WO2022179545A1 - 针对cd19和cd22的双靶点star - Google Patents
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Definitions
- the invention relates to the field of biomedicine, in particular to a synthetic T-cell receptor antigen receptor (Snythetic T-Cell Receptor and Antigen Receptor, STAR) for dual targets and applications thereof.
- a synthetic T-cell receptor antigen receptor Synthetic T-Cell Receptor and Antigen Receptor, STAR
- the present invention discloses a dual-target STAR against CD19 and CD22, T cells comprising the dual-target STAR, and uses thereof.
- CAR-T chimeric antigen receptor T cell
- CAR-T therapy is based on the expression of CAR molecules in T cells.
- the CAR molecule consists of three parts: the extracellular region is the antigen recognition domain derived from the antibody, which is responsible for recognizing the target antigen; the transmembrane region; the intracellular region is the signaling molecule and costimulatory signaling molecule derived from the T cell receptor, responsible for Transmits T-cell activation signals after stimulation.
- the CAR molecule When the CAR molecule is combined with its corresponding antigen, the CAR molecule will aggregate, activate the effector function of T cells, and kill the target tumor cells.
- TCR-T therapy is based on the T cell receptor (TCR).
- TCR is the identity characteristic of T cells, and T cells can be divided into ⁇ T cells and ⁇ T cells according to the type of TCR.
- 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 rearrangement of TCR ⁇ and TCR ⁇ chains, and then develop into ⁇ T cells.
- ⁇ T cells account for 90%-95% of peripheral blood T cells, while ⁇ T cells account for 5%-10% of peripheral blood T cells.
- Two types of T cells recognize antigens in an MHC-restricted and MHC-unrestricted manner, respectively, and play an important role in immunity to pathogens and tumors.
- T cell receptor (TCR) complex molecules contain multiple chains, TCR ⁇ chain and TCR ⁇ chain (or 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 ⁇ / delta chain) binding and function of signal transduction.
- the natural TCR complex contains a total of 10 ITAM signal sequences, which can theoretically transmit stronger signals than CAR. Using the signaling function of natural TCR, it will be possible to construct a novel receptor to alleviate T cell dysfunction and enable it to play a better role against solid tumors.
- the extracellular region of the TCR is very similar to the Fab domain of the antibody, so the TCR variable region sequence can be replaced with the antibody variable region sequence to obtain a synthetic T cell receptor antigen receptor (Synthetic TCR and Antigen Receptor, STAR), It has both the specificity of antibodies and the superior signaling function of natural TCR, which can mediate T cell activation.
- Synthetic TCR and Antigen Receptor Synthetic TCR and Antigen Receptor
- T cell therapies that have been put into clinical use in the field is still limited, so there is still a need for improved T cell therapies, such as STAR-based T cell therapies.
- the invention provides a synthetic T cell receptor antigen receptor (STAR) for CD19 and CD22 comprising an alpha chain and a beta chain, the alpha chain comprising a first constant region, the beta chain comprising a first constant region Two constant regions,
- the alpha chain comprises an antigen binding region that specifically binds CD19
- the beta chain comprises an antigen binding region that specifically binds CD22
- the alpha chain comprises an antigen binding region that specifically binds CD22
- the beta chain Contains an antigen-binding region that specifically binds CD19.
- the first constant region is a modified TCR ⁇ chain constant region.
- the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region in which the amino acid at position 48, eg, threonine T, is mutated, relative to the wild-type mouse TCR ⁇ chain constant region, to Cysteine C.
- the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region in which the amino acid at position 112, eg, serine S, is changed to leucine relative to the wild-type mouse TCR ⁇ chain constant region
- the amino acid at position 114 such as methionine M is changed to isoleucine I
- the amino acid at position 115 such as glycine G is changed to pyrimidine V.
- the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region in which the amino acid at position 48, eg, threonine T, is mutated, relative to the wild-type mouse TCR ⁇ chain constant region, to Cysteine C, amino acid at position 112 such as serine S is changed to leucine L, amino acid at position 114 such as methionine M is changed to isoleucine I, amino acid at position 115 such as Glycine G is changed to pyrimidine V.
- the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region, which relative to the wild-type mouse TCR ⁇ chain constant region, the amino acid at position 6, such as E, is substituted with D, position 13 The K was replaced by R, and amino acids 15-18 were deleted.
- the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region, which relative to the wild-type mouse TCR ⁇ chain constant region, the amino acid at position 6, such as E, is substituted with D, position 13
- the K is replaced by R, and amino acids 15-18 are deleted, and the amino acid at position 48, such as threonine T, is mutated to cysteine C, and the amino acid at position 112, such as serine S, is changed to light Amino acid L, 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 pyrimidine V.
- the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region that, relative to the wild-type mouse TCR ⁇ chain constant region, lacks the intracellular region of the constant region, eg, deletions 136-137 amino acid.
- the modified TCR alpha chain constant region comprises the amino acid sequence set forth in one of SEQ ID NOs: 3-4 and 39-41.
- the second constant region is a modified TCR ⁇ chain constant region.
- the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region in which the amino acid at position 56, eg, serine S, is mutated to a cysteine relative to the wild-type mouse TCR ⁇ chain constant region Amino acid C.
- the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region, which relative to the wild-type mouse TCR ⁇ chain constant region has an amino acid at position 3, such as R, substituted with K, and the 6th amino acid is substituted with K.
- the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region, which relative to the wild-type mouse TCR ⁇ chain constant region has an amino acid at position 3, such as R, substituted with K, and the 6th amino acid is substituted with K.
