive cells), they are usually formulated into injectable, unit-dose forms (solutions, suspensions, emulsions).

[0180] The formulations disclosed herein include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, percutaneous, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, cell populations are administered parenterally. As used herein, the term "parenterally" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, engineered cells are administered to a subject via peripheral systemic delivery, such as intravenous, intraperitoneal, or subcutaneous injection.

[0181] In some embodiments, the composition is provided as a sterile liquid article, such as an isotonic aqueous solution, suspension, emulsion, dispersant, or viscous composition, which may be buffered to a selected pH in some respects. Liquid articles are generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are more convenient to apply, especially by injection. Viscous compositions, on the other hand, can be formulated within a suitable viscosity range to provide a longer contact period with a specific tissue. Liquid or viscous compositions may contain a carrier, which may be a solvent or dispersion medium containing, for example, water, brine, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0182] Sterile injectable solutions can be prepared by incorporating engineered cells into a solvent, for example, by mixing with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, etc. Depending on the route of administration and the desired product, the composition may also contain excipients such as wetting agents, dispersants or emulsifiers (e.g., methylcellulose), pH buffers, gelling agents or viscosity-enhancing additives, preservatives, flavoring agents, and / or pigments. In some respects, suitable products can be prepared with reference to standard texts.

[0183] Various additives can be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid, can be used to ensure protection against microorganisms. Extended absorption of injectable drug forms can be achieved by using delayed-absorption agents such as aluminum monostearate and gelatin.

[0184] Formulations intended for internal administration are typically sterile. Sterility can be readily achieved, for example, by filtration through a sterile filter membrane.

[0185] The compositions or pharmaceutical compositions described herein may be included in containers, packages or dispensers together with the instructions for use.

[0186] Application method

[0187] Methods for applying engineered cells, populations, and compositions are also provided, as well as the use of such cells, populations, and compositions in the treatment or prevention of diseases, symptoms, and conditions, including cancer. In some embodiments, the methods described herein may reduce the risk of developing the diseases, symptoms, and conditions described herein.

[0188] In some embodiments, the engineered cells, populations, and compositions described herein are applied to a subject or patient suffering from a specific disease or condition, who is to be treated, for example, by adoptive cell therapy such as adoptive T-cell therapy. In some embodiments, cells and compositions prepared by the provided methods, such as engineered compositions and production termination compositions after incubation and / or other processing steps, are applied to a subject, such as a subject suffering from a disease or condition or at risk of suffering from a disease or condition. In some aspects, the methods thereby treat the disease or condition, for example, by improving one or more symptoms of the disease or condition, such as by reducing the tumor burden in cancers expressing antigens recognized by engineered cells (e.g., engineered T cells).

[0189] Methods for cell administration for adoptive cell therapy are known and can be used in conjunction with the methods and compositions provided. For example, adoptive T-cell therapy is described in the following literature: US 2003 / 0170238; US Patent No. 4,690,915; Rosenberg, “Cell transfer immunotherapy for metastatic solid cancer - what clinicians need to know.” Nature reviews Clinical oncology 8.10 (2011): 577; Themeli et al., “Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy.” Nature biotechnology 31.10 (2013): 928; Tsukahara et al., “CD19 target-engineered T-cells accumulate at tumor lesions in human B-cell lymphoma xenograft mouse models.” Biochemical and biophysical research communications 438.1 (2013): 84-89; Davila et al., “CD19 CAR-targeted T cells induce long-term remission and B Cell Aplasia in an immunocompetent mouse model of B cell acute “Lymphoblasticleukemia.” PloS one 8.4 (2013); each of these articles is incorporated into this paper in its entirety by reference.

[0190] In some implementations, cell therapy, such as adoptive T-cell therapy, is performed via autologous transfer, wherein the T cells are isolated and / or otherwise prepared from a subject to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, engineered cells are derived from a subject in need of treatment, such as a patient, and the engineered cells are administered to the same subject after isolation and processing.

[0191] In some embodiments, cell therapy, such as adoptive T-cell therapy, is performed via allogeneic transfer, wherein the T cells are isolated and / or otherwise prepared from a subject other than the subject to receive or ultimately receive the cell therapy (e.g., a first subject). In such embodiments, the engineered cells are subsequently administered to a different subject of the same species, such as a second subject. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0192] In some implementations, the subject's HLA class or HLA supertype has been identified. In some implementations, the subject is treated with a cell therapy that identifies antigens in the HLA class or HLA supertype background.

[0193] In some embodiments, the subject has been treated with a therapeutic agent targeting a disease or symptom, such as a tumor, prior to the administration of the engineered cells or a composition containing engineered cells. In some aspects, the subject is refractory or unresponsive to other therapeutic agents. In some embodiments, the subject experiences disease persistence or relapse, for example, after treatment with another therapeutic intervention, including chemotherapy, radiation therapy, and / or hematopoietic stem cell transplantation (HSCT), e.g., allogeneic HSCT. In some embodiments, the administration effectively treats the subject even though the subject has developed resistance to another therapy.

[0194] In some implementations, the subject responds to another therapeutic agent, and treatment with that agent reduces the disease burden. In some aspects, the subject initially responds to the therapeutic agent but exhibits a relapse of the disease or symptoms over time. In some implementations, the subject does not experience a relapse. In some such implementations, the subject is identified as having a risk of relapse, such as a high risk of relapse, and therefore engineered cells are administered prophylactically, for example, to reduce the likelihood of relapse or to prevent relapse. In some implementations, the subject has not received prior treatment with other therapeutic agents.

[0195] In some embodiments, engineered cells are administered at a desired dose, which in some aspects includes a desired dose or number of cells or cell types and / or a desired ratio of cell types. Thus, in some embodiments, the cell dose is based on the total number of cells (or cells per kilogram of body weight) and the desired ratio of individual populations or subtypes, such as the CD4+ to CD8+ ratio. In some embodiments, the cell dose is based on the desired total number of cells (or cells per kilogram of body weight) in a single population or single cell type. In some embodiments, the dose is based on a combination of these characteristics, such as the desired total number of cells, the desired ratio, and the desired total number of cells in a single population.

[0196] In some embodiments, cell populations or subtypes, such as CD8+ and CD4+ T cells, are administered at a desired dose of total cells (e.g., a desired dose of T cells) or within a permissible range of the desired dose. In some embodiments, the desired dose is the desired number of cells or the desired number of cells per unit body weight of the subject receiving the engineered cells, e.g., cells / kg. In some embodiments, the desired dose is equal to or greater than the minimum number of cells or the minimum number of cells per unit body weight. In some embodiments, in the total cells administered at the desired dose, the individual populations or subtypes are present at or close to the desired output ratio (e.g., the ratio of CD4+ to CD8+), e.g., within a specific permissible range or error for such ratios.

[0197] In some embodiments, engineered cells are administered at a desired dose for one or more individual cell populations or subtypes (e.g., a desired dose for CD4+ cells and / or a desired dose for CD8+ cells) or within permissible variations of the desired dose. In some embodiments, the desired dose is the desired number of cells for that subtype or population, or the number of such cells required per unit body weight of the subject receiving the engineered cells, for example, cells / kg. In some embodiments, the desired dose is equal to or greater than the minimum number of cells for that population or subtype, or the minimum number of cells for that population or subtype per unit body weight.

[0198] Therefore, in some embodiments, the dose is based on the required fixed dose and required ratio of total cells, and / or based on the required fixed dose of one or more (e.g., each) individual subtypes or subpopulations. Thus, in some embodiments, the dose is based on the required fixed or minimum dose of T cells, and the required ratio of CD4+ to CD8+ cells, and / or based on the required fixed or minimum dose of CD4+ and / or CD8+ cells.

