WO2021238903A1 - 增强型合成t细胞受体抗原受体 - Google Patents
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Definitions
- the present invention relates to the field of biomedicine, and in particular to an enhanced synthetic T-cell receptor antigen receptor (Snythetic T-Cell Receptor and Antigen Receptor, STAR) targeting CD19 and CD20, including the synthetic T-cell receptor antigen receptor.
- Synythetic T-Cell Receptor and Antigen Receptor, STAR enhanced synthetic T-cell receptor antigen receptor targeting CD19 and CD20, including the synthetic T-cell receptor antigen receptor.
- CAR-T chimeric antigen receptor T cell
- CAR-T therapy is based on the expression of CAR molecules in T cells.
- the CAR molecule contains three major parts: the extracellular domain is the antigen recognition domain derived from the antibody and is responsible for recognizing the target antigen; the transmembrane domain; the intracellular domain is the signal molecule and costimulatory signal molecule derived from the T cell receptor and is responsible for Transmit T cell activation signal after receiving the stimulation.
- the CAR molecule binds to its corresponding antigen, the CAR molecule will aggregate, start the effector function of T cells, and kill target tumor cells.
- TCR-T therapy is based on the T cell receptor (TCR).
- TCR is the identity feature of T cells.
- T cells can be divided into ⁇ T cells and ⁇ T cells.
- T precursor cells will first undergo VDJ rearrangement of TCR ⁇ and TCR ⁇ chains. If the rearrangement is successful, they will develop into ⁇ T cells. If the rearrangement fails, the precursor cells will undergo VDJ recombination of TCR ⁇ and TCR ⁇ chains, and then Develop into ⁇ T cells.
- ⁇ T cells account for 90%-95% of peripheral blood T cells
- ⁇ T cells account for 5%-10% of peripheral blood T cells.
- the two types of T cells recognize antigens in MHC restricted and MHC non-restricted ways, respectively, and play an important role in the immunity of pathogens and tumors.
- TCR T cell receptor
- TCR ⁇ chain and TCR ⁇ chain are responsible for recognizing MHC-polypeptide molecules, and the other 6 CD3 subunits are associated with TCR ⁇ / ⁇ chain (or TCR ⁇ / ⁇ chain) combined to play a signal transduction function.
- the natural TCR complex contains a total of 10 ITAM signal sequences, which can theoretically transmit stronger signals than CAR. Using the signal transduction function of natural TCR, it will be possible to construct a new type of receptor to alleviate the disability of T cells, so that it can better play the role of anti-solid tumors.
- TCR variable region sequence can be replaced with antibody variable region sequence to obtain synthetic T cell receptor antigen receptor (Synthetic TCR and Antigen Receptor, STAR), It has the specificity of antibodies and the superior signal transduction function of natural TCR, which can mediate T cell activation.
- Figure 1 Schematic diagram of optimization of STAR by constant region cysteine modification, transmembrane and intracellular region modification.
- Figure 2 Schematic diagram of optimization of STAR by adding co-stimulatory molecule receptor intracellular domains to alpha and/or beta chains.
- Figure 3 A schematic diagram of optimizing STAR by adding the intracellular domain of a costimulatory molecule receptor directly or through a linker after deleting the intracellular region of the ⁇ and/or ⁇ chain.
- Figure 4 Schematic diagram of optimization of STAR by adding the intracellular domain of the co-stimulatory molecule receptor to the CD3 subunit.
- Figure 5 Schematic diagram of optimizing STAR by adding intracellular signal transduction regions of cytokine receptors to alpha and/or beta chains.
- Figure 7 The effect of different costimulatory molecule receptor intracellular domains on the target killing ability of STAR-T cells under different effective target ratios.
- Figure 8 The effect of different co-stimulatory molecule receptor intracellular domains on the target killing ability of STAR-T cells under different co-cultivation times.
- Figure 9 The effect of OX40 intracellular domains added to ⁇ chain, ⁇ chain, ⁇ and ⁇ chain on the target killing ability of STAR-T cells under different effective target ratios.
- Figure 10 The effect of OX40 intracellular domains added to ⁇ chain, ⁇ chain, ⁇ and ⁇ chain on the target killing ability of STAR-T cells under different co-cultivation time.
- Figure 11 The effect of intracellular domains of different costimulatory molecule receptors on cytokine secretion in STAR-T cells.
- Figure 12 The effect of intracellular domains of different costimulatory molecule receptors on the cell proliferation of STAR-T cells.
- Figure 13 The effect of the addition of OX40 intracellular domains to the ⁇ chain, ⁇ chain, ⁇ and ⁇ chain on the cell proliferation of STAR-T cells.
- Figure 14 The tumor-killing ability of mut-STAR in which the intracellular domains of different costimulatory molecules are connected to the TCR ⁇ chain.
- Figure 15 The effect of adding different co-stimulatory molecule receptor intracellular domains to different CD3 subunit molecules on the target cell killing ability of STAR-T cells.
- Figure 16 The effect of adding different co-stimulatory molecule receptor intracellular domains to different CD3 subunit molecules on the cell proliferation of STAR-T cells.
- Figure 17 The effect of the OX40 intracellular domain connected to the ⁇ chain of the deleted intracellular region through different G4S linkers on the target killing ability of STAR-T cells.
- Figure 18 The effect of the OX40 intracellular domain connected to the ⁇ chain of the deleted intracellular region through different G4S linkers on the target killing ability of STAR-T cells.
- Figure 19 The effect of OX40 intracellular domains connected by different G4S linkers to delete the alpha and/or beta chains of the intracellular region on IL-2 secretion by STAR-T cells.
- Figure 20 The effect of OX40 intracellular domain connected through different G4S linkers to delete the ⁇ and/or ⁇ chains of the intracellular region on the secretion of IFN- ⁇ from STAR-T cells.
- Figure 21 The effect of the OX40 intracellular domain connected to the ⁇ chain of the deleted intracellular region through different linkers on the differentiation of central memory T cells.
- FIG. 22 The effect of OX40 intracellular domain connected to the alpha chain deleted intracellular region through different linkers on T cell differentiation.
- Figure 23 The effect of lysine modification in the transmembrane region or the intracellular terminal on the target killing ability of STAR-T cells.
- Figure 24 The effect of lysine modification in the transmembrane region or the intracellular terminal on the secretion of IFN- ⁇ from STAR-T cells.
- Figure 25 The effect of lysine modification in the transmembrane region or the intracellular terminal on IL-2 secretion by STAR-T cells.
- Figure 26 The effect of lysine modification in the transmembrane region or the intracellular terminal on the differentiation of central memory T cells.
- Figure 27 The effect of lysine modification in the transmembrane region or the intracellular terminal on T cell differentiation.
- Figure 28 The effect of linking the intracellular signal transduction regions of different cytokine receptors to the ⁇ and/or ⁇ chains on the target killing ability of STAR-T cells.
- Figure 29 Comparison of the killing effects of mutant STAR and ⁇ -del-(G4S)3-OX40-STAR connected to the stimulation region of different cytokine receptors.
- Figure 30 The effect of linking the intracellular signal transduction regions of different cytokine receptors to ⁇ and/or ⁇ chains on IL-2 secretion by STAR-T cells.
- Figure 31 The effect of linking the intracellular signal transduction regions of different cytokine receptors to ⁇ and/or ⁇ chains on IFN- ⁇ secretion by STAR-T cells.
- Figure 32 The effect of linking the intracellular signal transduction regions of different cytokine receptors to the alpha and/or beta chains on the differentiation of central memory T cells.
- Figure 33 The effect of linking the intracellular signal transduction regions of different cytokine receptors to ⁇ and/or ⁇ chains on T cell differentiation.
- Figure 34 In vivo anti-tumor effects of ⁇ OX40-STAR T, mut-STAR T and CAR-T in mouse tumor models.
- Figure 35 Survival curves of mice administered ⁇ OX40-STAR T, mut-STAR T and CAR-T.
- Figure 36 Proliferation of ⁇ OX40-STAR T, mut-STAR T and CAR-T in mice.
- Figure 37 In vivo anti-tumor effects of different STAR structures and CAR-T in mouse tumor models.
- Figure 38 Schematic diagram of optimization of STAR by constant region cysteine modification, transmembrane region and N-terminal rearrangement modification.
- Figure 39 An example of the connection position between the molecular domain of a costimulatory molecule and the STAR structure. Only the connection to the alpha chain is shown.
- Figure 40 Example of a STAR structure containing a co-stimulatory molecule domain.
- Figure 41 STAR and the killing ability of costimulatory factor STAR.
- Figure 42 The level of STAR and the proliferation signal RELB containing costimulatory factor STAR into the nucleus.
- Figure 44 Results of different STARs for CD19 and CD20, with costimulatory factors added to the ⁇ and ⁇ chains.
- Figure 45 STAR results for CD19 and CD20 with costimulatory factors added to the alpha chain.
- the term “and/or” encompasses all combinations of items connected by the term, and should be treated as if each combination has been individually listed herein.
- “A and/or B” encompasses “A”, “A and B”, and “B”.
- “A, B and/or C” encompasses "A”, “B”, “C”, “A and B”, “A and C”, “B and C”, and "A and B and C”.
- the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nuclei at one or both ends of the protein or nucleic acid. Glycolic acid, but still has the activity described in the present invention.
- methionine encoded by the start codon at the N-terminus of the polypeptide will be retained under certain actual conditions (for example, when expressed in a specific expression system), but does not substantially affect the function of the polypeptide.
- amino acid numbering refers to SEQ ID NO: x
- SEQ ID NO: x is a specific sequence listed herein
- amino acid correspondence can be determined according to sequence alignment methods known in the art. For example, the amino acid correspondence can be determined by the online comparison tool of EMBL-EBI (https://www.ebi.ac.uk/Tools/psa/), and the two sequences can use the Needleman-Wunsch algorithm, using the default parameters. Align.
- the amino acid in the polypeptide can also be described herein. It is "alanine at position 48 of the polypeptide, and the amino acid position refers to SEQ ID NO: x".
- the amino acid positions related to the constant region of the ⁇ chain refer to SEQ ID NO: 3.
- the amino acid positions related to the ⁇ chain constant region refer to SEQ ID NO: 4.
- the present invention provides a modified T cell receptor (TCR) complex, wherein
- the TCR may be ⁇ TCR, and the ⁇ TCR complex includes TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ ; wherein at least one of TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ is in its C At least one functional domain is attached to the end;
- the TCR ⁇ chain includes a first constant region and a first antigen binding region, and the TCR ⁇ chain includes a second constant region and a second antigen binding region;
- first antigen binding region specifically binds to the first antigen
- second antigen binding region specifically binds to the second antigen
- first antigen binding region and the second antigen binding region are combined with each other to be specific sexually binds the first antigen and the second antigen
- the first antigen is CD19 and the second antigen is CD20
- the first antigen is CD20 and the second antigen is CD19;
- the TCR may be ⁇ TCR, and the ⁇ TCR complex includes TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ ; wherein at least one of TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ is in its C At least one functional domain is attached to the end;
- the TCR ⁇ chain includes a first constant region and a first antigen binding region, and the TCR ⁇ chain includes a second constant region and a second antigen binding region;
- first antigen binding region specifically binds to the first antigen
- second antigen binding region specifically binds to the second antigen
- first antigen binding region and the second antigen binding region are combined with each other to be specific sexually binds the first antigen and the second antigen; preferably, the first antigen is CD19 and the second antigen is CD20, or the first antigen is CD20 and the second antigen is CD19.
- the antigen binding region is located at the N-terminus of the constant region, and the two can be connected directly or through a linker.
- the present invention provides a modified T cell receptor (TCR), wherein the TCR can be
- an ⁇ TCR comprising a TCR ⁇ chain and a TCR ⁇ chain, the TCR ⁇ chain and/or TCR ⁇ chain of the ⁇ TCR has at least one functional domain connected at its C terminal;
- the TCR ⁇ chain includes a first constant region and a first antigen binding region, and the TCR ⁇ chain includes a second constant region and a second antigen binding region;
- first antigen binding region specifically binds to the first antigen
- second antigen binding region specifically binds to the second antigen
- first antigen binding region and the second antigen binding region are combined with each other to be specific sexually binds the first antigen and the second antigen
- the first antigen is CD19 and the second antigen is CD20
- the first antigen is CD20 and the second antigen is CD19;
- a ⁇ TCR comprising a TCR ⁇ chain and a TCR ⁇ chain, wherein the TCR ⁇ chain and/or TCR ⁇ chain of the ⁇ TCR has at least one functional domain connected at its C terminal;
- the TCR ⁇ chain includes a first constant region and a first antigen binding region, and the TCR ⁇ chain includes a second constant region and a second antigen binding region;
- first antigen binding region specifically binds to the first antigen
- second antigen binding region specifically binds to the second antigen
- first antigen binding region and the second antigen binding region are combined with each other to be specific sexually binds the first antigen and the second antigen; preferably, the first antigen is CD19 and the second antigen is CD20, or the first antigen is CD20 and the second antigen is CD19.
- the natural intracellular region of at least one of TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ in the ⁇ TCR complex is deleted, or wherein the TCR ⁇ chain in the ⁇ TCR complex,
- the natural intracellular region of at least one of the TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ is deleted.
- the natural intracellular region of the TCR ⁇ chain and/or TCR ⁇ chain in the ⁇ TCR is deleted, or wherein the natural intracellular region of the TCR ⁇ chain and/or TCR ⁇ chain in the ⁇ TCR is deleted.
- the functional domain in the ⁇ TCR complex, is directly or through a linker connected to the TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ in which the natural intracellular region is deleted. At least one C-terminal.
- the functional domain is directly or via a linker connected to the C-terminus of the TCR ⁇ chain and/or TCR ⁇ chain where the natural intracellular region is deleted.
- the functional domain is directly or through a linker connected to the TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ in which the natural intracellular region is deleted. At least one C-terminal.
- the functional domain is directly or via a linker connected to the C-terminus of the TCR ⁇ chain and/or the TCR ⁇ chain where the natural intracellular region is deleted.
- the linker is a (G 4 S)n linker, where n represents an integer from 1-10.
- one of the TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ in the ⁇ TCR complex has at least one functional domain connected at its C-terminus.
- the TCR ⁇ chain and/or TCR ⁇ chain of the ⁇ TCR has at least one functional domain connected at its C-terminus.
- one of the TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ in the ⁇ TCR complex has at least one functional domain connected at its C-terminus.
- the TCR ⁇ chain and/or TCR ⁇ chain has at least one functional domain connected at its C-terminus.
- none of the CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ in the TCR complex includes at least one functional domain additionally connected to its C-terminus.
- the TCR ⁇ chain in the ⁇ TCR complex has at least one functional domain connected at its C-terminus.
- the TCR ⁇ chain of the ⁇ TCR has at least one functional domain attached to the C-terminus.
- the TCR ⁇ chain in the ⁇ TCR complex has at least one functional domain connected at its C-terminus.
- the TCR ⁇ chain of the ⁇ TCR has at least one functional domain connected at its C-terminus.