- the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region that, relative to the wild-type mouse TCR ⁇ chain constant region, lacks the intracellular region of the constant region, eg, deletions 167-172 amino acid.
- the modified TCR beta chain constant region comprises the amino acid sequence set forth in one of SEQ ID NOs: 7-8 and 42-44.
- the antigen binding region is a single chain antibody (eg, scFv) or a single domain antibody (eg, a camelid antibody), preferably the antigen binding region is a single chain antibody such as an scFv.
- the single chain antibody comprises a heavy chain variable region and a light chain variable region joined by a linker, eg, the linker is a (G4S)n linker, wherein n represents an integer from 1-10, Preferably, n is 1 or 3.
- the antigen binding region that specifically binds to CD19 comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising VH CDR1, SEQ ID NO: 25
- the VH CDR2 shown in NO: 26 and the VH CDR3 shown in SEQ ID NO: 27, and the light chain variable region comprises the VL CDR1 shown in SEQ ID NO: 28 and the VL CDR2 shown in SEQ ID NO: 29 , and the VL CDR3 shown in SEQ ID NO: 30,
- the antigen binding region that specifically binds to CD19 comprises the heavy chain variable region shown in SEQ ID NO:9 and the light chain variable region shown in SEQ ID NO:10.
- the antigen binding region that specifically binds to CD22 comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising VH CDR1, SEQ ID NO: 31
- the light chain variable region comprises the VL CDR1 shown in SEQ ID NO:34 and the VL CDR2 shown in SEQ ID NO:35
- the VL CDR3 shown in SEQ ID NO:36
- the antigen binding region that specifically binds to CD22 comprises the heavy chain variable region shown in SEQ ID NO:11, and the light chain variable region shown in SEQ ID NO:12.
- the antigen binding region of the alpha or beta chain is fused directly or indirectly to the N-terminus of the constant region.
- the antigen binding region of the alpha or beta chain is fused to the N-terminus of the constant region via a linker.
- the alpha chain or beta chain preferably the antigen binding region of the beta chain, is fused to the N-terminus of the constant region via a CD8 hinge region, eg, the CD8 hinge region comprises set forth in SEQ ID NO: 13 amino acid sequence.
- the alpha chain and/or beta chain has at least one exogenous intracellular functional domain attached to its C-terminus.
- the exogenous intracellular functional domain is linked to the C-terminus of the constant region of the alpha chain and/or beta chain, either directly or through a linker, eg, the linker is a (G4S)n linker, wherein n represents an integer of 1-10, preferably, n is 3.
- the exogenous intracellular functional domain is the intracellular domain of a costimulatory molecule, preferably the intracellular domain of OX40, for example, the intracellular domain of OX40 comprises SEQ ID NO: 14 amino acid sequence.
- the alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 16 or 18 or 37.
- the beta strand comprises the amino acid sequence set forth in SEQ ID NO: 17 or 19 or 38.
- the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:16 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:17.
- the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:18, and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:19.
- the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:37, and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:38.
- the present invention provides an isolated polynucleotide comprising a nucleotide sequence encoding the alpha chain and/or beta chain of a STAR directed against CD19 and CD22 of the present invention.
- the present invention provides an expression vector comprising a polynucleotide of the present invention operably linked to regulatory sequences.
- the present invention provides a method for producing therapeutic T cells, comprising expressing the STARs of the present invention directed against CD19 and CD22 in the T cells.
- the present invention provides a therapeutic T cell comprising the STAR directed against CD19 and CD22 of the present invention, or obtained by the method of the present invention for preparing a therapeutic T cell.
- the present invention provides a pharmaceutical composition
- a pharmaceutical composition comprising a therapeutic T cell of the present invention, and a pharmaceutically acceptable carrier.
- the present invention provides use of a therapeutic T cell of the present invention or a pharmaceutical composition of the present invention in the manufacture of a medicament for the treatment of a disease in a subject, eg, the disease is CD19 and/or CD22-related cancer.
- Figure 1 Shows that for M971 scFv, the killing effect on CD22 target cells is significantly increased after adding a CD8 hinge between it and the constant region.
- Figure 2 Shows that CD19-22 dual STAR (CT) also has a good killing effect on CD19 and CD22 single target cells.
- Figure 3 Shows that two chain constant region N-terminal modifications enhance the killing effect of CD19-22 dual STAR.
- Dual STAR-T cells have significantly elevated levels of IL-2 and IFN- ⁇ cytokines after co-culture with target cells.
- Figure 5 Shows that both FMC63 and M971 scFv are well expressed on the surface of T cells.
- Figure 6 Shows that dual STAR supplemented with OX40 costimulator has a stronger killing effect on CD19/CD22 target cells.
- Figure 7 Shows that in a mouse model, injection of single-target STAR-T cannot effectively inhibit tumor growth, while injection of dual STAR-T can effectively eliminate tumor cells.
- the term “and/or” covers all combinations of the items linked by the term, as if each combination had been individually listed herein.
- “A and/or B” covers “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 consist of the sequence or may have additional amino acids or nuclei at one or both ends of the protein or nucleic acid Glycosides, but still have the activity described in the present invention.
- the methionine encoded by the initiation codon at the N-terminus of the polypeptide is retained in some practical situations (eg, when expressed in a specific expression system), but does not substantially affect the function of the polypeptide.
- amino acid numbering with reference to SEQ ID NO:x refers to the position numbering of the particular amino acid being described as the amino acid in SEQ ID NO:x The position number of the corresponding amino acid above.