[0199] In some implementations, engineered cells are administered to the subject in numbers ranging from approximately 1 million to approximately 100 billion cells, for example, from 1 million to approximately 50 billion cells (e.g., approximately 5 million cells, approximately 25 million cells, approximately 500 million cells, approximately 1 billion cells, approximately 5 billion cells, approximately 20 billion cells, approximately 30 billion cells, approximately 40 billion cells, or a range defined by any two of the above values), for example, from approximately 10 million to approximately 100 billion cells (e.g., approximately 20 million cells, approximately 30 million cells, approximately 40 million cells, approximately 60 million cells, approximately 700 million cells, approximately 1 ... The number of cells is approximately 10 million, approximately 80 million, approximately 90 million, approximately 10 billion, approximately 25 billion, approximately 50 billion, approximately 75 billion, approximately 90 billion, or any two of the above values, and in some cases, approximately 100 million to approximately 50 billion (e.g., approximately 120 million, approximately 250 million, approximately 350 million, approximately 450 million, approximately 650 million, approximately 800 million, approximately 900 million, approximately 3 billion, approximately 30 billion, approximately 45 billion) or any value between these ranges.

[0200] In some implementations, the dose of total cells and / or the dose of individual cell subsets are in the range of 10. 4 Or about 10 4 Up to 10 9 Or about 10 9 Within the range of cells / kg body weight, for example, 10 5 Up to 10 6 Between cells / kg body weight, for example, at least or at least about or equal to or approximately 1 × 10⁻⁶. 5 Cells / kg body weight, 1.5×10 5 Cells / kg body weight, 2×10 5 Cells / kg body weight or 1×10 6 Cells / kg body weight. For example, in some embodiments, engineered cells are administered at the following dosage or within a specific error range of the following dosage: at 10 4 Or about 10 4 Up to 10 9 Or about 10 9 Between 10 T cells per kilogram (kg) of body weight, for example 10 5 Up to 10 6 Between 1 T cells / kg body weight, for example, at least or at least about or equal to or approximately 1 × 10⁻⁶. 5 T cells / kg body weight, 1.5×10 5 T cells / kg body weight, 2×10 5T cells / kg body weight or 1×10 6 T cells per kg of body weight.

[0201] In some implementations, engineered cells are administered at the following doses or within a specific error range of the following doses: at 10 4 Or about 10 4 Up to 10 9 Or about 10 9 Between 10 CD4+ and / or CD8+ cells per kilogram (kg) of body weight, for example 10 5 Up to 10 6 Between 100 CD4+ and / or CD8+ cells / kg body weight, for example, at least or at least about or equal to or about 1 × 10⁻⁶ cells / kg body weight. 5 CD4+ and / or CD8+ cells / kg body weight, 1.5×10 5 CD4+ and / or CD8+ cells / kg body weight, 2×10 5 CD4+ and / or CD8+ cells / kg body weight or 1×10⁻⁶ cells / kg body weight 6 CD4+ and / or CD8+ cells per kg body weight.

[0202] In some implementations, the engineered cells are administered at doses higher than and / or at least about 1 × 10⁻⁶. 6 Approximately 2.5 × 10 6 Approximately 5×10 6 Approximately 7.5 × 10 6 Or approximately 9×10 6 100 CD4+ cells, and / or at least about 1 × 10⁶ cells. 6 Approximately 2.5 × 10 6 Approximately 5×10 6 Approximately 7.5 × 10 6 Or approximately 9×10 6 100 CD8+ cells, and / or at least about 1000 CD8+ cells. 6 Approximately 2.5 × 10 6 Approximately 5×10 6 Approximately 7.5 × 10 6 Or approximately 9×10 6 T cells. In some implementations, the engineered cells are administered at the following dose or within a specific error range of the following dose: approximately 10 8 Up to 10 12 Or about 10 10 Up to 10 11 Between each T cell, approximately 10 8 Up to 10 12 Or about 10 10 Up to 10 11 Between CD4+ cells, and / or approximately 108 Up to 10 12 Or about 10 10 Up to 10 11 Between CD8+ cells.

[0203] In some implementations, engineered cells are administered at a desired output ratio or within permissible limits for multiple cell populations or subtypes (such as CD4+ and CD8+ cells or subtypes). In some aspects, the desired ratio may be a specific ratio or a range of ratios. For example, in some implementations, the desired ratio (e.g., the ratio of CD4+ to CD8+ cells) is between 1:5 or about 1:5 and 5:1 or about 5:1 (or greater than about 1:5 and less than about 5:1), or between 1:3 or about 1:3 and 3:1 or about 3:1 (or greater than about 1:3 and less than about 3:1), such as between 2:1 or about 2:1 and 1:5 or about 1:5 (or greater than about 1:5 and less than about 2:1), such as equal to or about 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1: 1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5. In some respects, the permissible difference is within approximately 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, or approximately 50% of the desired ratio, including any value between these ranges. In some respects, the TCRs described herein provide improved expression and activity, thereby providing therapeutic efficacy even in cases of low target:T ratios.

[0204] Optimal response to therapy may depend on the ability of engineered recombinant receptors, such as TCRs, to be persistently and reliably expressed and / or bind to target antigens on the surface of engineered cells. For example, in some cases, the nature of certain recombinant receptors (e.g., TCRs) can affect their expression and / or activity, especially when expressed in cells used in cell therapy, such as human T cells. In some cases, the expression level of a particular recombinant receptor (e.g., TCR) may be low, and the activity of engineered cells (e.g., human T cells) expressing such recombinant receptors may be limited due to poor expression or poor signaling activity. In some cases, the consistency and / or efficiency of recombinant receptor expression, and the activity of that receptor, are limited in certain cells or cell populations in which the therapeutic approach is available. In some cases, a large number of engineered cells (e.g., engineered T cells) (efficiency:target (E:T) ratio) are required to exhibit functional activity. In some embodiments, the required ratio (E:T ratio) is between 1:10 or about 1:10 and 10:1 or about 10:1 (or greater than about 1:10 and less than about 10:1), or between 1:1 or about 1:1 and 10:1 or about 10:1 (or greater than about 1:1 and less than about 5:1), such as between 2:1 or about 2:1 and 10:1 or about 10:1. In some embodiments, the E:T ratio is greater than or about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0205] For the prevention or treatment of a disease, the appropriate dosage may depend on the type of disease to be treated, the type of cell or recombinant receptor, the severity and course of the disease, whether the engineered cells are administered for preventative or therapeutic purposes, prior treatment, the subject's clinical history and response to the engineered cells, and the judgment of the attending physician. In some embodiments, the composition and cells are appropriately administered to the subject either once or as part of a series of treatments.

[0206] The engineered cells described herein may be administered by any suitable means, such as by bolus infusion, by injection, such as intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, intra-anterior chamber injection, subconjunctival injection, subconjuntival injection, sub-Tenon's injection, retroocular injection, periocular injection, or posterior juxtascleral delivery. In some embodiments, administration may be made via parenteral, intrapulmonary, and intranasal administration, and, if local treatment requires, via intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus infusion of engineered cells. In some embodiments, engineered cells are administered by, for example, multiple bolus infusions over a period not exceeding 3 days, or by continuous infusion of engineered cells.

[0207] In some embodiments, engineered cells are administered as part of a combination therapy, such as concurrently with or sequentially with another therapeutic intervention (e.g., an antibody or engineered cell or receptor or agent, such as a cytotoxic agent or therapeutic agent). In some embodiments, engineered cells are co-administered with one or more additional therapeutic agents, or in combination with another therapeutic intervention, said administration may be simultaneous or sequential in any order. In some cases, engineered cells are co-administered with another therapy in sufficiently close proximity such that the cell population enhances the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, engineered cells are administered prior to said one or more additional therapeutic agents. In some embodiments, engineered cells are administered after said one or more additional therapeutic agents. In some embodiments, said one or more additional therapeutic agents include cytokines, such as IL-2, for example, to enhance persistence. In some embodiments, the method includes administration of a chemotherapeutic agent.