- the TCR ⁇ chain in the ⁇ TCR complex has at least one functional domain connected at its C-terminus.
- the TCR ⁇ chain has at least one functional domain connected at its C-terminus.
- the TCR ⁇ chain in the ⁇ TCR complex has at least one functional domain connected at its C-terminus.
- the TCR ⁇ chain in the ⁇ TCR has at least one functional domain connected at its C-terminus.
- two of the TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ in the ⁇ TCR complex have at least one functional domain connected at their C-terminus.
- two of the TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ in the ⁇ TCR complex have at least one functional domain connected at their C-terminus.
- each of the TCR ⁇ chain and the TCR ⁇ chain in the ⁇ TCR complex has at least one functional domain connected at its C-terminus.
- each of the TCR ⁇ chain and the TCR ⁇ chain of the ⁇ TCR has at least one functional domain connected at its C-terminus.
- TCR alpha chain and TCR beta chain are deleted.
- the functional domain is directly or via a linker connected to the C-terminus of the TCR ⁇ chain and the TCR ⁇ chain where the natural intracellular region is deleted.
- each of the TCR ⁇ chain and the TCR ⁇ chain in the ⁇ TCR complex has at least one functional domain connected at its C-terminus.
- each of the TCR ⁇ chain and the TCR ⁇ chain in the ⁇ TCR has at least one functional domain connected at its C-terminus.
- the functional domain is directly or through a linker connected to the C-terminus of the TCR ⁇ chain and the TCR ⁇ chain where the natural intracellular region is deleted.
- two or more of TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ in the ⁇ TCR complex may be connected to the same or different functional domains.
- the TCR ⁇ chain and/or TCR ⁇ chain in the ⁇ TCR may be connected to the same or different functional domains.
- two or more of TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ in the ⁇ TCR complex may be connected to the same or different functional domains.
- the TCR ⁇ chain and/or TCR ⁇ chain in the ⁇ TCR may be connected to the same or different functional domains.
- At least one of the TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ in the ⁇ TCR complex has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains.
- the TCR ⁇ chain and/or TCR ⁇ chain of the ⁇ TCR has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains connected at its C-terminus .
- At least one of TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ in the ⁇ TCR complex has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains.
- the TCR ⁇ chain and/or TCR ⁇ chain has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains connected at its C-terminus .
- 1, 2, 3, 4, 5 or 6 of the TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ in the complex have at least one functional domain connected to the C-terminus, For example, the intracellular domain of costimulatory molecules.
- one of TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ in the complex has at least one functional domain, such as an intracellular domain of a costimulatory molecule, connected to its C-terminus.
- the TCR ⁇ chain in the complex has at least one functional domain connected at its C-terminus, such as the intracellular domain of a costimulatory molecule.
- the intracellular domain of the costimulatory molecule is OX40 or ICOS.
- the TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ do not include at least one functional domain, such as an intracellular domain of a costimulatory molecule, additionally connected to the C-terminus thereof.
- the CD3 ⁇ in the complex has at least one functional domain, such as an intracellular domain of a costimulatory molecule, attached to its C-terminus.
- TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ do not include at least one functional domain, such as an intracellular domain of a costimulatory molecule, additionally connected to the C-terminus thereof.
- two of the TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ in the complex have at least one functional domain, such as the intracellular domain of a costimulatory molecule, connected at the C-terminus.
- each of the TCR ⁇ chain and the TCR ⁇ chain in the complex has at least one functional domain, such as an intracellular domain of a costimulatory molecule, connected at its C-terminus.
- the intracellular domain of the costimulatory molecule is OX40 or ICOS.
- CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ do not include at least one functional domain, such as an intracellular domain of a costimulatory molecule, additionally connected to the C-terminus thereof.
- the functional domain is an exogenous functional domain. In some embodiments, the functional domain is an exogenous intracellular domain, such as a domain that plays a signal transduction role in the cell.
- exogenous refers to a protein or nucleic acid sequence from a foreign species, or if from the same species, refers to a protein or protein that has undergone significant changes in composition and/or location from its natural form through deliberate human intervention. Nucleic acid sequence.
- the "functional domain” can be the intracellular domain of a costimulatory molecule, such as the intracellular domain of CD40, OX40, ICOS, CD28, 4-1BB, CD27, and CD137; it can also be the intracellular domain of a co-inhibitory molecule.
- a costimulatory molecule such as the intracellular domain of CD40, OX40, ICOS, CD28, 4-1BB, CD27, and CD137; it can also be the intracellular domain of a co-inhibitory molecule.
- Domains such as the intracellular domains of TIM3, PD1, CTLA4, LAG3; can also be cytokine receptors such as interleukin receptors (such as IL-2 ⁇ receptor, IL-7 ⁇ receptor or IL-21 receptor) , Interferon receptors, tumor necrosis factor superfamily receptors, colony stimulating factor receptors, chemokine receptors, growth factor receptors or the intracellular domains of other membrane proteins; or intracellular proteins such as NIK.
- interleukin receptors such as IL-2 ⁇ receptor, IL-7 ⁇ receptor or IL-21 receptor
- Interferon receptors such as interleukin receptors (such as IL-2 ⁇ receptor, IL-7 ⁇ receptor or IL-21 receptor)
- Interferon receptors such as tumor necrosis factor superfamily receptors, colony stimulating factor receptors, chemokine receptors, growth factor receptors or the intracellular domains of other membrane proteins
- chemokine receptors such as growth factor receptors or the intracellular domains of other membrane proteins
- intracellular proteins
- the functional domain can also be the intracellular domain of the cytokine receptor directly or through a linker (for example (G4S)n linker, where n represents an integer from 1 to 10) and the human STAT5 activation module (amino acid sequence is shown in SEQ ID NO: 35 ) Fusion.
- a linker for example (G4S)n linker, where n represents an integer from 1 to 10.
- the functional domain is the intracellular domain of a costimulatory molecule, preferably the intracellular domain of OX40 or ICOS, and more preferably the intracellular domain of OX40.
- An exemplary CD40 intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 10.
- An exemplary intracellular domain of OX40 includes the amino acid sequence shown in SEQ ID NO: 11.
- An exemplary intracellular domain of ICOS includes the amino acid sequence shown in SEQ ID NO: 12.
- An exemplary CD28 intracellular domain includes the amino acid sequence shown in SEQ ID NO: 13.
- An exemplary 4-1BB intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 14.
- An exemplary CD27 intracellular domain includes the amino acid sequence shown in SEQ ID NO: 15.
- An exemplary IL-2 ⁇ receptor intracellular domain comprises the amino acid sequence shown in SEQ ID NO:32.
- An exemplary IL-17 ⁇ receptor intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 33.
- An exemplary IL-21 receptor intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 34.
- An exemplary fusion amino acid sequence of the IL-2 ⁇ receptor intracellular domain and the human STAT5 activation module is shown in SEQ ID NO: 36.
- the fusion amino acid sequence of an exemplary IL-17 ⁇ receptor intracellular domain and human STAT5 activation module is shown in SEQ ID NO: 37.
- the first constant region is a natural TCR ⁇ chain constant region, for example, a natural human TCR ⁇ chain constant region (an exemplary human TCR ⁇ chain constant region amino acid sequence is shown in SEQ ID NO:1) or a natural mouse TCR ⁇ Chain constant region (an exemplary mouse TCR ⁇ chain constant region amino acid sequence is shown in SEQ ID NO: 3); or the first constant region is a natural TCR ⁇ chain constant region, for example, a natural human TCR ⁇ chain constant region (exemplary human TCR ⁇ The chain constant region amino acid sequence is shown in SEQ ID NO: 50) or the natural mouse TCR gamma chain constant region (an exemplary mouse TCR gamma chain constant region amino acid sequence is shown in SEQ ID NO: 51).
- the first constant region is a modified TCR alpha chain constant region or a modified TCR gamma chain constant region.
- the modified TCR ⁇ chain constant region is derived from the mouse TCR ⁇ chain constant region, which is relative to the wild-type mouse TCR ⁇ chain constant region, the amino acid at position 48, such as threonine T, is mutated to Cysteine C.
- the modified TCR ⁇ chain constant region is derived from the mouse TCR ⁇ chain constant region, which is relative to the wild-type mouse TCR ⁇ chain constant region.
- the amino acid at position 112, such as serine S, is changed to leucine.
- the amino acid at position 114, such as methionine M, is changed to isoleucine I, and the amino acid at position 115, such as glycine G, is changed to amino acid V.
- the modified TCR ⁇ chain constant region is derived from the mouse TCR ⁇ chain constant region, which is relative to the wild-type mouse TCR ⁇ chain constant region, the amino acid at position 6 such as E is replaced by D, and the 13th K at position is replaced by R, and amino acids at positions 15-18 are deleted.
- the modified TCR ⁇ chain constant region is derived from the mouse TCR ⁇ chain constant region, which is relative to the wild-type mouse TCR ⁇ chain constant region, the amino acid at position 48, such as threonine T, is mutated to Cysteine C, the amino acid at position 112 such as serine S is changed to leucine L, the amino acid at position 114 such as methionine M is changed to isoleucine I, the amino acid at position 115, for example Glycine G is changed to glycine V.
- the modified TCR ⁇ chain constant region is derived from the mouse TCR ⁇ chain constant region, which is relative to the wild-type mouse TCR ⁇ chain constant region, the amino acid at position 6 such as E is replaced by D, and the 13th K was replaced by R, amino acids 15-18 were deleted, amino acids at position 48, such as threonine T, were mutated to cysteine C, and amino acids at position 112, such as serine S, were changed to leucine.
- the amino acid at position 114 such as methionine M
- the amino acid at position 115 such as glycine G
- the modified TCR ⁇ chain constant region is derived from the mouse TCR ⁇ chain constant region, which, relative to the wild-type mouse TCR ⁇ chain constant region, lacks the natural intracellular region of the constant region, for example, deletion of the 136th- 137 amino acid.
- the first constant region comprises an amino acid sequence shown in one of SEQ ID Nos: 1, 3, 5, 7, 8, 26, 41, 42, and 56.
- the second constant region is a natural TCR ⁇ chain constant region, for example, a natural human TCR ⁇ chain constant region (an exemplary human TCR ⁇ chain constant region amino acid sequence is shown in SEQ ID NO: 2) or a natural mouse TCR ⁇ Chain constant region (an exemplary mouse TCR ⁇ chain constant region amino acid sequence is shown in SEQ ID NO: 4); or wherein the second constant region is a natural TCR ⁇ chain constant region, for example, a natural human TCR ⁇ chain constant region (an exemplary human The amino acid sequence of the TCR ⁇ chain constant region is shown in SEQ ID NO: 52) or the natural mouse TCR ⁇ chain constant region (the exemplary mouse TCR ⁇ chain constant region amino acid sequence is shown in SEQ ID NO: 53).
- the second constant region is a modified TCR ⁇ chain constant region; or the second constant region is a modified TCR ⁇ chain constant region.
- the modified TCR ⁇ chain constant region is derived from the mouse TCR ⁇ chain constant region, which is relative to the wild-type mouse TCR ⁇ chain constant region, the amino acid at position 56 such as serine S is mutated to cysteine Acid C.
- the modified TCR ⁇ chain constant region is derived from the mouse TCR ⁇ chain constant region, which is relative to the wild-type mouse TCR ⁇ chain constant region in which the amino acid at position 3 such as R is replaced by K, and the sixth The amino acid at position such as T is replaced by F, the K at position 9 is replaced by E, the S at position 11 is replaced by A, the L at position 12 is replaced by V, and the amino acids at positions 17 and 21-25 are deleted.
- the modified TCR ⁇ chain constant region is derived from the mouse TCR ⁇ chain constant region, which is relative to the wild-type mouse TCR ⁇ chain constant region, the amino acid at position 56 such as serine S is mutated to cysteine
- the amino acid C, the amino acid at position 3 such as R is replaced by K
- the amino acid at position 6 such as T is replaced by F
- the K at position 9 is replaced by E
- the S at position 11 is replaced by A
- the L at position 12 is replaced V is substituted
- the 17th, 21-25th amino acids are deleted.
- the modified TCR ⁇ chain constant region is derived from the mouse TCR ⁇ chain constant region, which, relative to the wild-type mouse TCR ⁇ chain constant region, lacks the natural intracellular region of the constant region, for example, deletion of the 167th- 172 amino acid.
- the modified TCR ⁇ chain constant region comprises an amino acid sequence shown in one of SEQ ID NOs: 2, 4, 6, 9, 27, 43, and 49.
- Antigen binding region refers to a domain that can specifically bind to a target antigen alone or in combination with another antigen binding region.
- the antigen binding region is derived from an antibody that specifically binds to the target antigen.
- the first or second antigen-binding region can each independently specifically bind to the same or different target antigens, for example, the first antigen-binding region specifically binds to the first antigen, and the second antigen-binding region Specific binding to the second antigen.
- the antigen binding region is a single chain antibody (such as scFv) or a single domain antibody (such as a camel antibody), preferably the antigen binding region is a single chain antibody such as scFv.
- the single chain antibody comprises a heavy chain variable region and a light chain variable region connected by a linker.
- the linker is, for example, a (G 4 S)n linker, where n represents an integer from 1-10, preferably n is 1 or 3.
- the antigen binding region that specifically binds to CD19 comprises the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 44 and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 45.
- the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 44 and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 45 are connected by a linker.
- the linker is a (G4S)n linker, where n represents an integer from 1-10, preferably n is 1 or 3.
- the antigen binding region that specifically binds to CD19 comprises the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 46 and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 47.
- the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 46 and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 47 are connected by a linker.
- the linker is a (G4S)n linker, where n represents an integer from 1-10, preferably n is 1 or 3.
- the antigen binding region that specifically binds to CD19 comprises the scFv amino acid sequence shown in SEQ ID NO: 39.
- the antigen binding region that specifically binds to CD20 comprises the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 54 and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 55.
- the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 54 and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 55 are connected by a linker.
- the linker is a (G4S)n linker, where n represents an integer from 1-10, preferably n is 1 or 3.
- the antigen binding region that specifically binds to CD20 comprises the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 56 and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 57.
- the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 56 and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 57 are connected by a linker.
- the linker is a (G4S)n linker, where n represents an integer from 1-10, preferably n is 1 or 3.
- the antigen binding region that specifically binds to CD20 comprises the scFv amino acid sequence shown in SEQ ID NO: 38.
- the first antigen binding region and the second antigen binding region are combined with each other to specifically bind to the target antigen.
- the first antigen binding region contains the heavy chain of an antibody, while the second antigen binding region contains the light chain of the antibody, and vice versa.
- the first antigen binding region comprises a heavy chain variable region of an antibody that specifically binds to a first antigen and a heavy chain variable region of an antibody that specifically binds to a second antigen
- the second antigen The binding region includes the light chain variable region of the antibody that specifically binds to the first antigen and the light chain variable region of the antibody that specifically binds to the second antigen, so that the first and second antigen-binding regions combine with each other to specifically bind The first antigen and the second antigen.