- sequence alignment methods well known in the art. For example amino acid correspondences can be determined by the online alignment tool of EMBL-EBI (https://www.ebi.ac.uk/Tools/psa/), where two sequences can be determined using the Needleman-Wunsch algorithm using default parameters to Align.
- the amino acid in the polypeptide can also be described herein.
- Aminine at position 48 of the polypeptide the amino acid position being referenced to SEQ ID NO:x.
- SEQ ID NO: 2 for the amino acid positions related to the alpha chain constant region.
- SEQ ID NO:6 for the amino acid positions related to the beta chain constant region.
- the invention provides a synthetic T cell receptor antigen receptor (STAR) for CD19 and CD22 comprising an alpha chain and a beta chain, the alpha chain comprising a first constant region, the beta chain comprising a first constant region Two constant regions,
- the alpha chain comprises an antigen binding region that specifically binds CD19
- the beta chain comprises an antigen binding region that specifically binds CD22
- the alpha chain comprises an antigen binding region that specifically binds CD22
- the beta chain Contains an antigen-binding region that specifically binds CD19.
- the alpha and beta chains are capable of forming functional TCR complexes upon expression in T cells.
- the ⁇ chain and ⁇ chain can combine with endogenous CD3 molecules (CD3 ⁇ , CD3 ⁇ , CD3 ⁇ ) to form an 8-subunit TCR complex, which is displayed on the cell. surface and activate T cells upon binding to the target antigens CD19 and/or CD22.
- a functional TCR complex refers to its ability to activate T cells after specific binding to the target antigen.
- the first constant region is a native TCR ⁇ chain constant region, eg, a native human TCR ⁇ chain constant region or a native mouse TCR ⁇ chain constant region.
- An exemplary native human TCR alpha chain constant region comprises the amino acid sequence set forth in SEQ ID NO:1.
- An exemplary native mouse TCR alpha chain constant region comprises the amino acid sequence set forth in SEQ ID NO:2.
- the first constant region is a modified TCR ⁇ chain constant region.
- the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region in which the amino acid at position 48, eg, threonine T, is mutated, relative to the wild-type mouse TCR ⁇ chain constant region, to Cysteine C.
- the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region in which the amino acid at position 112, eg, serine S, is changed to leucine relative to the wild-type mouse TCR ⁇ chain constant region
- the amino acid at position 114 such as methionine M is changed to isoleucine I
- the amino acid at position 115 such as glycine G is changed to pyrimidine V.
- the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region, which relative to the wild-type mouse TCR ⁇ chain constant region has an amino acid at position 6, such as E, substituted with D, and at position 13 The K at position was substituted with R, and amino acids 15-18 were deleted.
- the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region in which the amino acid at position 48, eg, threonine T, is mutated, relative to the wild-type mouse TCR ⁇ chain constant region, to Cysteine C, amino acid at position 112 such as serine S is changed to leucine L, amino acid at position 114 such as methionine M is changed to isoleucine I, amino acid at position 115 such as Glycine G is changed to pyrimidine V.
- the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region, which relative to the wild-type mouse TCR ⁇ chain constant region has an amino acid at position 6, such as E, substituted with D, and at position 13
- the K at position is replaced by R
- the amino acids 15-18 are deleted
- 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
- the amino acid at position 114 such as methionine M is changed to isoleucine I
- the amino acid at position 115 such as glycine G is changed to pyrimidine V.
- the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region that, relative to the wild-type mouse TCR ⁇ chain constant region, lacks the intracellular region of the constant region, eg, deletions 136-137 amino acid.
- the modified TCR alpha chain constant region comprises the amino acid sequence set forth in one of SEQ ID NOs: 3-4 and 39-41.
- the second constant region is a native TCR ⁇ chain constant region, eg, a native human TCR ⁇ chain constant region or a native mouse TCR ⁇ chain constant region.
- An exemplary native human TCR beta chain constant region comprises the amino acid sequence set forth in SEQ ID NO:5.
- An exemplary native mouse TCR beta chain constant region comprises the amino acid sequence set forth in SEQ ID NO:6.
- the second constant region is a modified TCR ⁇ chain constant region.
- the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region in which the amino acid at position 56, eg, serine S, is mutated to a cysteine relative to the wild-type mouse TCR ⁇ chain constant region Amino acid C.
- the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region, which relative to the wild-type mouse TCR ⁇ chain constant region has an amino acid at position 3, such as R, substituted with K, and the 6th amino acid is substituted with K.
- Amino acids at positions such as T are substituted with F, K at position 9 is substituted with E, S at position 11 is substituted with A, L at position 12 is substituted with V, and amino acids 17 and 21-25 are deleted.
- the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region in which the amino acid at position 56, eg, serine S, is mutated to a cysteine relative to the wild-type mouse TCR ⁇ chain constant region Amino acid C, amino acid at position 3 such as R is replaced by K, amino acid at position 6 such as T is replaced by F, K at position 9 is replaced by E, S at position 11 is replaced by A, L at position 12 is replaced by V was substituted and amino acids 17, 21-25 were deleted.
- amino acid at position 56 eg, serine S
- the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region that, relative to the wild-type mouse TCR ⁇ chain constant region, lacks the intracellular region of the constant region, eg, deletions 167-172 amino acid.
- the modified TCR beta chain constant region comprises the amino acid sequence set forth in one of SEQ ID NOs: 7-8 and 42-44.
- antigen binding region means that, alone or in combination with another antigen binding region, it can specifically bind to a target antigen.