[0208] In some embodiments, the bioactivity of the engineered cell population is determined after administration of engineered cells, for example by any of a variety of known methods. Parameters to be evaluated include the specific binding of the engineered cells (e.g., engineered T cells) to the antigen in vivo (e.g., by imaging) or in vitro (e.g., by ELISA or flow cytometry). In some embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in the following literature: for example, Kochenderfer et al., “Construction and pre-clinical evaluation of an anti-CD19 chimeric antigen receptor.” Journal of Immunotherapy (Hagerstown, Md.: 1997) 32.7 (2009): 689 and Hermans et al., “The VITAL assay: a versatile fluorometric technique for assessing CTL-and NKT-mediated cytotoxicity against multiple targets in vitro and in vivo.” Journal of Immunological Methods 285.1 (2004): 25-40. In some embodiments, the bioactivity of engineered cells is determined by measuring the expression and / or secretion of one or more cytokines such as CD107a, IFNγ, IL-2, and TNF. In other aspects, bioactivity is determined by assessing clinical outcomes such as a reduction in tumor burden or load.

[0209] Example

[0210] Several embodiments are given below to describe the invention provided in this disclosure in more detail. Please note that these embodiments are for illustrative purposes only and should not be construed as imposing any limitation on the scope of the disclosure.

[0211] Example 1

[0212] In this embodiment, two implementation schemes of the engineered TGFβ receptor system provided above are described, namely "TGFβR1-IL9R-TGFβR2-IL2Rγ (TGFβR1 / R2 TM)" (Implementation Scheme 1). Figure 2A ) and "TGFβR1-IL9R-TGFβR2-IL2Rγ (IL9R / IL2Rγ TM)" (Implementation Scheme 2; Figure 2BFunctional studies were conducted and side-by-side comparisons were performed. The two implementation schemes have identical configurations in the two engineered TGFβ receptor chains regarding aspects such as the signal peptide (SP), extracellular domain (ECD), and intracellular domain (ICD), except for the use of different transmembrane domains (TM). More specifically, in implementation scheme 1, the two engineered TGFβ receptor chains use TMs from TGFβR1 and TGFβR2, respectively, while in implementation scheme 2, the engineered TGFβ receptor chains use TMs from IL9R and IL2Rγ, respectively.

[0213] 1.1 Evaluation of the expression of TGFβR1R2-IL9R (TR TM) and TGFβR1R2-IL9R (9R TM) transgenes in T cells and their effects on T cell proliferation and survival.

[0214] In summary, two constructs of the engineered TGFβ receptor system, TGFβR1R2-IL9R (TR™) and TGFβR1R2-IL9R (9R™), were transduced into peripheral blood mononuclear cells (PBMCs) via retrovirus, and cell counts and viability were performed every 2–3 days. Figure 3B and Figure 3C On day 3 post-transduction, cell counts were performed, and 200,000 cells were collected and stained with TGFβR2 antibody to assess chimeric receptor expression. Figure 3A As shown in the figure, the two chimeric receptors, TGFβR1R2-IL9R (TR™) and TGFβR1R2-IL9R (9R™), are well expressed in PBMCs. Figure 3A ), and they obviously do not affect cell proliferation ( Figure 3B ) or cell survival ( Figure 3C Furthermore, there was no significant difference in cell expansion or survival between PBMCs modified with these two engineered TGFβ receptors (i.e., "TR1 / 2-IL9R (TR TM) modified" and "TR1 / 2-IL9R (9R Tm) modified" in the figure) and untransduced control cells (i.e., "unmodified", or "UTD" in the figure).

[0215] 1.2 Comparison of TGFβ-stimulated JAK-STAT activation in T cells modified with two TGFβR1R2-IL9R receptors.

[0216] JAK-STAT is a major signal transduction pathway mediated by cytokine receptors, and it has been demonstrated that IL9R activates several signal transduction and activation (STAT) proteins, namely STAT1, STAT3, and STAT5, upon binding to its homologous IL-9 ligand, leading to their phosphorylation. To examine whether the engineered TGFβR1 / 2-IL9R chimeric receptor system can activate STAT phosphorylation (i.e., "pSTAT") upon binding to its homologous ligand TGFβ, and if so, to further examine whether there are any differences in JAK-STAT activation in response to TGFβ between PBMCs modified with these two engineered TGFβ receptors, the following experiments were performed.

[0217] In summary, after stimulation with TGFβ1 (10 ng / mL) for 30 minutes, untransduced or unmodified (“UT”) T cells and two types of TGFβR1R2-IL9R modified T cells were fixed with 4% paraformaldehyde and permeabilized with 100% methanol, followed by pSTAT flow cytometry staining. pSTAT activation was analyzed by flow cytometry, and STAT activation was quantified using the mean fluorescence intensity (MFI) of antibody staining.

[0218] like Figures 4A-4C As shown, unmodified T cells responded to TGFβ1 stimulation without STAT1 / 3 / 5 signaling. However, there was a surprisingly large difference between the two types of modified T cells in terms of JAK-STAT activation upon TGFβ stimulation. In TR1 / 2-IL9R (9R™) modified T cells stimulated with TGFβ1 for 30 min, the levels of phosphorylated STAT1 (pSTAT1), phosphorylated STAT3 (pSTAT3), and phosphorylated STAT5 (pSTAT5) were approximately 1.44 times higher than the levels of pSTAT1, pSTAT3, and pSTAT5 in the same cells in the absence of TGFβ stimulation. Figure 4A ), approximately 2.48 times ( Figure 4B ) and approximately 1.67 times ( Figure 4C In stark contrast, T cells modified with TR1 / 2-IL9R (TR™) stimulated with TGFβ1 showed much stronger pSTAT activation. The levels of pSTAT1, pSTAT3, and pSTAT5 in TR1 / 2-IL9R (TR™) modified T cells 30 minutes after TGFβ stimulation were approximately 1.85 times higher than the levels in the same cells without TGFβ stimulation. Figure 4A ), approximately 4.62 times ( Figure 4B ) and approximately 3.33 times ( Figure 4C ).

[0219] Therefore, this data indicates that TGFβ1 induces much stronger STAT1 / 3 / 5 signaling in TR1 / R2-IL9R (TR TM) modified T cells than in TR1 / R2-IL9R (9R TM) modified T cells.

[0220] 1.3 Comparison of the proliferative capacity of T cells modified with two TGFβR1R2-IL9R receptors in response to TGFβ.

[0221] In short, unmodified or untransduced (“UT”) and TR1 / R2-IL9R modified PBMCs were combined with IL-2 (300 IU / mL) Figure 5B and Figure 5D ) or not with IL-2 ( Figure 5A and Figure 5C Cells were cultured together and cell counts (top image) and cell viability (bottom image) were performed at specified time points to assess cell proliferation. Figure 5A and Figure 5B ) and survival ( Figure 5C and Figure 5D ).

[0222] Figure 5A and Figure 5C The comparison results are shown, in which all three groups of T cells were cultured in the absence of IL-2. As shown in the figure, both engineered TR1 / R2-IL9R receptors unexpectedly inhibited cell exhaustion in the absence of TGFβ1. Figure 5A and Figure 5C (Left figure) TR1 / R2-IL9R (TR™) modified T cells decreased to their initial level (i.e., day 0) by day 9, while TR1 / R2-IL9R (9R™) modified T cells decreased to their initial level around day 11. In contrast, the number of unmodified control cells decreased to their initial level around day 5. In the presence of TGFβ1, both TR1 / 2-IL9R (TR™) modified T cells and TR1 / 2-IL9R (9R™) modified T cells showed enhanced proliferative capacity compared to unmodified control cells, with the former showing a more significant enhancement than the latter. Figure 5A(See right figure). Furthermore, the number of unmodified control cells decreased to initial levels around day 5, while the number of TR1 / R2-IL9R (9R™) modified T cells decreased to initial levels around day 9, and the number of TR1 / R2-IL9R (TR™) modified T cells did not even decrease to initial levels around day 12. Further, regarding survival, in the absence of IL-2, there was no difference between unmodified T cells and TR1 / 2-IL9R (9R™) modified T cells, while TR1 / 2-IL9R (TR™) modified T cells unexpectedly showed significantly better survival than the other two groups of T cells. Figure 5C (See right figure). Specifically, on day 12 after IL-2 was withdrawn from the culture, the viability of both unmodified T cells and TR1 / 2-IL9R (9R TM) modified T cells decreased to <approximately 45%, while the viability of TR1 / 2-IL9R (TR TM) modified T cells remained as high as approximately 70%. This data suggests that, in the absence of IL-2, TGFβ stimulation resulted in surprisingly better durability (better survival and enhanced proliferation) for TR1 / 2-IL9R (TR TM) modified T cells compared to TR1 / 2-IL9R (9RTM) modified T cells.