- the first antigen binding region comprises a heavy chain variable region of an antibody that specifically binds to a first antigen and a light chain variable region of an antibody that specifically binds to a second antigen
- the second antigen The binding region includes the light chain variable region of the antibody that specifically binds to the first antigen and the heavy chain variable region of the antibody that specifically binds to the second antigen, so that the first and second antigen-binding regions can be combined with each other to be specific Combines the first antigen and the second antigen.
- the first antigen binding region comprises a light chain variable region of an antibody that specifically binds to a first antigen and a light chain variable region of an antibody that specifically binds to a second antigen
- the second antigen The binding region includes the heavy chain variable region of the antibody that specifically binds to the first antigen and the heavy chain variable region of the antibody that specifically binds to the second antigen, so that the first and second antigen-binding regions can be combined with each other to be specific Combines the first antigen and the second antigen.
- the first antigen is CD19 and the second antigen is CD20.
- the first antigen is CD20 and the second antigen is CD19.
- the heavy chain variable region of the antibody that specifically binds to CD19 includes the amino acid sequence shown in SEQ ID NO: 44, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 45.
- the heavy chain variable region of the antibody that specifically binds to CD19 includes the amino acid sequence shown in SEQ ID NO: 46, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 47.
- the heavy chain variable region of the antibody that specifically binds to CD20 includes the amino acid sequence shown in SEQ ID NO: 54, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 55.
- the heavy chain variable region of the antibody that specifically binds to CD20 includes the amino acid sequence shown in SEQ ID NO: 56, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 57.
- the CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and/or CD3 ⁇ are of human origin.
- the human CD3 ⁇ comprises the amino acid sequence shown in SEQ ID NO:28.
- the human CD3 ⁇ comprises the amino acid sequence shown in SEQ ID NO:29.
- the human CD3 epsilon comprises the amino acid sequence shown in SEQ ID NO:30.
- the human CD3 ⁇ comprises the amino acid sequence shown in SEQ ID NO:31.
- the modified T cell receptor (TCR) or TCR complex of the present invention includes the TCR ⁇ chain shown in SEQ ID NO: 59 and the TCR ⁇ chain shown in SEQ ID NO: 60.
- the present invention provides an isolated therapeutic immune cell comprising the modified T cell receptor (TCR) or TCR complex of the present invention.
- TCR modified T cell receptor
- the immune cells are T cells. In other embodiments, the immune cells are NK cells.
- the present invention provides an isolated polynucleotide comprising a core encoding at least one of TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ as defined above in the present invention
- at least one of the TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ has at least one exogenous intracellular functional domain connected to its C terminal.
- the present invention provides an isolated polynucleotide comprising a nucleotide sequence encoding the TCR defined above in the present invention.
- the isolated polynucleotide comprises a nucleotide sequence encoding a TCR ⁇ chain and/or TCR ⁇ chain, and the TCR ⁇ chain and/or TCR ⁇ chain has at least one costimulatory molecule attached at its C-terminus. Domain.
- the polynucleotide comprises i) a nucleotide sequence encoding the alpha chain, ii) a nucleotide sequence encoding the beta chain, and iii) in the same reading frame. And ii) the nucleotide sequence encoding the self-cleavable peptide.
- the nucleotide sequence encoding the ⁇ chain may be located at the 5'end or the 3'end of the nucleotide sequence encoding the ⁇ chain.
- the isolated polynucleotide comprises a nucleotide sequence encoding a TCR ⁇ chain and/or TCR ⁇ chain, the TCR ⁇ chain and/or TCR ⁇ chain having at least one costimulatory molecule attached to its C-terminus intracellularly Domain.
- the polynucleotide comprises i) a nucleotide sequence encoding the gamma chain, ii) a nucleotide sequence encoding the delta chain, and iii) in the same reading frame. And ii) the nucleotide sequence encoding the self-cleavable peptide.
- the nucleotide sequence encoding the ⁇ chain may be located at the 5'end or the 3'end of the nucleotide sequence encoding the ⁇ chain.
- self-cleaving peptide means a peptide that can achieve self-cleavage within a cell.
- the self-cleaving peptide may include a protease recognition site, so that it can be recognized and specifically cleaved by the protease in the cell.
- the self-cleaving peptide may be a 2A polypeptide.
- 2A polypeptide is a type of short peptide derived from viruses, and its self-cleavage occurs during translation. When 2A polypeptide is used to connect two different target proteins and expressed in the same reading frame, the two target proteins are almost produced at a ratio of 1:1.
- Commonly used 2A polypeptides can be P2A from porcine techovirus-1, T2A from Thosea asignis virus, and E2A from equine rhinitis A virus. And F2A from foot-and-mouth disease virus. Among them, P2A has the highest cutting efficiency and is therefore preferred.
- a variety of functional variants of these 2A polypeptides are also known in the art, and these variants can also be used in the present invention.
- the polynucleotide comprises a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO:58.
- the present invention provides an expression vector comprising the polynucleotide of the present invention operably linked to a regulatory sequence.
- the "expression vector" of the present invention can be a linear nucleic acid fragment, a circular plasmid, a viral vector, or can be an RNA (such as mRNA) that can be translated.
- the expression vector is a viral vector, such as a lentiviral vector.
- regulatory sequence and “regulatory element” are used interchangeably and refer to the upstream (5' non-coding sequence), middle or downstream (3' non-coding sequence) of the coding sequence, and affect the transcription, RNA processing or processing of the related coding sequence. Stability or translated nucleotide sequence.
- An expression control element refers to a nucleotide sequence capable of controlling the transcription, RNA processing or stability, or translation of a nucleotide sequence of interest. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, enhancers, and polyadenylation recognition sequences.
- operably linked refers to the connection of regulatory elements (for example, but not limited to, promoter sequences, transcription termination sequences, etc.) to nucleic acid sequences (for example, coding sequences or open reading frames) such that the nucleotides The transcription of the sequence is controlled and regulated by the transcription control element.
- regulatory elements for example, but not limited to, promoter sequences, transcription termination sequences, etc.
- nucleic acid sequences for example, coding sequences or open reading frames
- the present invention provides a method for preparing the therapeutic immune cell of the present invention, which comprises introducing the polynucleotide of the present invention or the expression vector of the present invention into the immune cell.
- the immune cells of the present invention such as T cells or NK cells can be obtained from many non-limiting sources by various non-limiting methods, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, ascites, pleural effusion , Spleen tissue and tumors.
- the cells can be derived from a healthy donor or from a patient diagnosed with cancer.
- the cells may be part of a mixed population of cells exhibiting different phenotypic characteristics.
- T cells can be obtained by isolating peripheral blood mononuclear cells (PBMC), then using specific antibodies to activate and expand them.
- PBMC peripheral blood mononuclear cells
- the immune cells are derived from autologous cells of the subject.
- autologous refers to cells, cell lines, or cell populations used to treat a subject derived from the subject.
- the immune cells, such as T cells are derived from allogeneic cells, such as from a donor that is compatible with the subject's human leukocyte antigen (HLA). Standard protocols can be used to transform cells from a donor into non-alloreactive cells and replicate as needed to produce cells that can be administered to one or more patients.
- HLA human leukocyte antigen
- the present invention provides a pharmaceutical composition
- a pharmaceutical composition comprising the therapeutic immune cell of the present invention and a pharmaceutically acceptable carrier.
- pharmaceutically acceptable carrier includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
- the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (such as by injection or infusion).
- the present invention provides the use of the therapeutic immune cell of the present invention or the pharmaceutical composition of the present invention in the preparation of a medicament for treating diseases such as cancer in a subject.
- Subject refers to an organism suffering from or susceptible to diseases (such as cancer) that can be treated by the cells, methods, or pharmaceutical compositions of the present invention.
- diseases such as cancer
- Non-limiting examples include humans, cattle, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammals.
- the subject is a human.
- the present invention provides a method of treating a disease such as cancer in a subject, comprising administering to the subject a therapeutically effective amount of the therapeutic immune cell of the present invention or the pharmaceutical composition of the present invention.
- terapéuticaally effective amount or “therapeutically effective dose” or “effective amount” refers to the amount of a substance, compound, material, or cell that is at least sufficient to produce a therapeutic effect after administration to a subject. Therefore, it is an amount necessary to prevent, cure, ameliorate, block or partially block the symptoms of a disease or disorder.
- an "effective amount" of the cells or pharmaceutical composition of the present invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or prevention of injury or disability due to the pain of the disease.
- an "effective amount" of the cells or pharmaceutical composition of the present invention preferably inhibits tumor cell growth or tumor growth by at least about 10%, preferably at least about 20%, relative to subjects not receiving treatment. It is preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%.
- the ability to inhibit tumor growth can be evaluated in an animal model system that predicts the efficacy of human tumors. Alternatively, it can also be evaluated by examining the ability to inhibit the growth of tumor cells, and this inhibition can be determined in vitro by a test well known to those skilled in the art.
- the dosage level of the cells in the pharmaceutical composition of the present invention may be changed to obtain an amount of the active ingredient that can effectively achieve the desired therapeutic response to a specific patient, composition and mode of administration without being toxic to the patient.
- the dosage level selected depends on a variety of pharmacokinetic factors, including the activity of the specific composition of the invention used, the route of administration, the time of administration, the excretion rate of the specific compound used, the duration of treatment, and the specific application
- Other drugs, compounds and/or materials used in combination with the composition the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors known in the medical field.
- the administration of therapeutic immune cells or pharmaceutical compositions or drugs according to the present invention can be carried out in any convenient way, including by injection, infusion, implantation or transplantation.
- Administration of the cells or compositions described herein can be by intravenous, intralymphatic, intradermal, intratumoral, intramedullary, intramuscular, or intraperitoneal administration.
- the cells or compositions of the invention are preferably administered by intravenous injection.
- the disease is a disease related to CD19 and/or CD20, for example a disease related to abnormal expression of CD19 and/or CD20, for example, a cancer related to CD19 and/or CD20.
- the cancer may be a B-cell malignant tumor, such as chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia), lymphocytic lymphoma, non-Hodgkin Chikin's lymphoma, and a combination of said cancers.
- the cancer is a CD19 and/or CD20 positive cancer.
- the secreted antibody (Ab) or B cell receptor (BCR) produced by B cells is very similar to the T cell receptor (TCR) in gene structure, protein structure and spatial conformation.
- Antibodies and TCRs are composed of variable regions and constant regions, where the variable region plays the role of antigen recognition and binding, while the constant region domain plays the role of structural interaction and signal transduction.
- VH variable region
- VL light chain variable region
- the STAR molecule has two chains.
- the first chain is the fusion of the antigen recognition sequence (such as the variable region of the antibody heavy chain VH) and the constant region (C ⁇ ) of the T cell receptor ⁇ chain (TCR ⁇ ).
- the second chain is derived from the fusion of an antigen recognition sequence (such as the light chain variable region VL of an antibody) and the constant region (C ⁇ ) of the T cell receptor ⁇ chain (TCR ⁇ ).
- the antigen recognition domains (such as VH, VL or scFv, etc.) and constant region domains (TCR ⁇ , ⁇ , ⁇ , and ⁇ constant regions) in the construct can be arranged and combined to form a variety of structures with different configurations but similar functions body.
- Immunoreceptor tyrosine activation motif (Immunoreceptor Tyrosine-based Activation Motif, ITAM) is a motif that plays a role in signal transduction in TCR molecules, and its conservative sequence is YxxL/V.
- the intracellular region of CD3 ⁇ , ⁇ , ⁇ , and ⁇ chains contains 1 ITAM sequence, and the intracellular region of CD3 ⁇ chain contains 3 ITAM sequences, so a complete STAR complex contains a total of 10 ITAM sequences.
- the intracellular ITAM sequence is successively phosphorylated, which in turn activates downstream signaling pathways, activates transcription factors such as NF- ⁇ B, NFAT and AP-1, and triggers the activation of T cells , Produce effect function.
- mutant STAR mut-STAR
- STAR ub-STAR
- the STAR prototype design uses the constant region sequence of human-derived TCR ⁇ / ⁇ chains (or TCR ⁇ and ⁇ chains) (wild-type human TCR ⁇ constant region, SEQ ID NO: 1; wild-type human TCR ⁇ constant region, SEQ ID NO: 2 ). Since the constant region sequences of human, primate, and murine TCR ⁇ / ⁇ chains (mouse TCRaC-WT, SEQ ID NO: 3; mouse TCRbC-WT, SEQ ID NO: 4) have high functional conservation, And the key amino acid sequence is the same, so they can be replaced with each other.
- the inventors replaced the constant region of the STAR molecule with a murine sequence to enhance the function of the STAR molecule after it was transferred to human T cells.
- cysteine point mutations on the STAR molecule to introduce intermolecular disulfide bonds, enhance the mutual pairing between the two chains of the STAR molecule, and reduce the mismatch with endogenous TCR.
- the 48th threonine T was mutated to cysteine C (mouse TCRac-Cys, SEQ ID NO: 5) in the constant region of the TCR ⁇ chain
- the 56th serine S was mutated to cysteine in the TCR ⁇ chain constant region C (mouse TCRbC-Cys, SEQ ID NO: 6).
- TCR After TCR is bound to the antigen and after the activation is completed, ubiquitin activates the enzyme, ubiquitin conjugating enzyme and ubiquitin ligase through a series of ubiquitination reactions to ubiquitin the lysine in the intracellular end and the transmembrane region of the TCR molecule
- Chemical modification causes the occurrence of T cell endocytosis, which causes TCR molecules to be endocytosed into the cell and is further degraded by lysosomes, thereby reducing the concentration of TCR molecules on the surface of T cell membranes, resulting in a continuous decline in the activation effect of T cells.
- the present inventors modified the amino acids in the transmembrane or intracellular regions of the alpha and beta chains in the above-mentioned mut-STAR molecule, including the intracellular region of the alpha chain constant region of the STAR molecule, and the transmembrane region of the beta chain constant region.
- the amino acid is mutated to arginine, and the constant region sequence is Mouse TCR ⁇ C-Arg mut (SEQ ID NO: 8) and the constant region sequence is Mouse TCR ⁇ C-Arg mut (SEQ ID NO: 9), which reduces the ubiquitination of lysine.
- the STAR molecule caused by the vegetarianization is endocytosed.
- the inventors designed a new structure and performed the mut-STAR complex. It can be modified and customized enhanced mut-STAR cells according to needs to improve the clinical response rate of TCR-T and achieve long-lasting curative effects.
- TCR is a special marker on the surface of all T cells. It is divided into two types: ⁇ TCR and ⁇ TCR. The corresponding T cells are ⁇ T cells and ⁇ T cells. The inventors modified the costimulatory signals of ⁇ -STAR and ⁇ -STAR to improve the performance of ⁇ T cells and ⁇ T cells.
- the TCR of ⁇ T cells is composed of two chains, TCR ⁇ and TCR ⁇ , which account for 90%-95% of the total T cells.
- ⁇ TCR is composed of variable regions and constant regions.
- the variable regions are extremely diverse and play the role of antigen recognition and binding, while the constant region domains play the role of structural interaction and signal transduction.