- the antigen binding region is derived from an antibody that specifically binds the target antigen, including any commercially available antibody.
- the antigen binding region may be a single chain antibody (eg, scFv) or a single domain antibody (eg, camelid antibody) that specifically binds the target antigen.
- the antigen binding region is a single chain antibody such as a scFv.
- the single chain antibody comprises a heavy chain variable region and a light chain variable region linked by a linker.
- the linker is a (G4S)n linker, wherein n represents an integer from 1-10, preferably, n is 1 or 3.
- the antigen binding region that specifically binds to CD19 comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising VH CDR1, SEQ ID NO: 25 The VH CDR2 shown in NO: 26 and the VH CDR3 shown in SEQ ID NO: 27, and the light chain variable region comprises the VL CDR1 shown in SEQ ID NO: 28 and the VL CDR2 shown in SEQ ID NO: 29 , and the VL CDR3 shown in SEQ ID NO:30.
- the antigen binding region that specifically binds CD19 comprises a heavy chain variable region set forth in SEQ ID NO:9, and a light chain variable region set forth in SEQ ID NO:10.
- the antigen binding region that specifically binds CD19 is an scFv, eg, an scFv derived from antibody FMC63.
- the antigen binding region that specifically binds to CD22 comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising VH CDR1, SEQ ID NO: 31 The VH CDR2 shown in NO:32 and the VH CDR3 shown in SEQ ID NO:33, the light chain variable region comprises the VL CDR1 shown in SEQ ID NO:34 and the VL CDR2 shown in SEQ ID NO:35 , and the VL CDR3 shown in SEQ ID NO:36.
- the antigen binding region that specifically binds CD22 comprises a heavy chain variable region set forth in SEQ ID NO:11, and a light chain variable region set forth in SEQ ID NO:12.
- the antigen binding region that specifically binds to CD22 comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising VH CDR1, SEQ ID NO: 58 VH CDR2 shown in NO:59 and VH CDR3 shown in SEQ ID NO:60, the light chain variable region comprises VL CDR1 shown in SEQ ID NO:55, VL CDR2 shown in SEQ ID NO:56 , and the VL CDR3 shown in SEQ ID NO:57.
- the antigen binding region that specifically binds CD22 comprises a heavy chain variable region set forth in SEQ ID NO:46, and a light chain variable region set forth in SEQ ID NO:45.
- the antigen binding region that specifically binds to CD22 comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising VH CDR1, SEQ ID NO: 65 VH CDR2 shown in NO:66 and VH CDR3 shown in SEQ ID NO:67, the light chain variable region comprises VL CDR1 shown in SEQ ID NO:61, VL CDR2 shown in SEQ ID NO:62 , and the VL CDR3 shown in SEQ ID NO:63.
- the antigen binding region that specifically binds CD22 comprises a heavy chain variable region set forth in SEQ ID NO:68, and a light chain variable region set forth in SEQ ID NO:64.
- the antigen binding region that specifically binds to CD22 comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising VH CDR1, SEQ ID NO: 73 VH CDR2 shown in NO:74 and VH CDR3 shown in SEQ ID NO:75, the light chain variable region comprises VL CDR1 shown in SEQ ID NO:69, VL CDR2 shown in SEQ ID NO:70 , and the VL CDR3 shown in SEQ ID NO:71.
- the antigen binding region that specifically binds CD22 comprises a heavy chain variable region set forth in SEQ ID NO:76, and a light chain variable region set forth in SEQ ID NO72.
- the antigen binding region that specifically binds to CD22 comprises a heavy chain variable region and a light chain variable region
- the heavy chain variable region comprises VH CDR1, SEQ ID NO: 81 VH CDR2 shown in NO: 82 and VH CDR3 shown in SEQ ID NO: 83
- the light chain variable region comprises VL CDR1 shown in SEQ ID NO: 77, VL CDR2 shown in SEQ ID NO: 78 , and the VL CDR3 shown in SEQ ID NO:79.
- the antigen binding region that specifically binds CD22 comprises a heavy chain variable region set forth in SEQ ID NO:84, and a light chain variable region set forth in SEQ ID NO80.
- the antigen binding region that specifically binds CD22 is an scFv, eg, an scFv derived from antibodies m971, HA22, BL22, M972, or HRFB4.
- the antigen binding region of the alpha or beta chain is fused directly or indirectly to the N-terminus of the constant region.
- the alpha or beta chain, preferably the antigen binding region of the beta chain is fused to the N-terminus of the constant region via a linker.
- the linker is a CD8 hinge region.
- the CD8 hinge region comprises the amino acid sequence set forth in SEQ ID NO:13.
- the alpha chain and/or beta chain has at least one exogenous intracellular functional domain attached to its C-terminus.
- the exogenous intracellular functional domain is linked directly or via a linker to the C-terminus of the constant region of the alpha and/or beta chains.
- the exogenous intracellular functional domain is linked via a linker to the C-terminus of the constant region of the alpha and/or beta chain deleted in the intracellular region.
- the linker is a (G4S)n linker, wherein n represents an integer from 1-10, preferably, n is 3.
- foreign means a protein or nucleic acid sequence from a foreign species, or if from the same species, a protein that has undergone significant changes in composition and/or location from its native form by deliberate human intervention or nucleic acid sequence.