[0223] Figure 5B and Figure 5D The comparison results are shown, in which all three groups of T cells were cultured in the presence of IL-2. Figure 5D As shown, in the presence of IL-2, neither of the two TR1 / 2-IL9R receptors appeared to have any effect on cell viability, regardless of the presence of TGFβ1. Further as... Figure 5B As shown, in the presence of TGFβ1, both TR1 / 2-IL9R receptors appear to confer enhanced proliferative capacity on T cells modified accordingly. Figure 5B (The image on the right).

[0224] 1.4 Comparison of the proliferative capacity of T cells modified with two TGFβR1R2-IL9R receptors in response to TGFβ after repeated antigen activation.

[0225] In short, in the presence of IL-2 supply (100 IU / mL) ( Figure 6B and Figure 6D ) or in the absence of IL-2 ( Figure 6A and Figure 6C Untransduced and TR1 / R2-IL9R modified PBMCs were co-cultured with fresh HeLa (anti-CD3) cells at a 1:1 ET ratio every 3–4 days (as indicated by repeated stimulation “STI” at each time point). Cell numbers were measured at specified time points. Figure 6Aand Figure 6B ) and cell viability ( Figure 6C and Figure 6D ) were counted. Here, HeLa (anti-CD3) cells were engineered to express the anti-CD3 scFv-B7.1 fusion protein on their surface, which has been reported to induce potent T cell activation and cytotoxicity (Judith Leitner et al., J Immunol Methods. 2010 Oct 31; 362(1-2):131–141).

[0226] Figure 6A and Figure 6C The comparison results are shown, with all three groups of T cells cultured in the absence of IL-2. As shown in the figure, when cultured in the presence of TGFβ1 and under repeated antigen stimulation, both TR1 / 2-IL9R (TR™) modified T cells and TR1 / 2-IL9R (9R™) modified T cells showed significantly better survival rates compared to unmodified T cells, with the former showing a slightly more pronounced effect (see Figure 1). Figure 6C (as shown in the right figure); further as... Figure 6A As shown in the right figure, TGFβ1 significantly inhibited the proliferation of unmodified T cells, while both TR1 / 2-IL9R (TR TM) modified T cells and TR1 / 2-IL9R (9R TM) modified T cells showed significantly enhanced proliferative capacity. Notably, the TR1 / 2-IL9R (TR TM) receptor conferred a significantly higher enhancement compared to the TR1 / 2-IL9R (9R TM) receptor (e.g., on day 12, the cell expansion ratio of TR1 / 2-IL9R (TR TM) modified T cells was approximately 3 times that of TR1 / 2-IL9R (9R TM) modified T cells).

[0227] like Figure 6B and Figure 6D As shown, all three groups of T cells were cultured in the absence of IL-2. Regardless of the presence of TGFβ1, there was essentially no difference in proliferation or survival between TR1 / 2-IL9R (TR TM) modified T cells and TR1 / 2-IL9R (9R TM) modified T cells.

[0228] The above data collectively demonstrate that transmembrane domains (TMs) have a significant impact on engineered TR1 / R2-IL9R receptors. Compared to TGFβR1R2-IL9R receptors with IL9R / IL2Rγ TMs, TGFβR1R2-IL9R receptors with both TGFβR1 and TGFβR2 TMs exhibit unexpectedly superior ability to activate JAK-STAT (especially STAT3 and STAT5) under TGFβ stimulation. This allows T cells modified with these TMs to avoid cell exhaustion in the presence of TGFβ but in the absence of IL-2, especially under repeated or continuous antigen stimulation.

[0229] Example 2

[0230] In this embodiment, implementation scheme 1 of the engineered TGFβ receptor system, namely "TGFβR1-IL9R-TGFβR2-IL2Rγ (TGFβR1 / R2TM)" (e.g.) Figure 2A As shown in the figure, further functional studies were conducted in tumor-infiltrating lymphocytes (TILs).

[0231] 2.1 Evaluation of the expression of TGFβR-IL9R transgene in TIL and its effects on proliferation and survival.

[0232] In short, a construct expressing the TGFβR1 / 2-IL9R chimeric receptor system was transduced into TILs via retrovirus. After transduction, cell counts were performed at specified time points during the amplification phase (e.g., days 1, 6, 8, 10, and 12), and 200,000 cells were collected every few days for staining with a TGFβR2 antibody to assess the expression of the engineered TGFβ receptor. Figures 7A-7C As shown, the engineered TGFβ receptor system (i.e., TGFβR1R2-IL9R) ​​can be well expressed in TILs (see...). Figure 7A Furthermore, when TGFβR1 / 2-IL9R-modified cells were compared with unmodified control cells, cell proliferation did not appear to be affected (see [link to relevant documentation]). Figure 7B ) or survive (see Figure 7C ).

[0233] 2.2 Evaluation of the response of TGFβR-IL9R modified TIL to TGFβ JAK-STAT activation.

[0234] TILs were transduced using a TGFβR1 / 2-IL9R construct. After stimulation with TGFβ1 (10 ng / mL) for 30 min, both unmodified and TGFβR1R2-IL9R-modified cells were fixed with 4% paraformaldehyde and permeabilized with 100% methanol, followed by pSTAT flow cytometry staining. pSTAT activation was analyzed by flow cytometry, and the mean fluorescence intensity (MFI) of antibody staining was used to quantify STAT activation. Figure 8 As shown, compared with unmodified TIL, TGFβ1 can induce strong pSTAT3 (approximately 1.38-fold) and pSTAT5 (approximately 1.39-fold) signaling in TGFβR1R2-IL9R modified TIL, but induces relatively weak pSTAT1 (approximately 1.11-fold) signaling.

[0235] 2.3 Assessment of the proliferative capacity of TGFβR-IL9R modified T cells in response to TGFβ.

[0236] To evaluate whether the engineered TGFβR1R2-IL9R system conferred proliferative capacity upon TILs modified with it, the following experiments were conducted. In short, untransduced (i.e., unmodified) and TGFβR1R2-IL9R-modified TILs were compared... Figure 9A and Figure 9B In the presence of IL-2 (3000 IU / mL) Figure 9B ) or IL-2 is not present ( Figure 9A The cells were cultured under the following conditions, and then the cell count was performed at specified time points (day 0, day 3, day 5, etc.).

[0237] like Figure 9A The results showed that, in the absence of IL-2, the number of TIL cells began to decline continuously, typically starting from day 3 after transduction. In the absence of TGFβ1, there was essentially no difference in cell number reduction between unmodified TILs and TGFβR1R2-IL9R modified TILs; however, in the presence of TGFβ1, TGFβR1R2-IL9R modified TILs showed a significantly slower reduction compared to unmodified control cells. Figure 9B (See right figure, from day 3 to day 12, except for day 9). This suggests that in the absence of IL-2, TGFβ1 makes TGFβR1R2-IL9R modified TILs have better durability than unmodified TILs.