- the present invention introduces the human costimulatory molecule receptor intracellular end sequence into the C-terminus of the ⁇ -STAR constant region (Figure 2) to explore its effect on the function of STAR T cells. Influence.
- the STAR constant region of the present invention includes an unmodified WT-STAR constant region, a cys-STAR constant region containing additional intermolecular disulfide bonds, a murine-derived hm-STAR constant region, and an integrated WT-STAR constant region.
- the co-stimulatory signal transduction structure includes intracellular signal transduction domains of CD40, OX40, ICOS, CD28, 4-1BB or CD27, and the sequences are respectively SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 , SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15.
- the costimulatory intracellular domain can be tandemly connected to the C-terminus of the TCR alpha chain, or the C-terminus of the beta chain, or the C-terminus of the alpha and beta chains (co-STAR).
- co-stimulatory molecule domain and the C-terminus of the TCR constant region can be directly connected or connected via G4S/(G4S)n (G4S linker sequences are SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, respectively.
- the TCR of ⁇ T cells is composed of two chains, TCR ⁇ and TCR ⁇ . According to the type of TCR ⁇ chains, ⁇ T cells can be divided into three subgroups: ⁇ 1, ⁇ 2 and ⁇ 3. The distribution of different subgroups in the human body is different. ⁇ T cells mainly recognize non-peptide antigens in a non-MHC restricted manner, and play an important role in the surveillance of pathogens and tumors. Experiments have proved that costimulatory signals such as CD28 or 4-1BB play a very important role in the activation and proliferation of ⁇ T cells. The present inventors introduced the intracellular end sequences of human costimulatory molecule receptors into the C ends of TCR ⁇ and TCR ⁇ ( Figure 2, right) to improve the performance of ⁇ T cells.
- CD3 subunits include ⁇ chain, ⁇ chain, ⁇ chain and ⁇ chain, and TCR molecules form a T cell receptor complex, which transmits signals from the outside of the cell to the inside of the cell, thereby regulating the state of the cell and responding to stimuli.
- TCR molecules form a T cell receptor complex, which transmits signals from the outside of the cell to the inside of the cell, thereby regulating the state of the cell and responding to stimuli.
- the inventors modified the CD3 molecule to introduce the intracellular domain of the human costimulatory molecule receptor into the CD3 ⁇ chain (SEQ ID NO: 28), ⁇ chain (SEQ ID NO: 29), ⁇ chain (SEQ ID NO: 30) and ⁇ chain (SEQ ID NO: 31) at the C end ( Figure 4).
- the modified CD3 molecules are expressed in mut-STAR T cells to improve their functions.
- Cytokines play an important role in the proliferation, anti-tumor, differentiation and other functions of T cells. Different cytokines bind to their receptors to transmit signals from the outside of the cell to the inside of the cell, thereby regulating the state of the cell and responding to stimuli. In addition, studies have shown that the intracellular end of the IL-2 receptor activates the downstream molecule STAT5 (SEQ ID NO: 35) through a cascade reaction, thereby enhancing the transcription of T cell proliferation-related molecules and enhancing the proliferation ability of CAR-T cells.
- STAT5 SEQ ID NO: 35
- the inventors modified the STAR molecule to transform the human-derived cytokine receptor intracellular signal transduction region (such as : IL-2 ⁇ receptor intracellular end IL2Rb, SEQ ID NO: 32; IL-7 ⁇ receptor intracellular end; SEQ ID NO: 33; IL-21 receptor intracellular end, SEQ ID NO: 34, etc.)) in series
- the STAT5 activation module can be further connected to the IL-2 ⁇ or IL-7R ⁇ receptor intracellular domain end (IL- 2RbQ, SEQ ID NO: 36; IL-7RbQ, SEQ ID NO: 37) ( Figure 5).
- the vectors used in the present invention including viral vectors, plasmid vectors, etc., are purchased from commercial companies or synthesized by commercial companies, and the full-length sequences of these vectors are obtained, with well-known restriction sites.
- the TCR mentioned in the present invention can be any functional TCR, including WT-STAR, mut-STAR, ub-STAR, co-STAR, co-linker-STAR, CK-STAR, co-CD3 used in the present invention -STAR etc.
- gene fragments such as the variable region of TCR, the constant region of TCR, the intracellular region of costimulatory molecule receptor, the intracellular signal transduction region of cytokine receptor, the tag sequence and the linker (linker), etc., are all derived from commercial sources. Chemical company synthesis. These gene fragments are connected by means of PCR.
- the lentiviral vector used in this statement is pHAGE-EF1 ⁇ -IRES-RFP.
- the linear vector is obtained by restriction endonuclease Not I/Nhe I, the gene fragment is obtained by synthesis and PCR methods, and the complete vector is obtained by homologous recombination.
- pHAGE can stably insert the target gene into the target cell genome, which is an important way to construct a stable cell line.
- Luciferase is an enzyme with catalytic activity that can catalyze the chemiluminescence of the substrate. By stably expressing luciferase in the target cell, the number of the target cell can be indicated after the substrate is added, thereby reflecting the function of the cell. The influence of the target cell.
- the PHAGE-EF1A vector carries restriction enzymes NotI/ClaI restriction sites. The two enzymes are used to cut the vector, and NCBI is used to obtain the luciferase and GFP sequences.
- the overlap PCR method first combines the luciferase gene and the GFP gene, and then connects the luciferase-GFP fragment into the pHAGE vector by the homologous recombination method.
- the lentiviral vector was packaged by Lenti-X-293T, the lentiviral solution was concentrated by the PEG8000 concentration method, the virus titer was determined by the gradient dilution method, and then the lymphoma cell line Raji was infected. 72 hours after infection, observe whether there are GFP-positive cells through a fluorescence microscope, and then sort out GFP-positive cells by a flow sorter, select a single clone, and build a bank for storage.
- the luciferase substrate is used to incubate with the target cells to detect the expression and detection level of luciferin to determine the expression level.
- TCR TCR ⁇ - ⁇ -Jurkat cell line
- Obtain the exon sequences of the constant regions of the TCR ⁇ and ⁇ chains at NCBI submit the exon 1 sequences of the constant regions of the ⁇ and ⁇ chains to the tools.genome-engineering.org website to design the guide sequence, synthesize the oligo sequence according to the result, and then construct it sgRNA-LentiCRISPR lentiviral vector (purchased from Aidi Gene).
- the guide sequence of the ⁇ chain is connected with LentiCRISPR-puro
- the guide sequence of the ⁇ chain is connected with LentiCRISPR-BSD.
- sgRNA-LentiCRISPR lentivirus packaging spread HEK-293T to 10cm dish in advance, and when the cells grow to 80%-90%, add the transfection system to HEK-293T, put the cells back into the 37 degree incubator and culture. Count as 0 hours; 12 hours after transfection, change into fresh 10% FBS-DMEM. The virus can be collected within 48 hours and 72 hours after transfection. Centrifuge and filter the virus-containing medium, add PEG8000 and mix well, let it stand at 4°C for more than 12 hours, then centrifuge at 3500 rpm for 30 minutes, discard the supernatant, and resuspend the pellet with a suitable volume of medium. -Store frozen at 80°C, or use directly.
- Jurkat T cells are seeded in a 12 or 24-well plate; at the same time, a suitable volume of ⁇ -chain and ⁇ -chain sgRNA-LentiCRISPR virus and polybrene (according to the total volume) are added. 1:1000 addition), mix well. Centrifuge infection, 1000 rpm, 32 degrees centrifugation for 90 minutes. Put it in a 37°C incubator and count it as 0 hours; change the medium after 10-12 hours; add Puromycin and BSD to a proper final concentration after 48 hours, and treat for 48 hours. It can be seen that all uninfected control cells die.
- the surviving cells were aspirated, centrifuged and cultured in complete medium to obtain TCR ⁇ - ⁇ -Jurkat cell bank.
- the TCR ⁇ - ⁇ -Jurkat cell bank was used to sort single cells into a 96-well plate by flow Aria. After two weeks of culture, the grown-up single cells were aspirated and cultured. Monoclonal cell lines were identified with TCR ⁇ chain and ⁇ chain antibodies, and cell lines deficient in both chains were expanded to obtain Jurkat-T cell lines with endogenous TCR knockout.
- Lentix-293T cells were inoculated into a 10 cm culture dish at a rate of 5 ⁇ 10 5 cells/mL, cultured in a 37°C, 5% CO 2 incubator, and transformed when the cell density reached about 80% (observed under a microscope) dye.
- Jurkat-C5 cells were seeded in a flat-bottom 96-well plate at 1.5 ⁇ 10 5 cells/mL, and 100 uL of 1640 medium containing 10% FBS and 0.2 ⁇ L of 1000 ⁇ polybrene was added to each well.
- 1640 complete medium for 10-fold dilution. If it is a virus stock solution, the amount of virus in the first hole is 100 ⁇ L, and if it is a concentrated solution, the amount of virus in the first hole is 1 ⁇ L.
- the diluted cells were added to the virus well, 100 ⁇ L/well, mixed, centrifuged at 32°C, 1500 rpm, centrifuged for 90 min, 37°C, 5% CO 2 incubator, and cultured for 72 hours.
- viruses with the following plasmids packaged by the above method pHAGE-EF1A-IRES-RFP, WT-STAR, mut-STAR, co-WT-STAR ( ⁇ -4-1BB-WT, ⁇ -CD27-WT, ⁇ -CD28- WT, ⁇ -ICOS-WT, ⁇ -OX40-WT, ⁇ -OX40-WT, ⁇ -OX40-WT), co-STAR ( ⁇ -4-1BB, ⁇ -CD27, ⁇ -CD28, ⁇ -ICOS, ⁇ -OX40, ⁇ -OX40, ⁇ -OX40), co-CD3-STAR (CD3 ⁇ -4-1BB, CD3 ⁇ -CD28, CD3 ⁇ -ICOS, CD3 ⁇ -OX40, CD3 ⁇ -4-1BB, CD3 ⁇ -CD28, CD3 ⁇ -ICOS, CD3 ⁇ -OX40, CD3 ⁇ -4-1BB, CD3 ⁇ -CD28, CD3 ⁇ -ICOS, CD3 ⁇ -OX40, CD3
- the Jurkat T cell line is cultured in RPMI1640 medium containing 10% FBS, and the culture density is 3*10 5 /ml, and the highest is not more than 3*10 6 /ml. Subculture is carried out every 1-2 days. After counting the cells, take the required amount of cells, supplement the medium to adjust to the above density, and place them in a CO 2 incubator for culture.
- Count the cells take 1*10 6 /ml cell centrifugation and change the medium, resuspend it with 1ml RPMI 1640 medium containing 10% FBS, add it to a 24-well plate, add an appropriate amount of virus solution, 1500rpm, centrifuge for 90min, and place it in CO 2 culture Box culture. After 12 hours of infection, the medium was completely changed to fresh RPMI 1640 medium containing 10% FBS, and the positive rate was detected 72 hours.
- T cells After obtaining primary T cells by the Ficoil separation method, they were cultured in X-VIVO medium containing 10% FBS and 100IU/ml IL-2 at an initial culture density of 1*10 6 /ml.
- CD3 and RetroNectin r-Fibronectin The final concentration is 5ug/ml) in a pre-coated orifice plate.
- the later culture density is 5*10 5 /ml, and the highest is not more than 3*10 6 /ml. Passage is carried out every 1-2 days.
- the cells were blown and counted, centrifuged at 5*10 5 /ml, the supernatant was discarded, the staining solution was PBS+2% FBS+2mM EDTA, the corresponding antibody was added for incubation, incubated for 30 minutes, and then washed twice with PBS. On-board testing.
- Target cells Raji-luciferase, Raji CD19KO -luciferase, Raji CD20KO -luciferase and Primary T cells are suspension cells, the cells take the appropriate amount of target cells using the culture medium was centrifuged to mix when co-incubated. The specific steps are: use the packaged and purified WT-STAR and mut-STAR T viruses to infect primary T cells, use flow cytometry the day before co-culture to determine the ratio of functional cells to target cells, generally 1:1 ratio Calculate the total number of T cells based on the infection efficiency, and the general use amount of target cells is 1 ⁇ 10 5 /well (96-well plate).
- Target antigen stimulates T cells
- the target antigen of the present invention is generally a cell surface protein, which can be directly used for the activation of T cells to detect the function of T cells. Usually 1 ⁇ 10 5 /well of positive T cells are added, centrifuged, and 24 hours after activation, T cells or target cells are collected to test T cell function.
- T cells and target cells are co-cultured for 24 hours, gently blow the cell suspension, add 150 ⁇ L of cell suspension from each well to a white 96-well plate, centrifuge at 1500rpm/min for 5min, take the supernatant, add cell lysate to lyse at room temperature for 15min Then, centrifuge at 4°C, 4000rpm/min for 15min, take the supernatant, take 2 replicate wells per well, add luciferase substrate (firefly luciferase detection reagent), use a multifunctional microplate reader to detect the chemiluminescence value, gain The value is fixed at 100.
- Cell killing calculation: killing efficiency 100%-(effector cell-target cell pore value/control cell-target cell pore value).
- mutant mut-STAR T cells showed stronger T cell killing ability, as shown in Figure 6 and Table 1, mut targeting CD19 and CD20 -STAR T cells Raji-luciferase, Raji CD19KO -luciferase and Raji CD20KO -luciferase after killing residual tumor survival rates were 2.41%, 13.94% and 24.40%, significantly higher than the 45.73% WT-STAR, 77.00% and 76.16% , Showing that the mutant mut-STAR has a more significant tumor-killing effect.
- the costimulatory intracellular domain is connected in series at the C-terminus of TCR ⁇ and ⁇ chains or at the C-terminus of ⁇ or ⁇ chains, which affects the function of STAR-T cells
- the target cell Raji-luciferase and the primary T cell are suspended cells.
- the specific steps are: use the packaged and purified co-STAR and mut-STAR T viruses to infect primary T cells, use flow cytometry the day before co-culture to determine the ratio of functional cells to target cells, generally 8:1 4:1, 2:1, 1:1, 1:2, 1:4, 1:8 ratios of co-incubation, in addition to co-incubation differences in time detection, the general detection time points are 6h, 12h, 24h, 36h, 48h.
- target cells Raji-luciferase and primary T cells were incubated for 7 days to observe the changes in the number of cell proliferation and IL-2 secretion, and were sorted by flow cytometry after 7 days Obtain positive T cells, culture them for two days without antigen stimulation, and then co-incubate them with target cells for 24 hours to detect the killing of T cells.
- the target cells are generally used at 1 ⁇ 10 5 /well (96-well plate).
- the target antigen of the present invention is generally a cell surface protein, which can be directly used for T cell activation to detect the function of T cells. Usually, 1 ⁇ 10 5 /well of positive T cells are added, centrifuged, and 24 hours after activation, the cell suspension is collected or The supernatant of the culture medium was tested for T cell function, or the killing function of T cells was tested 6h, 12h, 24h, 36h, 48h or 7 days after activation.