- an "exogenous intracellular functional domain” can be an intracellular domain of a costimulatory molecule such as the intracellular domain of CD40, OX40, ICOS, CD28, 4-1BB, CD27, CD137; it can also be a co-suppression Intracellular domains of molecules, such as those of TIM3, PD1, CTLA4, LAG3; also cytokine receptors such as interleukin receptors (such as IL-2 ⁇ receptors, IL-7 ⁇ receptors, or IL- 21 receptors), interferon receptors, tumor necrosis factor superfamily receptors, colony-stimulating factor receptors, chemokine receptors, growth factor receptors, or intracellular domains of other membrane proteins; or intracellular proteins such as NIK domain.
- a costimulatory molecule such as the intracellular domain of CD40, OX40, ICOS, CD28, 4-1BB, CD27, CD137
- Intracellular domains of molecules such as those of TIM3, PD1, CTLA4, L
- the exogenous intracellular functional domain is the intracellular domain of a costimulatory molecule, preferably the intracellular domain of OX40.
- the intracellular domain of OX40 comprises the amino acid sequence of SEQ ID NO:14.
- the alpha chain comprises an antigen binding region that specifically binds CD19 and a modified TCR alpha chain constant region, wherein
- the antigen-binding region that specifically binds to CD19 comprises the heavy chain variable region shown in SEQ ID NO:9 and the light chain variable region shown in SEQ ID NO:10 connected by a linker (G4S) 3 ,
- the modified TCR alpha chain constant region is derived from a mouse TCR alpha chain constant region in which the amino acid at position 48, e.g. threonine T, is mutated to cysteine C relative to the wild-type mouse TCR alpha chain constant region,
- 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 such as glycine G is changed to Jie Amino acid V.
- the alpha chain comprises an antigen binding region that specifically binds CD19 and a modified TCR alpha chain constant region, wherein
- the antigen-binding region that specifically binds to CD19 comprises the heavy chain variable region shown in SEQ ID NO:9 and the light chain variable region shown in SEQ ID NO:10 connected by a linker (G4S) 3 ,
- 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, the amino acid at position 6 such as E is replaced by D, and the K at position 13 is replaced by R, and amino acids 15-18 were deleted, and the amino acid at position 48 such as threonine T was mutated to cysteine C, the amino acid at position 112 such as serine S was changed to leucine L, and the amino acid at position 114 such as serine S was changed to leucine L.
- the amino acid at position 115, eg, methionine M is changed to isoleucine I, and the amino acid at position 115, eg, glycine G, is changed to pyrimidine V.
- the alpha chain comprises an antigen binding region that specifically binds CD19, a modified TCR alpha chain constant region, and an intracellular domain of OX40, wherein
- the antigen-binding region that specifically binds to CD19 comprises the heavy chain variable region shown in SEQ ID NO:9 and the light chain variable region shown in SEQ ID NO:10 connected by a linker (G4S) 3 ,
- the modified TCR alpha chain constant region is derived from a mouse TCR alpha chain constant region in which the amino acid at position 48, e.g. threonine T, is mutated to cysteine C relative to the wild-type mouse TCR alpha chain constant region,
- 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 such as glycine G is changed to Jie Amino acid V
- amino acids 136-137 were deleted.
- the alpha chain comprises an antigen binding region that specifically binds CD19, a modified TCR alpha chain constant region, and an intracellular domain of OX40, wherein
- the antigen-binding region that specifically binds to CD19 comprises the heavy chain variable region shown in SEQ ID NO:9 and the light chain variable region shown in SEQ ID NO:10 connected by a linker (G4S) 3 ,
- 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, the amino acid at position 6 such as E is replaced by D, and the K at position 13 is replaced by R, and amino acids 15-18 were deleted, and the amino acid at position 48 such as threonine T was mutated to cysteine C, the amino acid at position 112 such as serine S was changed to leucine L, and the amino acid at position 114 such as serine S was changed to leucine L.
- Amino acids at position 115 such as methionine M were changed to isoleucine I
- amino acids at position 115 such as glycine G were changed to pyrimidine V
- amino acids 136-137 were deleted.
- the alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 16, 18 or 37.
- the beta chain comprises an antigen binding region that specifically binds CD22 and a modified TCR beta chain constant region, wherein
- the antigen-binding region that specifically binds to CD22 comprises the heavy chain variable region shown in SEQ ID NO: 11 and the light chain variable region shown in SEQ ID NO: 12 connected by a linker (G4S) 3 ,
- the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region in which the amino acid at position 56, eg, serine S, is mutated to cysteine C relative to the wild-type mouse TCR ⁇ chain constant region.
- the beta chain comprises an antigen binding region that specifically binds CD22 and a modified TCR beta chain constant region, wherein
- the antigen-binding region that specifically binds to CD22 comprises the heavy chain variable region shown in SEQ ID NO: 11 and the light chain variable region shown in SEQ ID NO: 12 connected by a linker (G4S) 3 ,
- the modified TCR ⁇ chain constant region is derived from a mouse TCR ⁇ chain constant region, which, relative to the wild-type mouse TCR ⁇ chain constant region, has an amino acid at position 56, such as serine S, mutated to cysteine C,
- antigen binding region that specifically binds CD22 is linked to the modified TCR ⁇ chain constant region through a CD8 hinge region.
- the beta chain comprises an antigen binding region that specifically binds CD22 and a modified TCR beta chain constant region, wherein
- the antigen-binding region that specifically binds to CD22 comprises the heavy chain variable region shown in SEQ ID NO: 11 and the light chain variable region shown in SEQ ID NO: 12 connected by a linker (G4S) 3 ,
- the modified TCR ⁇ chain constant region is derived from the mouse TCR ⁇ chain constant region, and its amino acid at position 3, such as R, is substituted with K, and the amino acid at position 6, such as T, is substituted with respect to the wild-type mouse TCR ⁇ chain constant region.