[0238] Further as Figure 9B The results showed that when TILs were cultured in the presence of IL-2, the number of TIL cells typically increased continuously. Figure 9BAs shown, while TGFβR1R2-IL9R modified TILs exhibited slightly slower proliferation compared to unmodified control cells in the absence of TGFβ1, TGFβR1R2-IL9R modified TILs showed significantly enhanced proliferation in the presence of TGFβ1 compared to unmodified control cells (see right panel; for example, on day 12, the cell expansion rate of modified cells was greater than approximately 2.5 times compared to unmodified cells). More specifically, starting from day 7, the presence of TGFβ1 significantly inhibited the proliferation of unmodified TILs (compared to the absence of TGFβ1), while in stark contrast, the presence of TGFβ1 still significantly stimulated the proliferation of TGFβR1R2-IL9R modified TILs. Therefore, in the presence of IL-2, TGFβ1 significantly enhances the proliferative capacity of TGFβR1R2-IL9R modified TILs compared to unmodified T cells.

[0239] 2.4 Assessment of the proliferative capacity of TGFβR-IL9R modified T cells in response to TGFβ after repeated tumor attacks.

[0240] To further evaluate whether the engineered TGFβR1R2-IL9R system enhances T cell resistance to exhaustion under repeated or continuous antigen stimulation, the following experiments were performed. In short, under conditions of IL-2 supply (1000 IU / mL) ( Figure 10B ) or in the absence of IL-2 ( Figure 10A Untransduced (i.e., unmodified) and TGFβR1R2-IL9R-modified TILs were co-cultured with fresh HeLa (anti-CD3) cells at a 1:1 ET ratio every 3-4 days. Cell counts were performed at specified time points.

[0241] Figure 10A The results show the effects of experiments on unmodified TILs cultured under repeated antigen stimulation (STI) and in the absence of IL-2, or on TILs modified with the engineered TGFβR1R2-IL9R system. In the absence of TGFβ1 (see left panel), both modified and unmodified TILs were difficult to amplify (although modified TILs showed slightly better amplification). In the presence of TGFβ1 (see right panel), the proliferation of unmodified TILs cultured under repeated antigen stimulation and in the absence of IL-2 was strongly inhibited, while conversely, TGFβ1 strongly promoted the proliferation of TGFβR1R2-IL9R modified TILs.

[0242] Figure 10BThe results show the effects of experiments on unmodified TILs cultured under repeated antigen stimulation (STI) and in the presence of IL-2, or on TILs modified with the engineered TGFβR1R2-IL9R system. In the absence of TGFβ1 (see left panel), these cultured TILs proliferated well, with little difference between modified and unmodified T cells. In the presence of TGFβ1 (see right panel), while TGFβ1 strongly inhibited the proliferation of unmodified TILs under these culture conditions, this inhibitory effect was significantly reduced or mitigated in TGFβR1R2-IL9R modified TILs.

[0243] Other implementation plans

[0244] It should be understood that the present invention has been described in conjunction with specific embodiments, but the foregoing description is illustrative only and is not intended to limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are all within the scope of the appended claims.

Claims

1. An engineered transforming growth factor β (TGFβ) receptor chain, which, when expressed in immune cells, encodes a transmembrane polypeptide, wherein, The transmembrane polypeptide comprises an extracellular portion and an intracellular portion operatively linked by a transmembrane portion, wherein: The extracellular portion comprises the extracellular domain (ECD) of either TGFβR1 or TGFβR2, or a functional portion or functional variant thereof; and The intracellular portion contains an intracellular domain (ICD) of either IL9R or IL2Rγ, or a functional portion or functional variant thereof.

2. The engineered TGFβ receptor chain according to claim 1, wherein the transmembrane portion comprises a transmembrane domain (TM) of any one of TGFβR1, TGFβR2, IL2Rγ, IL4R, IL9R, IL7R, IL15R, IL10R, IL21R, CD28, CTLA4, PD1, TIM3, TIGIT, CD4, CD8, or 4-1BB, or a functional portion or functional variant thereof.

3. The engineered TGFβ receptor chain of claim 1, wherein the transmembrane portion comprises a transmembrane domain (TM) of one of TGFβR1, TGFβR2, IL2Rγ, or IL9R, or a functional portion or functional variant thereof.

4. The engineered TGFβ receptor chain according to any one of claims 1-3, wherein: The extracellular portion comprises the extracellular domain (ECD) of TGFβR1, the extracellular domain containing a sequence having at least 70% identity with SEQ ID NO: 1; and The intracellular portion contains an intracellular domain (ICD) of IL9R, the intracellular domain containing a sequence having at least 70% identity with SEQ ID NO:

3.

5. The engineered TGFβ receptor chain according to claim 4, wherein the transmembrane portion comprises a transmembrane domain (TM) of TGFβR1, the transmembrane domain comprising a sequence having at least 70% identity with SEQ ID NO:

5.

6. The engineered TGFβ receptor chain according to claim 5, comprising a sequence having at least 70% identity with SEQ ID NO:

9.

7. The engineered TGFβ receptor chain of claim 4, wherein the transmembrane portion comprises a transmembrane domain (TM) of IL9R, the transmembrane domain comprising a sequence having at least 70% identity with SEQ ID NO:

7.

8. The engineered TGFβ receptor chain according to claim 5, comprising a sequence having at least 70% identity with SEQ ID NO:

10.

9. The engineered TGFβ receptor chain according to any one of claims 1-3, wherein: The extracellular portion comprises the extracellular domain (ECD) of TGFβR1, the extracellular domain containing a sequence having at least 70% identity with SEQ ID NO: 1; and The intracellular portion contains an intracellular domain (ICD) of IL2Rγ, the intracellular domain containing a sequence having at least 70% identity with SEQ ID NO:

4.

10. The engineered TGFβ receptor chain of claim 7, wherein the transmembrane portion comprises a transmembrane domain (TM) of TGFβR1, the transmembrane domain comprising a sequence having at least 70% identity with SEQ ID NO:

5.

11. The engineered TGFβ receptor chain according to claim 5, comprising a sequence having at least 70% identity with SEQ ID NO:

11.

12. The engineered TGFβ receptor chain of claim 7, wherein the transmembrane portion comprises a transmembrane domain (TM) of IL2Rγ, the transmembrane domain comprising a sequence having at least 70% identity with SEQ ID NO:

8.

13. The engineered TGFβ receptor chain according to claim 5, comprising a sequence having at least 70% identity with SEQ ID NO:

12.

14. The engineered TGFβ receptor chain according to claim 1, wherein: The extracellular portion comprises the extracellular domain (ECD) of TGFβR2, the extracellular domain containing a sequence having at least 70% identity with SEQ ID NO: 2; and The intracellular portion contains an intracellular domain (ICD) of IL9R, the intracellular domain containing a sequence having at least 70% identity with SEQ ID NO:

3.

15. The engineered TGFβ receptor chain of claim 10, wherein the transmembrane portion comprises a transmembrane domain (TM) of TGFβR2, the transmembrane domain comprising a sequence having at least 70% identity with SEQ ID NO:

6.

16. The engineered TGFβ receptor chain according to claim 5, comprising a sequence having at least 70% identity with SEQ ID NO:

13.

17. The engineered TGFβ receptor chain of claim 10, wherein the transmembrane portion comprises a transmembrane domain (TM) of IL9R, the transmembrane domain comprising a sequence having at least 70% identity with SEQ ID NO:

7.

18. The engineered TGFβ receptor chain according to claim 5, comprising a sequence having at least 70% identity with SEQ ID NO:

14.

19. The engineered TGFβ receptor chain according to claim 1, wherein: The extracellular portion comprises the extracellular domain (ECD) of TGFβR2, the extracellular domain containing a sequence having at least 70% identity with SEQ ID NO: 2; and The intracellular portion contains an intracellular domain (ICD) of IL2Rγ, the intracellular domain containing a sequence having at least 70% identity with SEQ ID NO:

4.