- the results of the luciferase detection method to detect T cell killing function test show that the costimulatory intracellular domain is connected to the C-terminus of the TCR ⁇ chain and ⁇ chain. There is no significant difference in the survival rate of residual tumors in different effective target ratios, as shown in Figure 7 and Table 2.
- the results of tumor killing and killing at different time points showed that the mut-STAR ( ⁇ -OX40) and mut-STAR with the intracellular domain of OX40 connected to the C-terminus of the TCR ⁇ chain and ⁇ chain and mut-STAR were incubated with target cells Raji-luciferase for 48 hours.
- mut-STAR which connects the intracellular domain of OX40 to the C-terminus of TCR ⁇ chain ( ⁇ -OX40) or ⁇ chain ( ⁇ -OX40) separately, is tested by luciferase.
- TNF- ⁇ , IFN- ⁇ , and IL-2 are released to help T cells kill target cells or promote the expansion of T cells themselves.
- the common ones are TNF- ⁇ , IFN- ⁇ , and IL-2.
- TNF- ⁇ , IFN- ⁇ , IL-2ELISA kits use Human IL-2Uncoated ELISA, Human TNF- ⁇ Uncoated ELISA, Human IFN- ⁇ Uncoated ELISA (product numbers are 88-7025, 88-7346, 88-7316, respectively).
- the specific steps are: dilute the 10X Coating Buffer to 1X with ddH 2 O, add the coated antibody (250X), mix well and add to the 96-well plate, 100 ⁇ l/well. Seal the cling film overnight at 4°C, wash 3 times with 1X PBST (0.05% Tween 20 in 1X PBS), 260 ⁇ l/well each time, dilute 5XELISA/ELISPOT Diluent to 1X with ddH2O, add to 96-well plate, 200 ⁇ l/well , Let stand for 1h at room temperature. Wash once with PBST, dilute the standard song (range: 2 ⁇ 250, 4 ⁇ 500, 4 ⁇ 500), and use 1xDiluent to dilute the sample 20-50 times.
- the ELISA results showed that after the T cells and the target cells were incubated for 24 hours, the mut-STAR ( ⁇ -OX40) in which the intracellular domain of OX40 was connected to the C-terminus of the TCR ⁇ and ⁇ chains was significantly higher than other structures in the secretion of IL-2.
- ⁇ -OX40 is about 10000pg/ml
- the IL-2 secretion of other structure STAR is about mut-STAR: 7700pg/ml; ⁇ -41BB: 6450pg/ml; ⁇ -CD27: 6690pg/ml; ⁇ -CD28: 6000pg/ml; ⁇ -ICOS: 6050pg/ml; and the secretion of TNF ⁇ and IFN- ⁇ , ⁇ -OX40 also showed similar results from mut-STAR, while other structures were in the secretion of TNF ⁇ or IFN- ⁇ It shows a decline in different situations, as shown in Figure 11 and Table 6.
- T cell proliferation change detection flow cytometry counting
- T cell proliferation In the process of T cell activation, a large number of cytokines are released to help T cells kill target cells or promote the expansion of T cells themselves.
- the mut-STAR-T cells and Raji-luciferase cells of each structure were initially co-cultured according to the effective target ratio of 1:3, which was recorded as day 0, and then the cells were collected on day 1 and day 7 for flow cytometric analysis.
- the medium used is 1640 complete medium without IL2
- the initial TCR T cell is 1 ⁇ 10 5 cells
- the samples at each time point are incubated independently, and the remaining co-incubated samples are half-changed every other day
- Add target cells The cells used for flow cytometry are first stained with anti-human CD3 antibody, and the cells of a specified volume are collected and recorded on the computer, and the number and proportion of T cells in the system are obtained by conversion.
- the TCR ⁇ chain ( ⁇ -OX40) showed that the proliferation of T cells was 11.75 times after 7 days, while the other structures of mut-STAR, ⁇ -OX40, and ⁇ -OX40 were 2.755 times, 4.128 times and 6.744 times, respectively.
- the proliferation effect is significantly higher than other structures.
- Combining the above results of tumor killing effect, ELISA results, and T cell proliferation results showed that mut-STAR, in which the intracellular domains of OX40 were individually connected to the TCR ⁇ chain ( ⁇ -OX40), exhibited stronger proliferation and tumor clearance capabilities than other results.
- the costimulatory molecule OX40 connected to the TCR- ⁇ chain has the best proliferation effect and does not affect the killing effect of T cells. Therefore, the intracellular domains of different costimulatory molecules are connected in tandem with the mut-STAR of the TCR ⁇ chain.
- the results of different T cell and target cell effect-to-target ratios show that the killing effect of the intracellular domain of the costimulatory molecule OX40 connected to mut-STAR is better than the killing effect of STAR in other costimulatory regions. Under the conditions of 1:2 and 1:4, the intracellular domain of costimulatory molecule OX40 is connected to mut-STAR T cells ( ⁇ -OX40).
- the effect is similar to that of ⁇ -OX40, and is more effective than other co-stimulators.
- the effect of stimulating molecules in tandem with mut-STAR T cells is good.
- the results are shown in Figure 14 and Table 9. Based on the above killing results, it shows that the intracellular domain of the costimulatory molecule OX40 is connected in tandem with the TCR ⁇ chain ( ⁇ -OX40) alone, but has a tumor clearance and proliferation ability that is significantly higher than that of mut-STAR.
- the target cell Raji-luciferase and the primary T cell are suspended cells.
- the specific steps are: use packaged and purified co-STAR and mut-STAR T viruses to infect primary T cells, use flow cytometry the day before co-culture to determine the ratio of functional cells to target cells, generally 1:1 or Co-incubation was carried out at a ratio of 2:1.
- the target cell Raji-luciferase was incubated with primary T cells for 7 days to observe the changes in the number of cell proliferation. Calculate the total number of T cells based on the infection efficiency, and the general use amount of target cells is 1 ⁇ 10 5 /well (96-well plate).
- Target antigen stimulates T cells
- the target antigen of the present invention is generally a cell surface protein, which can be directly used for the activation of T cells to detect the function of T cells. Usually 1 ⁇ 10 5 /well of positive T cells are added, centrifuged, and 24 hours after activation, the cell suspension or culture supernatant is collected to test the T cell function.
- the results of the T cell killing function test by the luciferase detection method show that the costimulatory intracellular domain is tandem with the C-terminus of CD3 ⁇ or CD3 ⁇ or CD3 ⁇ or CD3 ⁇ chain, and is co-expressed on mut-STAR T, in different T cells and target cells
- the result of an effective target ratio of 1:1 showed that all co-CD3-STAR did not show a better target cell killing effect than ⁇ -OX40, but compared with mut-STAR, CD3 ⁇ -OX40, CD3 ⁇ -41BB, CD3 ⁇ -CD28, CD3 ⁇ -ICOS, and CD3 ⁇ -OX40 showed similar tumor-killing effects, and there was no significant difference in the survival rate of residual tumors in the ratio of 1:1 efficiency, as shown in Figure 15 and Table 10.
- the costimulatory intracellular domain connected to the C-terminus of different CD3 chains did not significantly improve the tumor killing effect of mut-STAR, but the mut-STAR effect of the costimulatory intracellular domain connected to the C-terminus of CD3 ⁇ was not significant. decline.
- T cell proliferation change detection flow cytometry counting
- the mut-STAR-T cells and Raji-luciferase cells of each structure were initially co-cultured according to the effective target ratio of 1:3, which was recorded as day 0, and then the cells were collected on day 1 and day 7 for flow cytometric analysis.
- the medium used is 1640 complete medium without IL2
- the initial TCR T cell is 1 ⁇ 10 5 cells
- the samples at each time point are incubated independently, and the remaining co-incubated samples are half-changed every other day
- Add target cells The cells used for flow cytometry are first stained with anti-human CD3 antibody, and the cells of a specified volume are collected and recorded on the computer, and the number and proportion of T cells in the system are obtained by conversion.
- the target cell Raji-luciferase and the primary T cell are suspended cells.
- the specific steps are: use the packaged and purified co-STAR and mut-STAR T viruses to infect primary T cells, use flow cytometry the day before co-culture to determine the ratio of functional cells to target cells, generally 1:1 or Co-incubation was carried out at a ratio of 2:1.
- the target cell Raji-luciferase was incubated with primary T cells for 7 days to observe the changes in the number of cell proliferation. Calculate the total number of T cells based on the infection efficiency, and the general use amount of target cells is 1 ⁇ 10 5 /well (96-well plate).
- Target antigen stimulates T cells
- the target antigen of the present invention is generally a cell surface protein, which can be directly used for T cell activation to detect the function of T cells. Usually, 1 ⁇ 10 5 /well of positive T cells are added, centrifuged, and 24 hours after activation, the cell suspension is collected or The supernatant of the culture fluid was tested for T cell function.
- the results of the T cell killing function test by the luciferase detection method showed that the costimulatory intracellular domain containing different lengths of G4S linker is connected to the intracellular end of the deleted ⁇ or ⁇ constant region, and the effective target ratio between T cells and target cells is 1:
- the results of 1 show that the linker is connected to the intracellular end of the ⁇ constant region, and the killing effect of ⁇ -del-OX40, ⁇ -OX40, ⁇ -del-G4S-OX40, ⁇ -del-(G4S)3-OX40 and ⁇ -OX40 Similar, both are better than ⁇ -del-(G4S)7-OX40 and ⁇ -del-(G4S)10-OX40, and the longer the linker, the weaker the T cell killing effect, and the linker is connected to the intracellular end of the ⁇ constant region, ⁇ -del-OX40, ⁇ -ox40, ⁇ -del-(
- the effect of connecting OX40 ( ⁇ -del-OX40 or ⁇ -del-OX40) is not much different than that of not removing it, but after the addition of linker (the number of linkers does not exceed 3), ⁇ -del-(G4S)1-3-OX40 or ⁇ -del-(G4S)1-3-OX40, the effect will be better than ⁇ -del-OX40 or ⁇ -del-OX40.
- the costimulatory intracellular domain containing G4S linker connected to the intracellular end of the ⁇ or ⁇ constant region does not affect the tumor-killing effect of mut-STAR, and the effect of connecting to the intracellular end of the ⁇ constant region is more effective than The connection to the intracellular end of the ⁇ constant region is good, but the longer the linker length, the weaker the tumor killing effect.
- TNF- ⁇ , IFN- ⁇ , and IL-2 are released to help T cells kill target cells or promote the expansion of T cells themselves.
- the common ones are TNF- ⁇ , IFN- ⁇ , and IL-2.
- TNF- ⁇ , IFN- ⁇ , IL-2ELISA kits use Human IL-2Uncoated ELISA, Human TNF- ⁇ Uncoated ELISA, Human IFN- ⁇ Uncoated ELISA (product numbers are 88-7025, 88-7346, 88-7316, respectively).
- the specific steps are: dilute 10X Coating Buffer to 1X with ddH2O, add coating antibody (250X), mix well and add to 96-well plate (for ELISA), 100 ⁇ l/well. Seal the cling film overnight at 4°C, wash 3 times with 1X PBST (also known as Wash Buffer, 0.05% Tween 20 in 1X PBS), 260 ⁇ l/well each time, dilute 5X ELISA/ELISPOT Diluent to 1X with ddH2O, add 96 Orifice plate, 200 ⁇ l/well, stand at room temperature for 1h.
- 1X PBST also known as Wash Buffer, 0.05% Tween 20 in 1X PBS
- wash once with PBST dilute the standard song (range: 2 ⁇ 250, 4 ⁇ 500, 4 ⁇ 500), and use 1xDiluent to dilute the sample 20-50 times.
- Add sample and standard song 100 microliters per well, two duplicate wells, after 2h incubation at room temperature, wash three times with PBST, add 1xDiluent diluted Detection antibody, after 1h incubation, wash three times with PBST, then add 1xDiluent diluted HRP, After incubating for 30 minutes, wash 6 times, add TMB for color development, the color development time does not exceed 15 minutes, add 2N H 2 SO 4 to stop, 450nm light absorption to detect light absorption.
- ELISA results showed that after T cells and target cells were incubated for 24 hours, the secretion of IL-2 and IFN- ⁇ was significantly lower than that of mut-STAR except for mut-STAR whose structure is ⁇ -del-(G4S)7-OX40.
- the results of comprehensive ELISA show that, with the deletion of the intracellular end of the ⁇ or ⁇ constant region, the deletion of the intracellular end of the ⁇ chain will affect the secretion of IFN- ⁇ , but does not affect the secretion of IL-2, while the cell of the ⁇ chain
- the deletion of the inner end does not affect the secretion of IL-2 and IFN- ⁇ ; at the same time, after the deletion of the intracellular end of the ⁇ chain, the linker is used to connect the intracellular domain of OX40. When the linker length is less than 7, it also does not affect IL. -2 secretion, but reduce the secretion of IFN- ⁇ .
- T cell activation In the process of T cell activation, a large number of cytokines and other chemokines are released, and signals are transduced into the nucleus through cytokines or chemokine receptors to regulate changes in T cell differentiation.
- the differentiation of T cells is from naive T cells (naive) to central memory T cells (Tcm) to effector memory T cells (Tem) and finally to effector T cells (Teff).
- Tcm central memory T cells
- Tefff effector T cells
- the proliferation and persistence of T cells in vivo are affected by the number of T cells that differentiate into central memory T cells (Tcm) to effector memory T cells (Tem).
- the classification of memory T cells is roughly divided into stem cell state T cells, central memory T cells and effector memory T cells.
- the differentiation ratio of central memory T cells affects the continuous killing effect of T cells in the body.
- the ratio of primitive T cells to effector T cells affects the killing effect and persistence of T cells on tumors in vivo.
- Using flow cytometry to detect the expression of CD45RA and CCR7 on the surface of T cells can know the differentiation of T cells. After the T cells and the target cells were incubated for 7 days, centrifuged, stained the T cells with anti-human-CD45RA-Percp-cy5.5 and anti-human-CCR7-APC flow cytometry antibodies for 30 minutes, and centrifuged. After washing once with PBS, it was fixed with 4% paraformaldehyde solution, and T cell differentiation was detected by flow cytometry.
- mut-STAR deletes the intracellular end and connects the OX40 intracellular domain in series, and OX40 deletes the intracellular end of the ⁇ or ⁇ constant region through the (G4S) 3 linker connection.
- STAR without affecting the tumor killing effect and IL-2 secretion, can significantly improve the differentiation of the memory cell population of T cells. Table 12
- the target cell Raji-luciferase and the primary T cell are suspended cells.
- the specific steps are: use packaged and purified co-STAR and mut-STAR T viruses to infect primary T cells, use flow cytometry the day before co-culture to determine the ratio of functional cells to target cells, generally 1:1 or Co-incubation was carried out at a ratio of 2:1.
- the target cell Raji-luciferase was incubated with primary T cells for 7 days to observe the changes in the number of cell proliferation. Calculate the total number of T cells based on the infection efficiency, and the general use amount of target cells is 1 ⁇ 10 5 /well (96-well plate).