- F is substituted
- K at position 9 is substituted with E
- S at position 11 is substituted with A
- L at position 12 is substituted with V
- amino acids 17, 21-25 are deleted
- the amino acid at position 56 is e.g. Serine S is mutated to cysteine C,
- antigen binding region that specifically binds CD22 is linked to the modified TCR ⁇ chain constant region through a CD8 hinge region.
- the beta chain comprises an antigen binding region that specifically binds CD22, a modified TCR beta chain constant region, and an intracellular domain of OX40, wherein
- the antigen-binding region that specifically binds to CD22 comprises the heavy chain variable region shown in SEQ ID NO: 11 and the light chain variable region shown in SEQ ID NO: 12 connected by a linker (G4S) 3 ,
- the modified TCR ⁇ chain constant region is derived from the mouse TCR ⁇ chain constant region, and its amino acid at position 3, such as R, is substituted with K, and the amino acid at position 6, such as T, is substituted with respect to the wild-type mouse TCR ⁇ chain constant region.
- F is substituted
- K at position 9 is substituted with E
- S at position 11 is substituted with A
- L at position 12 is substituted with V
- amino acids 17, 21-25 are deleted
- the amino acid at position 56 is e.g. Serine S was mutated to cysteine C, amino acids 167-172 were deleted
- antigen binding region that specifically binds CD22 is linked to the modified TCR ⁇ chain constant region through a CD8 hinge region.
- the beta strand comprises the amino acid sequence set forth in SEQ ID NO: 17, 19 or 38.
- the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:16 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:17. In some specific embodiments, the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:18, and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:19. In some specific embodiments, the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:37, and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:38.
- the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:48, and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:47. In some specific embodiments, the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:50, and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:49. In some specific embodiments, the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:52, and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:51. In some specific embodiments, the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:54, and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:53.
- the present invention provides an isolated polynucleotide comprising a nucleotide sequence encoding the alpha chain and/or beta chain of the synthetic T cell receptor antigen receptor (STAR) of the present invention.
- STAR synthetic T cell receptor antigen receptor
- the polynucleotide comprises i) the nucleotide sequence encoding the alpha chain, ii) the nucleotide sequence encoding the beta chain, and iii) located in the same reading frame in i) and ii) between the nucleotide sequences encoding the self-cleaving peptides.
- the nucleotide sequence encoding the alpha chain may be located at the 5' end or the 3' end of the nucleotide sequence encoding the beta chain.
- Self-cleaving peptide as used herein means a peptide that can achieve self-cleavage within a cell.
- the self-cleaving peptide may contain a protease recognition site so that it is recognized and specifically cleaved by proteases within the cell.
- the self-cleaving peptide can be a 2A polypeptide.
- 2A polypeptides are a class of short peptides from viruses whose self-cleavage occurs during translation. When the 2A polypeptide is used to link two different target proteins and express them in the same reading frame, the two target proteins are produced in an almost 1:1 ratio.
- Commonly used 2A polypeptides can be P2A from porcine techovirus-1, T2A from Thosea aigna virus, 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.
- Various functional variants of these 2A polypeptides are also known in the art, and these variants may also be used in the present invention.
- the present invention provides an expression vector comprising a polynucleotide of the present invention operably linked to regulatory sequences.
- the "expression vector" of the present invention may be a linear nucleic acid fragment, a circular plasmid, a viral vector, or may be an RNA capable of translation (eg, mRNA).
- the expression vector is a viral vector, such as a lentiviral vector.
- regulatory sequence and “regulatory element” are used interchangeably and refer to a coding sequence upstream (5' non-coding sequence), intermediate or downstream (3' non-coding sequence) and affecting transcription, RNA processing or Stability or translated nucleotide sequence.
- An expression regulatory 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 can include, but are not limited to, promoters, translation leader sequences, introns, enhancers, and polyadenylation recognition sequences.
- operably linked refers to regulatory elements (eg, but not limited to, promoter sequences, transcription termination sequences, etc.) are linked to a nucleic acid sequence (eg, a coding sequence or open reading frame) such that nucleotides Transcription of the sequence is controlled and regulated by the transcriptional regulatory elements.
- a nucleic acid sequence eg, a coding sequence or open reading frame
- the present invention provides a method of making therapeutic T cells comprising expressing the synthetic T cell receptor antigen receptor (STAR) of the present invention in the T cells.
- the method comprises introducing a polynucleotide of the invention or an expression vector of the invention into a T cell.
- the present invention provides a therapeutic T cell comprising the synthetic T cell receptor antigen receptor (STAR) of the present invention, or obtained by the method of the present invention.
- STAR synthetic T cell receptor antigen receptor
- T cells of the present invention can be obtained by various non-limiting methods from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical 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 (PBMCs) followed by activation and expansion with specific antibodies.
- PBMCs peripheral blood mononuclear cells
- the T cells are derived from autologous cells of the subject.
- autologous means that a cell, cell line, or population of cells used to treat a subject is derived from the subject.
- the T cells are derived from allogeneic cells, eg, from a donor compatible with the subject's human leukocyte antigen (HLA). Cells from the donor can be transformed into non-aloreactive cells using standard protocols and replicated as needed, resulting in cells that can be administered to one or more patients.
- HLA human leukocyte antigen
- the present invention provides a pharmaceutical composition comprising the therapeutic T cells of the present invention, and a pharmaceutically acceptable carrier.
- pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
- the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion).
- the present invention provides the use of a therapeutic T cell of the present invention or a pharmaceutical composition of the present invention in the manufacture of a medicament for the treatment of a disease such as cancer in a subject.
- Subject refers to an organism suffering from or susceptible to a disease (eg, cancer) that can be treated by the cells, methods, or pharmaceutical compositions of the present invention.
- a disease eg, cancer
- Non-limiting examples include humans, cows, 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 a therapeutic T cell of the present invention or a pharmaceutical composition of the present invention.
- a “therapeutically effective amount” or “therapeutically effective dose” or “effective amount” refers to an amount of a substance, compound, material, or cell that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is an amount necessary to prevent, cure, ameliorate, retard or partially retard the symptoms of a disease or disorder.
- an "effective amount" of a cell or pharmaceutical composition of the invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of disease, or prevention of injury or disability due to disease distress.
- an "effective amount" of a cell or pharmaceutical composition of the invention preferably inhibits tumor cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably, relative to an untreated subject. 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 animal model systems that predict efficacy against human tumors. Alternatively, it can also be assessed by examining the ability to inhibit tumor cell growth, which can be measured in vitro by assays well known to those skilled in the art.
- the dosage level of cells in the pharmaceutical compositions of the present invention may be varied to obtain an amount of active ingredient effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration, without toxicity to the patient.
- the dose level selected depends on a variety of pharmacokinetic factors, including the activity of the particular composition of the invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of treatment, and the specificity of the
- the other drugs, compounds and/or materials with which the composition is used in combination the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical arts.
- Administration of the therapeutic T cells or pharmaceutical compositions or medicaments according to the present invention may be carried out in any convenient manner, 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 CD19 and/or CD22-related disease, eg, a disease associated with abnormal expression of CD19 and/or CD22, eg, a CD19 and/or CD22-related cancer.
- the cancer may be a B cell malignancy such as chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia), lymphocytic Chikin's lymphoma, and combinations of such cancers.
- the cancer is a CD19 and/or CD22 positive cancer.
- the lentiviral vectors and lentiviral packaging plasmids used in the examples of this application were purchased from or synthesized by commercial companies.
- the gene fragments used in the examples of this application including signal peptides, antibody binding regions, hinge regions, TCR constant regions, tag proteins, etc., are all synthesized by commercial companies.
- the corresponding functional sequences are obtained by ligating one or more target fragments by means of synthetic primer PCR.
- the lentiviral vector used in the present invention is pHAGE-hEF1 ⁇ -RFP
- pHAGE-hEF1A-WPRE-AMP vector is obtained by restriction endonuclease SpeI/SalI
- the fragment gene is obtained by synthesis and PCR method, and by homologous recombination method, in The complete vector is obtained under the action of recombinase.
- CD19-22 CAR CD19-22 STAR(CT), CD19-22 STAR-OX40, CD19-STAR(CT), CD22-STAR(CT), M971(BspEI)ba , M971(CD8h)ba, M971(EcoRI)ba, M971(G4S)ba, M971ba.
- the map of each construct is shown in Figure 8.
- the amino acid sequence of the CD19-22 CAR as a control is shown in SEQ ID NO:24.
- the scFv of M971 was selected for the antigen binding region targeting CD22, wherein the amino acid sequence of the heavy chain variable region (VH) of M971 is shown in SEQ ID NO: 11; the amino acid sequence of the light chain variable region (VL) is shown in SEQ ID NO: 12.
- the scFv of FMC63 was selected for the antigen binding region targeting CD19, wherein the amino acid sequence of FMC63 heavy chain variable region (VH) is shown in SEQ ID NO:9; the amino acid sequence of light chain variable region (VL) is shown in SEQ ID NO: 10.
- the linker linking VH and VL is GGGGSGGGGSGGGGS (ie (G 4 S) 3 ).
- the CD8 hinge region amino acid sequence is shown in SEQ ID NO:13.
- TRAC The ⁇ chain constant region is of mouse origin, the wild type is named TRAC, and the sequence is shown in SEQ ID NO: 2.
- TRAC(CT) refers to a murine constant region mutated from threonine T at position 48 to cysteine C and comprising a mutation of LSVMGLRIL to LLVIVLRIL, the amino acid sequence of which is shown in SEQ ID NO:3.
- TRAC(NCT) refers to N-terminal modification on the basis of TRAC(CT), and the amino acid sequence of TRAC(NCT) is shown in SEQ ID NO:4.
- TRBC The beta chain constant region is of mouse origin, the wild type is named TRBC, and the sequence is shown in SEQ ID NO:6.
- TRBC (CT) refers to a murine constant region mutated from serine S at position 56 to cysteine C, the amino acid sequence of which is shown in SEQ ID NO:7.
- TRBC(NCT) refers to N-terminal modification on the basis of TRBC(CT), and the amino acid sequence of TRBC(NCT) is shown in SEQ ID NO:8.
- the amino acid sequence of the cytoplasmic region of the costimulatory molecule OX40 is shown in SEQ ID NO:14.
- Lentix-293T cells were seeded into a 10cm culture dish at 5 ⁇ 10 5 cells/mL, and cultured in a 37°C, 5% CO2 incubator, and transfection was carried out when the cell density reached about 80% (observed under a microscope). .
- the Jurkat T cell line was cultured in RPMI 1640 medium containing 10% FBS.
- the culture density is 3*10 5 /ml, and the maximum is not more than 3*10 6 /ml. Passage 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 in CO2 for culture. box cultivation.