20. The engineered TGFβ receptor chain of claim 13, wherein the transmembrane portion comprises a transmembrane domain (TM) of TGFβR2, the transmembrane domain comprising a sequence having at least 70% identity with SEQ ID NO:

6.

21. The engineered TGFβ receptor chain according to claim 5, comprising a sequence having at least 70% identity with SEQ ID NO:

15.

22. The engineered TGFβ receptor chain of claim 13, wherein the transmembrane portion comprises a transmembrane domain (TM) of IL2Rγ, the transmembrane domain comprising a sequence having at least 70% identity with SEQ ID NO:

8.

23. The engineered TGFβ receptor chain according to claim 5, comprising a sequence having at least 70% identity with SEQ ID NO:

16.

24. An engineered transforming growth factor β (TGFβ) receptor system comprising at least one engineered TGFβ receptor chain, each engineered TGFβ receptor chain being based on an engineered TGFβ receptor chain according to any one of claims 1-23.

25. The engineered TGFβ receptor system of claim 24, comprising a first engineered TGFβ receptor chain and a second engineered TGFβ receptor chain, wherein: The extracellular portion of the first engineered TGFβ receptor chain and the extracellular portion of the second engineered TGFβ receptor chain respectively contain the extracellular domain (ECD) of TGFβR1 or a functional portion or functional variant thereof, and the extracellular domain (ECD) of TGFβR2 or a functional portion or functional variant thereof, or vice versa; and The intracellular portion of the first engineered TGFβ receptor chain and the intracellular portion of the second engineered TGFβ receptor chain respectively contain an intracellular domain (ICD) of IL9R or a functional portion or functional variant thereof, and an intracellular domain (ICD) of IL2Rγ or a functional portion or functional variant thereof, or vice versa.

26. The engineered TGFβ receptor system according to claim 24 or claim 25, wherein: The extracellular and intracellular portions of the first engineered TGFβ receptor chain each comprise: The extracellular domain (ECD) of TGFβR1 or a functional portion or variant thereof, said extracellular portion comprising a sequence having at least 70% identity with SEQ ID NO: 1; and The intracellular domain (ICD) of IL9R or a functional portion or variant thereof, wherein the intracellular portion comprises a sequence having at least 70% identity with SEQ ID NO: 3; Furthermore, the extracellular and intracellular portions of the second engineered TGFβ receptor chain respectively include: The extracellular domain (ECD) of TGFβR2 or a functional portion or variant thereof, said extracellular portion comprising a sequence having at least 70% identity with SEQ ID NO: 2; and The intracellular domain (ICD) of IL2Rγ or a functional portion or variant thereof, wherein the intracellular portion comprises a sequence having at least 70% identity with SEQ ID NO:

4.

27. The engineered TGFβ receptor system according to claim 26, wherein: The transmembrane portion of the first engineered TGFβ receptor chain comprises a transmembrane domain (TM) of TGFβR1 or a functional portion or variant thereof, said transmembrane portion comprising a sequence having at least 70% identity with SEQ ID NO: 5; and The transmembrane portion of the second engineered TGFβ receptor chain comprises a transmembrane domain (TM) of TGFβR2 or a functional portion or variant thereof, the transmembrane portion comprising a sequence having at least 70% identity with SEQ ID NO:

6.

28. The engineered TGFβ receptor system according to claim 27, wherein: The first engineered TGFβ receptor chain contains a sequence with at least 70% identity to SEQ ID NO: 9; and The second engineered TGFβ receptor chain contains a sequence that has at least 70% identity with SEQ ID NO:

15.

29. The engineered TGFβ receptor system according to claim 26, wherein: The transmembrane portion of the first engineered TGFβ receptor chain comprises a transmembrane domain (TM) of IL9R or a functional portion or variant thereof, said transmembrane portion comprising a sequence having at least 70% identity with SEQ ID NO: 7; and The transmembrane portion of the second engineered TGFβ receptor chain contains a transmembrane domain (TM) of IL2Rγ or a functional portion or variant thereof, the transmembrane portion containing a sequence having at least 70% identity with SEQ ID NO:

8.

30. The engineered TGFβ receptor system according to claim 29, wherein: The first engineered TGFβ receptor chain contains a sequence having at least 70% identity with SEQ ID NO: 10; and The second engineered TGFβ receptor chain contains a sequence that has at least 70% identity with SEQ ID NO:

16.

31. The engineered TGFβ receptor system according to claim 24 or claim 25, wherein: The extracellular and intracellular portions of the first engineered TGFβ receptor chain each comprise: The extracellular domain (ECD) of TGFβR1 or a functional portion or variant thereof, said extracellular portion comprising a sequence having at least 70% identity with SEQ ID NO: 1; and The intracellular domain (ICD) of IL2Rγ or a functional portion or variant thereof, wherein the intracellular portion comprises a sequence having at least 70% identity with SEQ ID NO: 4; Furthermore, the extracellular and intracellular portions of the second engineered TGFβ receptor chain respectively include: The extracellular domain (ECD) of TGFβR2 or a functional portion or variant thereof, said extracellular portion comprising a sequence having at least 70% identity with SEQ ID NO: 2; and The intracellular domain (ICD) of IL9R or a functional portion or variant thereof, wherein the intracellular portion contains a sequence having at least 70% identity with SEQ ID NO:

3.

32. The engineered TGFβ receptor system according to claim 31, wherein: The transmembrane portion of the first engineered TGFβ receptor chain comprises the transmembrane region (TM) of TGFβR1 or a functional portion or variant thereof, said transmembrane portion comprising a sequence having at least 70% identity with SEQ ID NO: 5; and The transmembrane portion of the second engineered TGFβ receptor chain comprises the transmembrane region (TM) of TGFβR2 or a functional portion or functional variant thereof, the transmembrane portion comprising a sequence having at least 70% identity with SEQ ID NO:

6.

33. The engineered TGFβ receptor system according to claim 32, wherein: The first engineered TGFβ receptor chain contains a sequence having at least 70% identity with SEQ ID NO: 11; and The second engineered TGFβ receptor chain contains a sequence that has at least 70% identity with SEQ ID NO:

13.

34. The engineered TGFβ receptor system according to claim 31, wherein: The transmembrane portion of the first engineered TGFβ receptor chain comprises the transmembrane region (TM) of IL2Rγ or a functional portion or variant thereof, said transmembrane portion comprising a sequence having at least 70% identity with SEQ ID NO: 8; and The transmembrane portion of the second engineered TGFβ receptor chain comprises the transmembrane region (TM) of IL9R or a functional portion or functional variant thereof, the transmembrane portion comprising a sequence having at least 70% identity with SEQ ID NO:

7.

35. The engineered TGFβ receptor system according to claim 34, wherein: The first engineered TGFβ receptor chain contains a sequence having at least 70% identity with SEQ ID NO: 12; and The second engineered TGFβ receptor chain contains a sequence that has at least 70% identity with SEQ ID NO:

14.

36. The engineered TGFβ receptor system according to any one of claims 24-35, wherein, When the engineered TGFβ receptor system is expressed in immune cells, the immune cells stimulated with TGFβ exhibit activation of the JAK-STAT signaling pathway compared to those without TGFβ stimulation.

37. The engineered TGFβ receptor system according to claim 36, wherein, When the engineered TGFβ receptor system is expressed in immune cells, the immune cells stimulated with TGFβ show increased phosphorylation of at least one of STAT1, STAT3, or STAT5 compared to those without TGFβ stimulation.

38. The engineered TGFβ receptor system according to claim 37, wherein, When the engineered TGFβ receptor system is expressed in immune cells, the phosphorylation levels of STAT3 and / or STAT5 in the immune cells stimulated with TGFβ are at least 50% higher than those without TGFβ stimulation.