- Target antigen stimulates T cells
- the target of the present invention is generally a cell surface protein, which can be directly used for the activation of T cells to detect the function of T cells. Usually, 1 ⁇ 10 5 /well of positive T cells are added, centrifuged, and 24 hours after activation, the cell suspension is collected or The supernatant of the culture fluid was tested for T cell function.
- the results of the T cell killing function test by the luciferase detection method showed that the transmembrane region or the intracellular end of the lysine related to TCR endocytosis was modified to arginine mut-STAR (ub-STAR), in different T cells and The results of a target cell-to-target ratio of 2:1 or 1:1 showed that the killing effect of ub-STAR on tumor cells was significantly lower than that of mut-STAR T cells, as shown in Figure 23 and Table 18.
- T cell activation In the process of T cell activation, a large number of cytokines are released to help T cells kill target cells or promote the expansion of T cells themselves.
- the common ones are TNF- ⁇ , IFN- ⁇ , and IL-2.
- the T cells After the T cells are stimulated with target cells or antigens, the T cells are collected, centrifuged, and the supernatant is taken.
- the IFN- ⁇ and IL-2ELISA kits use Human IL-2Uncoated ELISA and Human IFN- ⁇ Uncoated ELISA (product numbers are 88-7025, 88-7346, 88-7316, respectively).
- the specific steps are: dilute 10X Coating Buffer to 1X with ddH2O, add coating antibody (250X), mix well and add to 96-well plate (for ELISA), 100 ⁇ l/well. Seal the cling film overnight at 4°C, wash 3 times with 1X PBST (also known as Wash Buffer, 0.05% Tween 20 in 1X PBS), 260 ⁇ l/well each time, dilute 5X ELISA/ELISPOT Diluent to 1X with ddH2O, add 96 Orifice plate, 200 ⁇ l/well, stand at room temperature for 1h.
- 1X PBST also known as Wash Buffer, 0.05% Tween 20 in 1X PBS
- wash once with PBST dilute the standard song (range: 2 ⁇ 250, 4 ⁇ 500, 4 ⁇ 500), and use 1xDiluent to dilute the sample 20-50 times.
- Add sample and standard song 100 microliters per well, two duplicate wells, after 2h incubation at room temperature, wash three times with PBST, add 1xDiluent diluted Detection antibody, after 1h incubation, wash three times with PBST, then add 1xDiluent diluted HRP, After incubating for 30 minutes, wash 6 times, add TMB for color development, the color development time does not exceed 15 minutes, add 2N H 2 SO 4 to stop, 450nm light absorption to detect light absorption.
- ELISA results showed that the transmembrane region or the intracellular end of the lysine related to TCR endocytosis was modified to arginine mut-STAR (ub-STAR), and the effective target ratio of T cells and target cells was 2:1 or After a total of 1:1 or 1:2 incubation for 24 hours, the secretion of ub-STAR in IL-2 and IFN- ⁇ was significantly lower than that of mut-STAR T cells, as shown in Figures 24 and 25 and Tables 19 and 20.
- T cell activation In the process of T cell activation, a large number of cytokines and other chemokines are released, and signals are transduced into the nucleus through cytokines or chemokine receptors to regulate changes in T cell differentiation.
- the proliferation and persistence of T cells in the body are affected by the number of T cells that differentiate into memory T cell types.
- the classification of memory T cells is roughly divided into stem cell state T cells, central memory T cells and effector memory T cells. Using flow cytometry to detect the expression of CD45RA and CCR7 on the surface of T cells can know the differentiation of T cells.
- T cells and the target cells were incubated for 7 days, centrifuged, stained the T cells with anti-human-CD45RA-Percp-cy5.5 and anti-human-CCR7-APC flow cytometry antibodies for 30 minutes, and centrifuged. After washing once with PBS, it was fixed with 4% paraformaldehyde solution, and T cell differentiation was detected by flow cytometry.
- mut-STAR in which the lysine related to TCR endocytosis in the transmembrane region or the intracellular end was modified to arginine (ub-STAR) in the central memory T cell differentiation and There is no significant difference between the ratios of T cells and effector T cells and mut-STAR, indicating that the transmembrane region or the intracellular end of the lysine related to TCR endocytosis is modified to arginine and has a significant effect on the effect of mut-STAR T cells. There was no significant effect on differentiation, as shown in Figures 26 and 27 and Tables 21 and 22.
- mut-STAR ub-STAR
- mut-STAR paired with mut-STAR in which the transmembrane region or intracellular end is related to TCR endocytosis modified to arginine The transformation does not have an effective promotion effect.
- the target cell Raji-luciferase and the primary T cell are suspended cells.
- the specific steps are: use packaged and purified co-STAR and mut-STAR T viruses to infect primary T cells, use flow cytometry the day before co-culture to determine the ratio of functional cells to target cells, generally 1:1 or Co-incubation was carried out at a ratio of 2:1.
- the target cell Raji-luciferase was incubated with primary T cells for 7 days to observe the changes in the number of cell proliferation. Calculate the total number of T cells based on the infection efficiency, and the general use amount of target cells is 1 ⁇ 10 5 /well (96-well plate).
- Target antigen stimulates T cells
- the target of the present invention is generally a cell surface protein, which can be directly used for the activation of T cells to detect the function of T cells. Usually, 1 ⁇ 10 5 /well of positive T cells are added, centrifuged, and 24 hours after activation, the cell suspension is collected or The supernatant of the culture fluid was tested for T cell function.
- the results of the T cell killing function test by the luciferase detection method showed that the stimulation region of different cytokine receptors is connected to the intracellular end of the ⁇ or ⁇ constant region of the mutant STAR, and the effective target ratio between T cells and target cells is 2:1. It was shown that ⁇ -IL-2Rb, ⁇ -IL-2Rb, ⁇ -IL7RA, and ⁇ -IL21R all showed similar killing effects of mut-STAR, but ⁇ -IL2RbQ, ⁇ -IL2RbQ and ⁇ -IL7RAQ had tumor killing effects. The effect is significantly lower than mut-STAR, as shown in Figure 28 and Table 23.
- T cell activation In the process of T cell activation, a large number of cytokines are released to help T cells kill target cells or promote the expansion of T cells themselves.
- the common ones are TNF- ⁇ , IFN- ⁇ , and IL-2.
- the T cells After the T cells are stimulated with target cells or antigens, the T cells are collected, centrifuged, and the supernatant is taken.
- the IFN- ⁇ and IL-2ELISA kits use Human IL-2Uncoated ELISA and Human IFN- ⁇ Uncoated ELISA (product numbers are 88-7025, 88-7346, 88-7316, respectively).
- the specific steps are: dilute 10X Coating Buffer to 1X with ddH2O, add coating antibody (250X), mix well and add to 96-well plate (for ELISA), 100 ⁇ l/well. Seal the cling film overnight at 4°C, wash 3 times with 1X PBST (also known as Wash Buffer, 0.05% Tween 20 in 1X PBS), 260 ⁇ l/well each time, dilute 5X ELISA/ELISPOT Diluent to 1X with ddH 2 O. Add a 96-well plate, 200 ⁇ l/well, and let stand at room temperature for 1h.
- 1X PBST also known as Wash Buffer, 0.05% Tween 20 in 1X PBS
- wash once with PBST dilute the standard song (range: 2 ⁇ 250, 4 ⁇ 500, 4 ⁇ 500), and use 1xDiluent to dilute the sample 20-50 times.
- Add sample and standard song 100 microliters per well, two duplicate wells, after 2h incubation at room temperature, wash three times with PBST, add 1xDiluent diluted Detection antibody, after 1h incubation, wash three times with PBST, then add 1xDiluent diluted HRP, After incubating for 30 minutes, wash 6 times, add TMB for color development, the color development time does not exceed 15 minutes, add 2N H 2 SO 4 to stop, 450nm light absorption to detect light absorption.
- ELISA results showed that after T cells and target cells were incubated for 24 hours, the effective target ratio was 1:1 or 1:2.
- ⁇ -IL-2Rb showed a more significant IL2 secretion than mut-STAR, while ⁇ - IL-2Rb, ⁇ -IL2RbQ, and ⁇ -IL7RAQ showed similar IL-2 secretion.
- the IL-2 secretion of ⁇ -IL2RbQ was significantly lower than that of mut-STAR, as shown in Figure 30 and Table 25.
- ⁇ -IL-2Rb showed more significant IFN- ⁇ secretion than mut-STAR, while other structures showed that the secretion of IFN- ⁇ was significantly lower than that of mut-STAR, as shown in the figure 31 and Table 26.
- T cell differentiation change detection flow cytometry analysis
- T cell activation In the process of T cell activation, a large number of cytokines and other chemokines are released, and signals are transduced into the nucleus through cytokines or chemokine receptors to regulate changes in T cell differentiation.
- the proliferation and persistence of T cells in the body are affected by the number of T cells that differentiate into memory T cell types.
- the classification of memory T cells is roughly divided into stem cell state T cells, central memory T cells and effector memory T cells. Using flow cytometry to detect the expression of CD45RA and CCR7 on the surface of T cells can know the differentiation of T cells.
- T cells and the target cells were incubated for 7 days, centrifuged, stained the T cells with anti-human-CD45RA-Percp-cy5.5 and anti-human-CCR7-APC flow cytometry antibodies for 30 minutes, and centrifuged. After washing once with PBS, it was fixed with 4% paraformaldehyde solution, and T cell differentiation was detected by flow cytometry.
- Different cytokine receptor stimulation regions are connected to the intracellular end of the ⁇ or ⁇ constant region of mutant STAR in the differentiation of central memory T cells and The ratios of T cells and effector T cells show different differences. There is no significant difference between ⁇ -IL-2Rb, ⁇ -IL-2Rb, ⁇ -IL-2RbQ, ⁇ -IL-2RbQ and mut-STAR, while ⁇ -IL-2Rb, ⁇ -IL-2Rb, ⁇ -IL-2RbQ, ⁇ -IL-2RbQ and mut-STAR are not significantly different.
- IL-7RA, ⁇ -IL-7RAQ, ⁇ -IL-21R have no significant difference compared with mut-STAR, as shown in Figure 32, 33 and Table 27, 28. Based on the above results, the connection of different cytokine receptor stimulation regions to the intracellular end of the ⁇ or ⁇ constant region of mutant STAR has a significant effect on the differentiation of mut STAR T cells.
- mice are NSG immunodeficient mice as a model.
- the mouse genotype is NOD-Prkdcem26Il2rgem26/Nju, lacks T cells, B cells, and NK cells, and its macrophages and dendritic cells are also defective.
- NSG mice are currently the most immunodeficient mouse strain. Because they will not reject transplanted tumors and T cells, they are widely used in preclinical research on T cell therapy. In this experiment, female NSG mice aged 6 to 8 weeks will be used, and the weight difference of mice in each batch of experiments will be controlled within 2g. Mice are raised in independent ventilated cages in a clean-level barrier free of specific pathogens (SPF), and provide normal diet and drinking water with acidic pH to prevent pathogen contamination.
- SPPF specific pathogens
- this experiment uses human Burkitt's lymphoma cell line Raji cells for xenotransplantation.
- Raji cells are cell lines expressing the luciferase gene through lentiviral vectors.
- the development and changes of Raji tumors are monitored in real time in mice through fluorescein chemiluminescence and in vivo imaging.
- Raji-luciferase cells mixed with matrigel of different doses usually about 1 ⁇ 3 ⁇ 10 6 cells
- Raji cells grow fast in mice and form solid tumors in the abdominal cavity, causing symptoms such as weight loss in mice; without treatment, the burden of Raji tumors leads to death of mice in about 40 days.
- This experiment mainly uses in vivo fluorescence imaging method: tumor cells with luciferase gene are injected into animals to colonize. Inject the fluorescein potassium salt solution into the abdominal cavity of the mouse. The substrate emits light of a specific wavelength under the action of the enzyme, and the fluorescent signal of the tumor cells in the body is detected by the sensitive CCD device of the in vivo imaging instrument. Quantitative analysis of fluorescence signals and drawing heat maps can quantitatively reflect tumor growth.
- T cells in the body are directly related to their ultimate anti-tumor effect.
- this experiment regularly collects blood from mice, and analyzes the proportion of STAR-T cells in the peripheral blood, cell status and cell grouping.
- the specific operation is: every 3-4 days, the mice are anesthetized with isoflurane, and about 100ul of blood is collected from the orbit of the mice.
- the remaining cells are subjected to flow staining to detect the ratio of CD4 and CD8, and CCR7, CD45RA, PD-1, LAG-3, TIM-3 And other molecules to perform T cell subpopulation analysis and cell state analysis.
- the absolute number of STAR-T cells in the peripheral blood of the mouse was obtained by the method of flow cytometry or digital PCR.
- the mice can be dissected and the proportion of T cells in the other immune organs of the mice can be detected.
- T cells In order to evaluate the toxicity and safety of STAR-T cells, whether the cells cause side effects on experimental animals can be detected. By observing the behavior of the mouse, performing pathological analysis of the mouse, and analyzing the slices of the important organs of the mouse, it is possible to evaluate whether the reinfused T cells are significantly toxic. At the same time, through the analysis of T cell infiltration in the non-tumor tissues of mice, it is also possible to determine whether T cells have off-target killing effects on them. In addition, by detecting the levels of cytokines in the blood of mice, such as IL-2, IFN- ⁇ , TNF ⁇ or IL-6, it can be determined whether T cells will cause a systemic cytokine storm.
- cytokines such as IL-2, IFN- ⁇ , TNF ⁇ or IL-6
- T cells to infiltrate tumors is its core ability to challenge solid tumors.
- the tumor tissue can be separated, digested and ground to obtain single cells, and the proportion of T cells in the tumor tissue can be detected by flow staining.
- density gradient centrifugation such as Percoll gradient, Ficoll gradient, etc.
- mice in group B/C/D were injected with 5 ⁇ 10 5 TCR T cells via tail vein, group A was injected with an equal volume of 200 ⁇ L PBS.
- group A was injected with an equal volume of 200 ⁇ L PBS.
- the ⁇ OX40-STAR T and mut-STAR T cells constructed in this example can significantly prolong the survival time of tumor-bearing mice. Inject the tumor cells into the mice again. Both ⁇ OX40-STAR T and mut-STAR T can eliminate tumor cells better, and the effect of ⁇ OX40-STAR T is better than that of mut-STAR T.
- the mice are in ⁇ OX40-STAR T.
- the survival time in the group was higher than that in the STAR-T group and the CAR-T group.
- the effect of ⁇ OX40-STAR T in vivo proliferation is better than that of STAR-T cells, and each time the tumor is reinfused, ⁇ OX40-STAR T cells can have Slight proliferation, but STAR-T cells did not have this phenomenon.
- the in vivo and in vitro effects of the structure of ⁇ OX40-STAR are better than that of mut-STAR.
- mice in group B/C/D/E/F/G were injected with 2 ⁇ 10 6 TCR T cells via tail vein, group A was injected with an equal volume of 200 ⁇ L PBS.