39. The engineered TGFβ receptor system of claim 38, wherein the extracellular and transmembrane portions of either the first engineered TGFβ receptor chain or the second engineered TGFβ receptor chain are derived from the same protein, wherein, When the engineered TGFβ receptor system is expressed in immune cells, the phosphorylation levels of STAT3 and / or STAT5 in the immune cells stimulated with TGFβ are at least 2.5 times higher than those without TGFβ stimulation.

40. The engineered TGFβ receptor system of claim 39, wherein the extracellular, transmembrane, and intracellular portions of the first engineered TGFβ receptor chain respectively comprise: the extracellular domain (ECD) of TGFβR1 or a functional portion or variant thereof, the transmembrane domain (TM) of TGFβR1 or a functional portion or variant thereof, and the intracellular domain (ICD) of IL9R or a functional portion or variant thereof; and the extracellular, transmembrane, and intracellular portions of the second engineered TGFβ receptor chain comprise the extracellular domain (ECD) of TGFβR2 or a functional portion or variant thereof, the transmembrane domain (TM) of TGFβR2 or a functional portion or variant thereof, and the intracellular domain (ICD) of IL2Rγ or a functional portion or variant thereof, wherein, When the engineered TGFβ receptor system is expressed in immune cells, the phosphorylation level of STAT3 in the immune cells stimulated with TGFβ is at least 4.0 times that without TGFβ stimulation.

41. The engineered TGFβ receptor system according to any one of claims 24-35, wherein, When the engineered TGFβ receptor system is expressed in immune cells cultured in the presence of IL-2, the immune cells stimulated with TGFβ exhibit enhanced proliferative capacity compared to immune cells in which the engineered TGFβ receptor system is not expressed.

42. The engineered TGFβ receptor system according to any one of claims 24-35, wherein, When the engineered TGFβ receptor system is expressed in immune cells cultured in the absence of IL-2, the immune cells stimulated with TGFβ exhibit slower proliferation reduction in the presence of TGFβ1 compared to immune cells without expression of the engineered TGFβ receptor system.

43. The engineered TGFβ receptor system according to any one of claims 24-35, wherein, When the engineered TGFβ receptor system is expressed in immune cells cultured in the absence of IL-2, the immune cells stimulated with TGFβ exhibit better viability in the presence of TGFβ1 compared to immune cells in which the engineered TGFβ receptor system is not expressed.

44. The engineered TGFβ receptor system of claim 43, wherein the extracellular and transmembrane portions of either the first engineered TGFβ receptor chain or the second engineered TGFβ receptor chain are derived from the same protein, wherein, When the engineered TGFβ receptor system is expressed in a population of immune cells cultured in the absence of IL-2, the population of immune cells stimulated with TGFβ has a viability of at least 50% 12 days after IL-2 is withdrawn.

45. The engineered TGFβ receptor system of claim 44, wherein the extracellular, transmembrane, and intracellular portions of the first engineered TGFβ receptor chain respectively comprise: the extracellular domain (ECD) of TGFβR1 or a functional portion or variant thereof, the transmembrane domain (TM) of TGFβR1 or a functional portion or variant thereof, and the intracellular domain (ICD) of IL9R or a functional portion or variant thereof; and the extracellular, transmembrane, and intracellular portions of the second engineered TGFβ receptor chain comprise the extracellular domain (ECD) of TGFβR2 or a functional portion or variant thereof, the transmembrane domain (TM) of TGFβR2 or a functional portion or variant thereof, and the intracellular domain (ICD) of IL2Rγ or a functional portion or variant thereof, wherein, When the engineered TGFβ receptor system is expressed in a population of immune cells cultured in the absence of IL-2, the population of immune cells stimulated with TGFβ has a viability of at least 65% 12 days after IL-2 is withdrawn.

46. ​​The engineered TGFβ receptor system according to any one of claims 24-35, wherein, When the engineered TGFβ receptor system is expressed in immune cells cultured in the absence of IL-2 and under repeated antigen stimulation, the immune cells stimulated with TGFβ exhibit enhanced proliferative capacity compared to immune cells in the absence of the engineered TGFβ receptor system.

47. The engineered TGFβ receptor system of claim 46, wherein the extracellular and transmembrane portions of either the first engineered TGFβ receptor chain or the second engineered TGFβ receptor chain are derived from the same protein, wherein... When the engineered TGFβ receptor system is expressed in immune cells cultured in the absence of IL-2 and under repeated antigen stimulation, the immune cells stimulated with TGFβ expand at least 300-fold.

48. The engineered TGFβ receptor system of claim 47, wherein the extracellular, transmembrane, and intracellular portions of the first engineered TGFβ receptor chain respectively comprise: the extracellular domain (ECD) of TGFβR1 or a functional portion or variant thereof, the transmembrane domain (TM) of TGFβR1 or a functional portion or variant thereof, and the intracellular domain (ICD) of IL9R or a functional portion or variant thereof; and the extracellular, transmembrane, and intracellular portions of the second engineered TGFβ receptor chain comprise the extracellular domain (ECD) of TGFβR2 or a functional portion or variant thereof, the transmembrane domain (TM) of TGFβR2 or a functional portion or variant thereof, and the intracellular domain (ICD) of IL2Rγ or a functional portion or variant thereof, wherein, When the engineered TGFβ receptor system is expressed in immune cells cultured in the absence of IL-2 and under repeated antigen stimulation, the immune cells stimulated with TGFβ expand at least 500-fold.

49. The engineered TGFβ receptor system according to any one of claims 24-35, wherein, When the engineered TGFβ receptor system is expressed in a population of immune cells cultured in the absence of IL-2 and under repeated antigen stimulation, the population of immune cells stimulated with TGFβ exhibits enhanced viability compared to immune cells in which the engineered TGFβ receptor system is not expressed.

50. The engineered TGFβ receptor system according to claim 49, wherein, When the engineered TGFβ receptor system is expressed in a population of immune cells cultured in the absence of IL-2 and under repeated antigen stimulation, the immune cell population stimulated with TGFβ has a survival rate of at least 70% 9 days after IL-2 is withdrawn.

51. A vector system comprising at least one vector encoding an engineered TGFβ receptor system according to any one of claims 24-50, wherein, When the aforementioned vector system is used to transduce immune cells, these immune cells are able to activate JAK-STAT signaling upon TGFβ stimulation.

52. The vector system of claim 51, wherein the engineered TGFβ receptor system comprises a first engineered TGFβ receptor chain and a second engineered TGFβ receptor chain, wherein the extracellular, transmembrane, and intracellular portions of the first engineered TGFβ receptor chain respectively comprise: the extracellular domain (ECD) of TGFβR1 or a functional portion or variant thereof, the transmembrane domain (TM) of TGFβR1 or a functional portion or variant thereof, and the intracellular domain (ICD) of IL9R or a functional portion or variant thereof; and the extracellular, transmembrane, and intracellular portions of the second engineered TGFβ receptor chain comprise the extracellular domain (ECD) of TGFβR2 or a functional portion or variant thereof, the transmembrane domain (TM) of TGFβR2 or a functional portion or variant thereof, and the intracellular domain (ICD) of IL2Rγ or a functional portion or variant thereof, or vice versa.

53. The vector system of claim 52, wherein the first engineered TGFβ receptor chain and the second engineered TGFβ receptor chain in the engineered TGFβ receptor system are encoded by a vector.

54. The vector system of claim 53, wherein the first engineered TGFβ receptor chain and the second engineered TGFβ receptor chain are encoded by an open reading frame (ORF) in one of the vectors.

55. The vector system of claim 54, wherein the open reading frame (ORF) comprises a polynucleotide encoding a self-cleaving peptide, the polynucleotide being operatively linked to a polynucleotide encoding the first engineered TGFβ receptor chain and a polynucleotide encoding the second engineered TGFβ receptor chain, wherein, The self-cleaving peptide is selected from T2A, P2A, F2A or E2A.