- group A was injected with an equal volume of 200 ⁇ L PBS.
- the ⁇ -del-(G4S)3-OX40-STAR-T cells constructed in this example can significantly kill the tumor cells of tumor-bearing mice, and the effect is better than other groups.
- the in vivo and in vitro effects of the structure of ⁇ -del-(G4S)3-OX40-STAR are better than that of mut-STAR.
- hSTAR refers to STAR including the constant region of human TCR.
- hmct STAR refers to the STAR of the constant region including the cysteine substitution shown in Example 1 and the modification of the transmembrane region.
- the mouse-derived TCRa chain constant region is hmct STAR TCRa (SEQ ID NO: 41), and the mouse-derived TCRb chain constant region is hmct STAR TCRb (SEQ ID NO: 6).
- the specific structure is shown in Figure 38.
- a specific rearrangement of the N-terminus of the constant region of the STAR molecule was performed on the basis of murineization of the constant region, cysteine point mutations and mutations of hydrophobic amino acids in the constant region of the ⁇ chain to obtain better results.
- Rearrangement means to delete part of the sequence and make humanized mutations to part of the sequence at the same time.
- the significance of humanized mutations is to reduce the non-human sequences in the STAR molecules as much as possible while ensuring the function of the STAR molecule, so as to avoid the possibility of STAR-T cells being rejected by the receptor in clinical applications.
- mouse and human sequences are homologous amino acid substitutions at E6D, K13R, R16K, and Q18S.
- the non-polar amino acid at the P15S site is replaced with a polar amino acid, so it can be considered that the properties of the protein near this site are not conservative and can be modified without affecting the function.
- the amino acid sequence at positions 1-14 was retained and humanized, and the amino acids at positions 15-18 were deleted.
- mouse and human sequences only belong to the same nature amino acid substitutions at the R3K and L12V positions, while in T6F, K9E , S11A, K17E, A21S, N22H, and K23T sites are all substitutions of different amino acid properties. Therefore, it can be considered that the properties of the protein near this site are not conservative and can be modified without affecting function.
- the amino acid sequence at positions 1-16 was retained and humanized, and the amino acids at positions 17, 21-25 were deleted.
- the TCRa chain constant region obtained by N-terminal rearrangement is Nrec STAR TCRa (SEQ ID NO: 42), and the TCRb chain constant region is Nrec STAR TCRb (SEQ ID NO: 43), and the specific structure is shown in Figure 38.
- the original non-optimized STAR structure (human TCR ⁇ / ⁇ STAR, hSTAR) was selected, and on this basis the modified C region murineization, Cystine modification and transmembrane modification hmct STAR , And Nrec STAR with N-terminal modification on hmct STAR.
- the present invention introduces the human costimulatory molecule receptor intracellular end sequence into the C-terminus of the STAR constant region (see Figure 39) to explore its effect on STAR-T cells. Functional impact.
- the STAR constant region structure of the present invention selects the above-mentioned STAR structure (human TCR ⁇ / ⁇ STAR, hSTAR) that is not optimized, and on this basis includes the modified hmct STAR and the N-terminal modification performed on the hmct STAR Nrec STAR has three structures.
- the costimulatory signal transduction structure includes the intracellular signal transduction domains of CD40, OX40, ICOS, CD28, 4-1BB, and CD27.
- the modification can occur in the TCR alpha chain, beta chain and alpha beta chain.
- costimulation The molecular modification occurs on the TCR ⁇ chain, see Figure 40.
- CD19 antibody heavy chain variable region (anti-CD19FMC63-VH, SEQ ID NO: 44) and antibody light chain variable region (anti-CD19FMC63-VL, SEQ ID NO: 45) select the published scFv sequence FMC63.
- STAR contains two polypeptide chains.
- the anti-CD19FMC63-VL and the TCRbC chain of hSTAR/hmct STAR/Nrec STAR are fused into the first polypeptide segment, and the anti-CD19FMC63VH is respectively combined with the TCRaC chain of hSTAR/hmct STAR/Nrec STAR. Fusion is the second polypeptide segment. Both chains use the GM-CSFR signal peptide signal peptide.
- the two chain gene sequences of hSTAR/hmct STAR/Nrec STAR are connected by the peptide segment of the Furin-SGSG-p2A protease cleavage site, and the two polypeptide chains will be transcribed and translated into protein together, and then by the protease corresponding to furin and p2A Cut, and eventually become two independent protein chains.
- the entire gene was inserted into the lentiviral expression vector pHAGE through restriction endonuclease sites NheI and NotI.
- the vector carries ampicillin resistance, EF1 ⁇ promoter and IRES-RFP fluorescent reporter gene.
- the following plasmids were obtained through the cloning and assembly of gene fragments, transformation, sequencing, and plasmid extraction: FMC63-hSTAR, FMC63-hmct STAR, FMC63-Nrec STAR.
- STAR vector containing costimulatory factors The three STAR vectors targeting CD19 FMC63-hSTAR, FMC63-hmct STAR, FMC63-Nrec STAR were constructed on the basis of costimulatory factors CD40, OX40, ICOS, CD28, 41BB, CD27, The above sequence was obtained by gene synthesis method.
- the costimulatory factors are added to the FMC63-hSTAR, FMC63-hmct STAR, FMC63-Nrec STAR vectors by PCR and homologous recombination methods.
- the connection method of the costimulatory factors in the present invention is to add the same costimulatory factors to the TCR ⁇ and ⁇ chains at the same time .
- Uninfected T cells (NC group), FMC63-hSTAR, FMC63-hmct STAR, FMC63-Nrec STAR, FMC63-hSTAR-CD40, FMC63-hSTAR-OX40, FMC63-hSTAR-ICOS, FMC63-hSTAR-CD28, FMC63 -hSTAR-41BB, FMC63-hSTAR-CD27, FMC63-hmct STAR-CD40, FMC63-hmct STAR-OX40, FMC63-hmct STAR-ICOS, FMC63-hmct STAR-CD28, FMC63-hmct STAR-41BB, FMC63-hmct STAR -CD27, FMC63-Nrec STAR-CD40, FMC63-Nrec STAR-OX40, FMC63-Nrec STAR-ICOS, FMC63-Nrec STAR-CD28, FMC63-Nrec STAR-ICOS, FMC
- the number of T cells was adjusted to 4E5 according to the positive rate of RFP, and the number of RAJI-luc cells was 4E5, a total of 1 mL co-culture system. After 24 hours of co-cultivation, mix the co-cultured cells, draw 150 ⁇ L of suspension, add 70 ⁇ L of luciferase substrate, and shake at low speed for 10 minutes in the dark. The fluorescence value of the multi-functional microplate reader is measured to calculate the killing of target cells in each group. The results show that hSTAR is significantly lower in killing compared to hmct STAR and Nrec STAR, and Nrec STAR has the highest killing efficiency.
- viruses will be obtained: FMC63-hSTAR, FMC63-hmctSTAR, FMC63-NrecSTAR, FMC63-hSTAR-CD40, FMC63-hSTAR-OX40, FMC63-hSTAR-ICOS, FMC63-hSTAR-CD28, FMC63-hSTAR-41BB, FMC63 -hSTAR-CD27, FMC63-hmct STAR-CD40, FMC63-hmct STAR-OX40, FMC63-hmct STAR-ICOS, FMC63-hmct STAR-CD28, FMC63-hmct STAR-41BB, FMC63-hmct STAR-CD27, FMC63-Nrec STAR-CD40, FMC63-NrecSTAR-OX40, FMC63-Nrec After 4 days, a 12-well plate was used to co-culture the T cell line and CD19 protein (2 ⁇ g/mL, 500 ⁇ L coated 12-well plate, 4° refrigerator coated
- CD19 antibody heavy chain variable region (anti-CD19 334-VH, SEQ ID NO: 46) and antibody light chain variable region (anti-CD19 334-VL, SEQ ID NO: 47) Choose our own developed 334 antibody sequence.
- the anti-CD19 334-VL is fused with the TCRbC chain of hmct STAR/Nrec STAR structure into the first polypeptide segment
- the anti-CD19 334VH is fused with the TCRbC chain of hmct STAR/Nrec STAR respectively.
- the second polypeptide segment. Both chains use the GM-CSFR signal peptide signal peptide.
- the two chain gene sequences of hmct STAR/Nrec STAR are connected by the Furin-SGSG-p2A protease cleavage site polypeptide segment.
- the two polypeptide chains will be transcribed and translated into protein together, and then cleaved by furin and p2A corresponding protease. Eventually become two independent protein chains.
- the entire gene was inserted into the lentiviral expression vector pHAGE through restriction endonuclease sites NheI and NotI.
- the vector carries ampicillin resistance, EF1 ⁇ promoter and IRES-RFP fluorescent reporter gene.
- the following plasmids were obtained by cloning and assembly of gene fragments, transformation, sequencing, and plasmid extraction: 334-hmct STAR, 334-Nrec STAR.
- the STAR vector 334-hmct STAR and 334-Nrec STAR targeting CD19 are constructed to contain costimulatory factor OX40, and the above sequence is obtained by gene synthesis.
- the costimulatory factors are added to the 334-hmct STAR and 334-Nrec STAR vectors through PCR and homologous recombination methods.
- the connection method of the costimulatory factors in the present invention is to add the same costimulatory factors to the TCR ⁇ and ⁇ chains at the same time.
- 334-hmct STAR-OX40 and 334-Nrec STAR-OX40 were constructed.
- Uninfected T cells (NC group), 334-hmct STAR, 334-Nrec STAR, 334-hmct STAR-OX40, 334-Nrec STAR-OX40 and RAJI-luc cells were co-cultured for 24 hours, and co-cultured in a 24-well plate Among them, the number of T cells was adjusted to 4E5 according to the positive rate of RFP, and the number of RAJI-luc cells was 4E5, a total of 1 mL co-culture system. After 24 hours of co-cultivation, mix the co-cultured cells, draw 150 ⁇ L of suspension, add 70 ⁇ L of luciferase substrate, and shake at low speed for 10 minutes in the dark.
- the fluorescence value of the multi-functional microplate reader is measured to calculate the killing of target cells in each group.
- the results show that compared with hmct STAR and Nrec STAR, OX40 can significantly improve the killing efficiency.
- OX40 can significantly increase the proliferation ability of STAR-T cells and the level of RELB into the nucleus. See Figure 43.
- Targeting CD19 antibody heavy chain variable region (anti-CD19FMC63-VH, SEQ ID NO: 44) and antibody light chain variable region (anti-CD19FMC63-VL, SEQ ID NO: 45), targeting CD20 antibody heavy chain can be The variable region (anti-CD202C6-VH, SEQ ID NO: 54) and the antibody light chain variable region (anti-CD20 2C6-VL, SEQ ID NO: 55).
- STAR contains two polypeptide chains.
- the anti-CD20 2C6VL-(G4S)3-VH and the TCRbC chain of the hmct STAR/Nrec STAR structure are fused into the first polypeptide segment, and the anti-CD19FMC63VL-(G4S)3-VH respectively Fusion with the TCRAC chain of hmct STAR/Nrec STAR is the second polypeptide segment. Both chains use the GM-CSFR signal peptide signal peptide.
- the two chain gene sequences of hmct STAR/Nrec STAR are connected by the Furin-SGSG-p2A protease cleavage site polypeptide segment.
- the two polypeptide chains will be transcribed and translated into protein together, and then cleaved by furin and p2A corresponding protease. Eventually become two independent protein chains.
- the entire gene was inserted into the lentiviral expression vector pHAGE through restriction endonuclease sites NheI and NotI.
- the vector carries ampicillin resistance, EF1 ⁇ promoter and IRES-RFP fluorescent reporter gene.
- the following plasmids were obtained through the cloning and assembly of gene fragments, transformation, sequencing, and plasmid extraction: FMC63-2C6-hmct STAR, FMC63-2C6-Nrec STAR.
- the target CD19 and CD20STAR vectors FMC63-2C6-hmct STAR and FMC63-2C6-Nrec STAR are constructed on the basis of constructing costimulatory factor OX40, and the above sequences are obtained by gene synthesis methods.
- the costimulatory factors are added to the FMC63-2C6-hmct STAR and FMC63-2C6-Nrec STAR vectors by PCR and homologous recombination methods.
- the co-stimulatory factors are connected by adding the same costimulatory factors to the TCR ⁇ and ⁇ chains at the same time.
- FMC63-2C6-hmct STAR-OX40 and FMC63-2C6-Nrec STAR-OX40 were constructed.
- Co-culture uninfected T cells (NC group), FMC63-2C6-hmct STAR, FMC63-2C6-Nrec STAR, FMC63-2C6-hmct STAR-OX40, FMC63-2C6-Nrec STAR-OX40 and RAJI-luc cells for 24 hours , Co-cultured in a 24-well plate, in which the number of T cells was adjusted to 4E5 according to the positive rate of RFP, and the number of RAJI-luc cells was 4E5, a total of 1mL co-culture system.
- OX40 can significantly improve the killing efficiency.
- OX40 can significantly increase the proliferation ability of STAR-T cells and the level of RELB into the nucleus, as shown in Figure 44.
- the costimulatory factor is only connected to the TCR ⁇ chain to enhance the proliferation ability of STAR targeting CD19 and CD20
- the anti-CD20 2C6VL-(G4S)3-VH is fused with the TCRbC (SEQ ID NO: 6) of the hmct STAR structure as the first polypeptide segment, and the anti-CD19FMC63VL-(G4S)3-VH is further removed from the natural intracellular region
- the TCRAC constant region (SEQ ID NO: 26) of hmct STAR of hmct STAR was fused and the OX40 costimulatory domain was added to the C-terminus of the constant region as the second polypeptide segment. Both chains use GM-CSFR signal peptide.
- the two chain gene sequences of STAR are connected by the Furin-SGSG-p2A protease cleavage site polypeptide segment.
- the two polypeptide chains will be transcribed and translated into protein (SEQ ID NO: 58), and then furin and p2A corresponding Protease cleaves and eventually becomes two independent protein chains.
- the costimulatory factor is only connected to the TCR ⁇ chain STAR: a(G4S) 3 OX40 targeting CD19 and CD20.
- This STAR compares the killing and proliferation abilities of the hmct STAR abOX40 targeting CD19 and CD20, which is linked to the above-mentioned costimulatory factor on the TCR ⁇ chain and ⁇ chain.
- the results are shown in Figure 45, a(G4S) 3 OX40 is better than abOX40.