56. The vector system according to claim 52, comprising two vectors encoding the first engineered TGFβ receptor chain and the second engineered TGFβ receptor chain, respectively.

57. The carrier system according to any one of claims 52-56, wherein, A first polynucleotide encoding a first signal peptide is operatively linked to a polynucleotide encoding a first engineered TGFβ receptor chain, and a second polynucleotide encoding a second signal peptide is operatively linked to a polynucleotide encoding a second engineered TGFβ receptor chain, wherein the extracellular portions of the first signal peptide and the first engineered TGFβ receptor chain are derived from the same protein, and the extracellular portions of the second signal peptide and the second engineered TGFβ receptor chain are derived from the same protein.

58. An immune cell that expresses the engineered TGFβ receptor system according to any one of claims 24-50.

59. The immune cell according to claim 58, wherein, Compared to no TGFβ stimulation, the immune cells showed activation of the JAK-STAT signaling pathway when stimulated with TGFβ.

60. The immune cell according to claim 59, wherein, Compared with no TGFβ stimulation, when stimulated with TGFβ, the immune cells showed increased phosphorylation of at least one of STAT1, STAT3, or STAT5.

61. The immune cell according to claim 60, wherein, When the immune cells were stimulated with TGFβ, the phosphorylation levels of STAT3 and / or STAT5 in the immune cells were at least 50% higher than when there was no TGFβ stimulation.

62. The immune cell of claim 61, wherein the extracellular and transmembrane portions of either the first engineered TGFβ receptor chain or the second engineered TGFβ receptor chain are derived from the same protein, wherein, When the immune cells are stimulated with TGFβ, the phosphorylation levels of STAT3 and / or STAT5 in the immune cells are at least 2.5 times higher than when there is no TGFβ stimulation.

63. The immune cell of claim 62, wherein the extracellular, transmembrane, and intracellular portions of the first engineered TGFβ receptor chain respectively comprise: the extracellular domain (ECD) of TGFβR1 or a functional portion or variant thereof, the transmembrane domain (TM) of TGFβR1 or a functional portion or variant thereof, and the intracellular domain (ICD) of IL9R or a functional portion or variant thereof; and the extracellular, transmembrane, and intracellular portions of the second engineered TGFβ receptor chain comprise the extracellular domain (ECD) of TGFβR2 or a functional portion or variant thereof, the transmembrane domain (TM) of TGFβR2 or a functional portion or variant thereof, and the intracellular domain (ICD) of IL2Rγ or a functional portion or variant thereof; wherein, When the immune cells were stimulated with TGFβ, the phosphorylation level of STAT3 in the immune cells was at least 4.0 times that without TGFβ stimulation.

64. The immune cells according to any one of claims 58-63, wherein, When cultured in the presence of IL-2, the TGFβ-stimulated immune cells exhibited enhanced proliferative capacity compared to immune cells lacking the engineered TGFβ receptor system.

65. The immune cells according to any one of claims 58-63, wherein, When cultured in the absence of IL-2, the TGFβ-stimulated immune cells exhibited slower proliferation reduction in the presence of TGFβ1 compared to immune cells without the engineered TGFβ receptor system.

66. The immune cells according to any one of claims 58-63, wherein, When cultured in the absence of IL-2, the immune cells stimulated with TGFβ showed better viability in the presence of TGFβ1 compared to immune cells without the engineered TGFβ receptor system.

67. The immune cell of claim 66, wherein the extracellular and transmembrane portions of either the first engineered TGFβ receptor chain or the second engineered TGFβ receptor chain are derived from the same protein, wherein, When the immune cells stimulated by TGFβ were repeatedly stimulated by the antigen without the administration of exogenous IL-2, the population of TGFβ-stimulated immune cells had a survival rate of at least 50% 12 days after IL-2 withdrawal.

68. The immune cell of claim 67, wherein the extracellular, transmembrane, and intracellular portions of the first engineered TGFβ receptor chain respectively comprise: the extracellular domain (ECD) of TGFβR1 or a functional portion or variant thereof, the transmembrane domain (TM) of TGFβR1 or a functional portion or variant thereof, and the intracellular domain (ICD) of IL9R or a functional portion or variant thereof; and the extracellular, transmembrane, and intracellular portions of the second engineered TGFβ receptor chain comprise the extracellular domain (ECD) of TGFβR2 or a functional portion or variant thereof, the transmembrane domain (TM) of TGFβR2 or a functional portion or variant thereof, and the intracellular domain (ICD) of IL2Rγ or a functional portion or variant thereof; wherein, When the immune cells stimulated by TGFβ were repeatedly stimulated by the antigen without the administration of exogenous IL-2, the population of TGFβ-stimulated immune cells had a survival rate of at least 65% 12 days after IL-2 withdrawal.

69. The immune cells according to any one of claims 58-63, wherein, When repeatedly stimulated by antigens without the administration of exogenous IL-2, the TGFβ-stimulated immune cells exhibited enhanced proliferative capacity compared to immune cells lacking the engineered TGFβ receptor system.

70. The immune cell of claim 69, wherein the extracellular and transmembrane portions of either the first engineered TGFβ receptor chain or the second engineered TGFβ receptor chain are derived from the same protein, wherein, When the immune cells stimulated by TGFβ were repeatedly stimulated without the administration of exogenous IL-2, the cells expanded at least 300-fold.

71. The immune cell of claim 70, wherein the extracellular, transmembrane, and intracellular portions of the first engineered TGFβ receptor chain respectively comprise: the extracellular domain (ECD) of TGFβR1 or a functional portion or variant thereof, the transmembrane domain (TM) of TGFβR1 or a functional portion or variant thereof, and the intracellular domain (ICD) of IL9R or a functional portion or variant thereof; and the extracellular, transmembrane, and intracellular portions of the second engineered TGFβ receptor chain comprise the extracellular domain (ECD) of TGFβR2 or a functional portion or variant thereof, the transmembrane domain (TM) of TGFβR2 or a functional portion or variant thereof, and the intracellular domain (ICD) of IL2Rγ or a functional portion or variant thereof; wherein, When the immune cells stimulated by TGFβ were repeatedly stimulated without the administration of exogenous IL-2, the cells expanded at least 500-fold.

72. The immune cells according to any one of claims 58-63, wherein, When repeatedly stimulated by antigens and cultured without IL-2, the immune cells exhibited enhanced viability upon stimulation with TGFβ compared to immune cells lacking the engineered TGFβ receptor system.

73. The immune cell according to claim 72, wherein, When the immune cells stimulated by TGFβ were repeatedly stimulated by the antigen without the administration of exogenous IL-2, the population of TGFβ-stimulated immune cells had a survival rate of at least 70% 9 days after IL-2 withdrawal.

74. The immune cell according to any one of claims 58-73, wherein the immune cell is a T cell, a natural killer (NK) cell, or a tumor-infiltrating lymphocyte (TIL).

75. The immune cell of claim 74, wherein the immune cell further expresses a target recognition receptor capable of binding to a target molecule on a target cell.

76. The immune cell of claim 75, wherein the target recognition receptor is selected from chimeric antigen receptor (CAR), T cell receptor (TCR) or Aspire-TCR.

77. A method of treating a tumor in a patient in need, comprising administering a group of immune cells according to any one of claims 58-77.

78. The method of claim 77, wherein the immune cells further express a target recognition receptor capable of binding to a target molecule on a target cancer cell in the tumor, wherein the target recognition receptor is selected from chimeric antigen receptor (CAR), T-cell receptor (TCR), or Aspire-TCR.

79. The method of claim 77 or claim 78, wherein the tumor is a solid tumor characterized by high expression of TGFβ.

80. The method of claim 79, wherein the solid tumor is selected from hepatocellular carcinoma (HCC), breast cancer, prostate cancer, glioma, bladder cancer, or thymic epithelial tumor.

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