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Abstract
Description
Claims (62)
- 一种经修饰的T细胞受体(TCR),其中所述TCR是i)包含TCRα链和TCRβ链的αβTCR,所述αβTCR的TCRα链和/或TCRβ链在其C末端连接有至少一个功能结构域;其中所述TCRα链包含第一恒定区和第一抗原结合区,所述TCRβ链包含第二恒定区和第二抗原结合区;其中所述第一抗原结合区特异性结合第一抗原,且所述第二抗原结合区特异性结合第二抗原;或者所述第一结合抗原区和所述第二抗原结合区相互组合而特异性结合第一抗原和第二抗原;或者,ii)包含TCRγ链和TCRδ链的γδTCR,所述γδTCR的TCRγ链和/或TCRδ链在其C末端连接有至少一个功能结构域;其中所述TCRγ链包含第一恒定区和第一抗原结合区,所述TCRδ链包含第二恒定区和第二抗原结合区;其中所述第一抗原结合区特异性结合第一抗原,且所述第二抗原结合区特异性结合第二抗原;或者所述第一结合抗原区和所述第二抗原结合区相互组合而特异性结合第一抗原和第二抗原;优选地,所述第一抗原是CD19而所述第二抗原是CD20,或者所述第一抗原是CD20而所述第二抗原是CD19。
- 权利要求1的经修饰的T细胞受体,其中所述抗原结合区衍生自抗体。
- 权利要求1或2的经修饰的T细胞受体,其中i)所述αβTCR中TCRα链和/或TCRβ链的天然胞内区被缺失,或ii)所述γδTCR中TCRγ链和/或TCRδ链的天然胞内区被缺失。
- 权利要求3的经修饰的T细胞受体,其中在i)所述αβTCR中,所述功能结构域直接或通过接头连接至所述天然胞内区被缺失的TCRα链和/或TCRβ链的C端;或在ii)所述γδTCR中,所述功能结构域直接或通过接头连接至所述天然胞内区被缺失的TCRγ链和/或TCRδ链的C端。
- 权利要求4的经修饰的T细胞受体,其中所述接头是(G 4S)n接头,其中n代表1-10的整数,优选n=3。
- 权利要求1-5中任一项的经修饰的T细胞受体,其中i)所述αβTCR中TCRα链和TCRβ链中的仅一种在其C端连接有至少一个功能结构域,例如共刺激分子胞内结构域;或ii)所述γδTCR中TCRγ链和TCRδ链中的仅一种在其C端连接有至少一个功能结构域。
- 权利要求6的经修饰的T细胞受体,其中i)所述αβTCR中TCRα链在其C端连接有至少一个功能结构域。
- 权利要求7的经修饰的T细胞受体,其中所述TCRα链的天然胞内区被缺失。
- 权利要求8的经修饰的T细胞受体,其中所述功能结构域直接或通过接头连接至所述天然胞内区被缺失的TCRα链的C端。
- 权利要求9的经修饰的T细胞受体,其中所述接头例如是(G4S)n接头,其中n代表1-10的整数,优选地,n是3。
- 权利要求6的经修饰的T细胞受体,其中i)所述αβTCR中TCRβ链在其C端连接有至少一个功能结构域。
- 权利要求11的经修饰的T细胞受体,其中所述TCRβ链的天然胞内区被缺失。
- 权利要求12的经修饰的T细胞受体,其中所述功能结构域直接或通过接头连接至所述天然胞内区被缺失的TCRβ链的C端。
- 权利要求13的经修饰的T细胞受体,其中所述接头例如是(G4S)n接头,其中n代表1-10的整数,优选地,n是3。
- 权利要求6的经修饰的T细胞受体,其中ii)所述γδTCR中TCRγ链在其C端连接有至少一个功能结构域。
- 权利要求15的经修饰的T细胞受体,其中所述TCRγ链的天然胞内区被缺失。
- 权利要求16的经修饰的T细胞受体,其中所述功能结构域直接或通过接头连接至所述天然胞内区被缺失的TCRγ链的C端。
- 权利要求17的经修饰的T细胞受体,其中所述接头例如是(G4S)n接头,其中n代表1-10的整数,优选地,n是3。
- 权利要求6的经修饰的T细胞受体,其中ii)所述γδTCR中TCRδ链在其C端连接有至少一个功能结构域。
- 权利要求19的经修饰的T细胞受体,其中所述TCRδ链的天然胞内区被缺失。
- 权利要求20的经修饰的T细胞受体,其中所述功能结构域直接或通过接头连接至所述天然胞内区被缺失的TCRδ链的C端。
- 权利要求21的经修饰的T细胞受体,其中所述接头例如是(G4S)n接头,其中n代表1-10的整数,优选地,n是3。
- 权利要求1-5中任一项的经修饰的T细胞受体,其中i)所述αβTCR中TCRα链和TCRβ链各自在其C端连接有至少一个功能结构域。
- 权利要求23的经修饰的T细胞受体,其中所述TCRα链和TCRβ链的天然胞内区被缺失。
- 权利要求24的经修饰的T细胞受体,其中所述功能结构域直接或通过接头连接至所述天然胞内区被缺失的TCRα链和TCRβ链的C端。
- 权利要求25的经修饰的T细胞受体,其中所述接头例如是(G4S)n接头,其中n代表1-10的整数,优选地,n是3。
- 权利要求1-5中任一项的经修饰的T细胞受体,其中ii)所述γδTCR中TCRγ链和TCRδ链各自在其C端连接有至少一个功能结构域。
- 权利要求27的经修饰的T细胞受体,其中所述TCRγ链和TCRδ链的天然胞内区被缺失。
- 权利要求28的经修饰的T细胞受体,其中所述功能结构域直接或通过接头连接至所述天然胞内区被缺失的TCRγ链和TCRδ链的C端。
- 权利要求29的经修饰的T细胞受体,其中所述接头例如是(G4S)n接头,其中n代表1-10的整数,优选地,n是3。
- 权利要求1-5中任一项的经修饰的T细胞受体,其中i)所述αβTCR中TCRα链和TCRβ链连接至相同的或不同的功能结构域;或ii)所述γδTCR中TCRγ链和TCRδ链连接至相同的或不同的功能结构域。
- 权利要求1-31中任一项的经修饰的T细胞受体,其中i)所述αβTCR中TCRα链和/或TCRβ链在其C端连接有1、2、3、4、5、6、7、8、9、10个或更多个功能结构域;或ii)所述γδTCR中TCRγ链和/或TCRδ链在其C端连接有1、2、3、4、5、6、7、8、9、10个或更多个功能结构域。
- 权利要求1-32中任一项的经修饰的T细胞受体,其中所述至少一个功能结构域选自共刺激分子的胞内结构域诸如CD40、OX40、ICOS、CD28、4-1BB、CD27、CD137的胞内结构域,或选自共抑制分子的胞内结构域诸如TIM3、PD1、CTLA4、LAG3的胞内结构域,或选自细胞因子受体如白细胞介素受体(如IL-2受体)、干扰素受体、肿瘤坏死因子超家族受体、集落刺激因子受体、趋化因子受体、生长因子受体或其他膜蛋白的胞内域,或者胞内蛋白如NIK的结构域。
- 权利要求1-33中任一项的经修饰的T细胞受体,其中所述共刺激分子的胞内结构域是OX40或ICOS,优选OX40。
- 权利要求1-34中任一项的经修饰的T细胞受体,其中所述第一恒定区是天然TCRα链恒定区,例如,天然人TCRα链恒定区或天然小鼠TCRα链恒定区;或所述第一恒定区是天然TCRγ链恒定区,例如,天然人TCRγ链恒定区或天然小鼠TCRγ链恒定区。
- 权利要求1-34中任一项的经修饰的T细胞受体,其中所述第一恒定区是经修饰的TCRα链恒定区或经修饰的TCRγ链恒定区。
- 权利要求36的经修饰的T细胞受体,其中所述经修饰的TCRα链恒定区衍生自小鼠TCRα链恒定区,其相对于野生型小鼠TCRα链恒定区,在第48位的氨基酸例如苏氨酸T被突变为半胱氨酸C。
- 权利要求36的经修饰的T细胞受体,其中所述经修饰的TCRα链恒定区衍生自小鼠TCRα链恒定区,其相对于野生型小鼠TCRα链恒定区,在第112位的氨基酸例如丝氨酸S被变成亮氨酸L,在114位的氨基酸例如甲硫氨酸M被变成异亮氨酸I,在第115位的氨基酸例如甘氨酸G被变成颉氨酸V。
- 权利要求36的经修饰的T细胞受体,其中所述经修饰的TCRα链恒定区衍生自小鼠TCRα链恒定区,其相对于野生型小鼠TCRα链恒定区,其第6位的氨基酸如E被D取代,第13位的K被R取代,且第15-18位氨基酸被缺失。
- 权利要求36的经修饰的T细胞受体,其中所述经修饰的TCRα链恒定区衍生自小鼠TCRα链恒定区,其相对于野生型小鼠TCRα链恒定区,在第48位的氨基酸例如苏氨酸T被突变为半胱氨酸C,在第112位的氨基酸例如丝氨酸S被变成亮氨酸L,在114位的氨基酸例如甲硫氨酸M被变成异亮氨酸I,在第115位的氨基酸例如甘氨酸G被变成颉氨酸V。
- 权利要求36的经修饰的T细胞受体,其中所述经修饰的TCRα链恒定区衍生自小鼠TCRα链恒定区,其相对于野生型小鼠TCRα链恒定区,其第6位的氨基酸如E被D取代,第13位的K被R取代,第15-18位氨基酸被缺失,在第48位的氨基酸例如苏氨酸T被突变为半胱氨酸C,在第112位的氨基酸例如丝氨酸S被变成亮氨酸L,在114位的氨基酸例如甲硫氨酸M被变成异亮氨酸I,在第115位的氨基酸例如甘氨酸G被变成颉氨酸V。
- 权利要求1-34中任一项的经修饰的T细胞受体,其中所述第一恒定区包含SEQ ID NO:1、3、5、7、8、26、41、42和48之一所示氨基酸序列。
- 权利要求1-42中任一项的经修饰的T细胞受体,其中所述第二恒定区是天然TCRβ链恒定区,例如,天然人TCRβ链恒定区或天然小鼠TCRβ链恒定区;或者其中所述第二恒定区是天然TCRδ链恒定区,例如,天然人TCRδ链恒定区或天然小鼠TCRδ链恒定区。
- 权利要求1-42中任一项的经修饰的T细胞受体,其中所述第二恒定区是经修饰的TCRβ链恒定区;或所述第二恒定区是经修饰的TCRδ链恒定区。
- 权利要求44的经修饰的T细胞受体,其中所述经修饰的TCRβ链恒定区衍生自小鼠TCRβ链恒定区,其相对于野生型小鼠TCRβ链恒定区,在第56位的氨基酸例如丝氨酸S被突变为半胱氨酸C。
- 权利要求45的经修饰的T细胞受体,其中所述经修饰的TCRβ链恒定区衍生自小鼠TCRβ链恒定区,其相对于野生型小鼠TCRβ链恒定区,其第3位的氨基酸如R被K取代,第6位的氨基酸如T被F取代,第9位的K被E取代,第11位的S被A取代,第12位的L被V取代,且第17、21-25位氨基酸被缺失。
- 权利要求44的经修饰的T细胞受体,其中所述经修饰的TCRβ链恒定区衍生自小鼠TCRβ链恒定区,其相对于野生型小鼠TCRβ链恒定区,在第56位的氨基酸例如丝氨酸S被突变为半胱氨酸C,第3位的氨基酸如R被K取代,第6位的氨基酸如T被F取代,第9位的K被E取代,第11位的S被A取代,第12位的L被V取代,且第17、21-25位氨基酸被缺失。
- 权利要求1-42中任一项的经修饰的T细胞受体,其中所述经修饰的TCRβ链恒定区包含SEQ ID NO:2、4、6、9、27、43和49之一所示氨基酸序列。
- 权利要求1-48中任一项的经修饰的T细胞受体,所述抗原结合区衍生自抗体。
- 权利要求1-49中任一项的经修饰的T细胞受体,其中所述抗原结合区包含单链抗体或单域抗体,例如,所述单链抗体包含通过接头相连接的重链可变区和轻链可变区,所述接头例如是(G 4S)n接头,其中n代表1-10的整数,优选地,n是1或3。
- 权利要求1-50中任一项的经修饰的T细胞受体,所述特异性结合CD19的抗原结合区包含SEQ ID NO:44所示重链可变区氨基酸序列和SEQ ID NO:45所示轻链可变区氨基酸序列。
- 权利要求1-50中任一项的经修饰的T细胞受体,所述特异性结合CD19的抗原结合区包含SEQ ID NO:46所示重链可变区氨基酸序列和SEQ ID NO:47所示轻链可变区氨基酸序列。
- 权利要求1-50中任一项的经修饰的T细胞受体,所述特异性结合CD19的抗原结合区包含SEQ ID NO:39所示scFv氨基酸序列。
- 权利要求1-53中任一项的经修饰的T细胞受体,所述特异性结合CD20的抗原结合区包含SEQ ID NO:54所示重链可变区氨基酸序列和SEQ ID NO:55所示轻链可变区氨基酸序列。
- 权利要求1-53中任一项的经修饰的T细胞受体,所述特异性结合CD20的抗原结合区包含SEQ ID NO:56所示重链可变区氨基酸序列和SEQ ID NO:57所示轻链可变区氨基酸序列。
- 权利要求1-53中任一项的经修饰的T细胞受体,所述特异性结合CD20的抗原结合区包含SEQ ID NO:38所示scFv氨基酸序列。
- 权利要求1的经修饰的T细胞受体,其中所述TCRα链包含SEQ ID NO:59所示氨基酸序列,所述TCRβ链包含SEQ ID NO:60所示氨基酸序列。
- 权利要求1-49中任一项的经修饰的T细胞受体,其中i)所述第一抗原结合区包含特异性结合第一抗原的抗体的重链可变区和特异性结合第二抗原的抗体的重链可变区,而所述第二抗原结合区包含特异性结合第一抗原的抗体的轻链可变区和特异性结合第二抗原的抗体的轻链可变区,从而所述第一和第二抗原结合区相互组合而特异性结合第一抗原和第二抗原;或ii)所述第一抗原结合区包含特异性结合第一抗原的抗体的重链可变区和特异性结合第二抗原的抗体的轻链可变区,而所述第二抗原结合区包含特异性结合第一抗原的抗体的轻链可变区和特异性结合第二抗原的抗体的重链可变区,从而所述第一和第二抗原结合区可以相互组合而特异性结合第一抗原和第二抗原;或iii)所述第一抗原结合区包含特异性结合第一抗原的抗体的轻链可变区和特异性结合CD20的抗体的轻链可变区,而所述第二抗原结合区包含特异性结合第一抗原的抗体的重链可变区和特异性结合第二抗原的抗体的重链可变区,从而所述第一和第二抗原结合区可以相互组合而特异性结合第一抗原和第二抗原;优选地,所述第一抗原是CD19而所述第二抗原是CD20,或者所述第一抗原是CD20而所述第二抗原是CD19。
- 一种分离的治疗性免疫细胞,其包含权利要求1-58中任一项的经修饰的T细胞受体。
- 权利要求59的治疗性免疫细胞,所述免疫细胞是T细胞或NK细胞。
- 一种药物组合物,其包含权利要求59或60的治疗性免疫细胞,和药物可接受的载体。
- 权利要求59或60的治疗性免疫细胞或权利要求61的药物组合物在制备用于在对象中治疗疾病例如癌症的药物中的用途。
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