- the primary T cells were cultured in X-VIVO medium containing 10% FBS and 100IU/ml IL-2, and the initial culture density was 1*10 6 /ml. 5 ⁇ g/ml) in pre-coated well plates. In the later stage, the culture density is 5*10 5 /ml, and the maximum does not exceed 3*10 6 /ml, and passages are 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 for 30 min, and then PBS was added to wash twice, On-board detection.
- the positive T cells were co-cultured with target cells expressing Luciferase (Raji cells, CD19-KO Raji cells or CD22-KO Raji cells) at the specified effect-target ratio.
- Luciferase Raji cells, CD19-KO Raji cells or CD22-KO Raji cells
- the model was constructed using NSG immunodeficient mice.
- the mouse is genotype NOD-Prkdc em26 Il2rg em26 /Nju, lacks T cells, B cells, NK cells, and is also deficient in macrophages and dendritic cells.
- mice Female NSG mice aged 6 to 8 weeks were used, and the weight difference of the mice in each batch of experiments was controlled within 2g.
- Mice were housed in specific pathogen-free (SPF) individually ventilated cages provided with a normal diet and drinking water with an acidic pH to prevent pathogen contamination.
- SPPF pathogen-free
- the tumor model was established by xenotransplantation of human Burkitt's lymphoma cell line Raji cells.
- Raji cells are cell lines that express the luciferase gene through a lentiviral vector.
- the development and changes of Raji tumors are monitored in real time in mice by means of luciferin chemiluminescence and in vivo imaging.
- 1-3 ⁇ 10 6 cells of Raji-luciferase cells were inoculated into 6-8 week-old female NSG mice through tail vein infusion.
- fluorescein potassium salt solution By intraperitoneal injection of fluorescein potassium salt solution into mice, the fluorescence signal of tumor cells in vivo was detected by in vivo imaging.
- the secretory antibody (Antibody, Ab) or B cell receptor (BCR) produced by B cells has great similarity in gene structure, protein structure and spatial conformation with T cell receptor (TCR).
- Both antibodies and TCRs are composed of variable and constant regions, wherein 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.
- a synthetic chimeric molecule can be constructed by substituting the variable regions of the TCR alpha and beta chains (or TCR gamma and delta chains) with the variable heavy (VH) and light (VL) domains of the antibody. , known as the synthetic T-cell receptor antibody receptor (Synthetic T-Cell Receptor and Antibody Receptor, STAR).
- the STAR molecule has two chains.
- the first chain is obtained by fusing the antigen recognition sequence (such as the variable region VH of the heavy chain of an antibody) with the constant region (C ⁇ ) of the T cell receptor ⁇ chain (TCR ⁇ ).
- the chain is obtained by fusing an antigen recognition sequence (eg, the variable region VL of the light chain of an antibody) to the constant region (C ⁇ ) of the T cell receptor ⁇ chain (TCR ⁇ ).
- the antigen recognition domains (such as VH, VL or scFv, etc.) and the constant region domains (constant regions of TCR ⁇ and ⁇ ) in the construct can be arranged and combined to form various constructs with different configurations but similar functions.
- first and second chains of STAR molecules After the first and second chains of STAR molecules are expressed in T cells, they will combine with the endogenous CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ chains in the endoplasmic reticulum to form a complex of 8 subunits, which is displayed in the form of a complex. on the cell membrane surface.
- Immunoreceptor Tyrosine-based Activation Motif (ITAM) is a signal transduction motif in TCR molecule, and its conserved sequence is YxxL/V.
- ITAM Immunoreceptor Tyrosine-based Activation Motif
- the intracellular region of CD3 ⁇ , ⁇ , ⁇ , and ⁇ chains contains 1 ITAM sequence
- 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 When the antigen recognition sequence of the STAR receptor binds to its specific antigen, the intracellular ITAM sequence will be phosphorylated one by one, thereby activating the downstream signaling pathway, activating transcription factors such as NF- ⁇ , NFAT and AP-1, and triggering the activation of T cells. , which produces an effector function.
- STAR is a structure designed according to TCR, because T cells themselves express TCR, STAR can mismatch with endogenous TCR, resulting in a new structure with no function.
- the function of STAR is improved by murineization of the constant region, cysteine point mutation, N-terminal modification, and hydrophobic amino acid mutation of the ⁇ chain constant region.
- the murine modification of the constant region Since the constant region sequences of human, primate and murine TCR ⁇ / ⁇ chains are highly functionally conserved and have the same key amino acid sequences, they can be replaced with each other. After the exchange, on the one hand, it increases the efficiency of correct pairing of STAR molecules, on the other hand, it reduces the possibility of unknown specificity caused by mismatching, and increases the safety.
- cysteine point mutation introduces a disulfide bond: in the constant region of the murine TCR ⁇ chain, the threonine T at position 48 is mutated to cysteine C, and in the constant region of the murine TCR ⁇ chain, the serine S at position 56 is mutated to Cysteine C.
- the two newly added cysteines will form a disulfide bond between the two STAR chains, reducing the mismatch between the two STAR chains and the endogenous TCR chain, helping the STAR molecules to form a more stable complex.
- the rearrangement scheme of 18 amino acids at the N-terminus of the constant region of the TCR ⁇ chain (the mouse sequence is DIQNPEPAVYQLKDPRSQ), through the analysis of amino acid properties, it is found that the mouse and human sequences are all amino acid substitutions of the same nature at these positions of E6D, K13R, R16K and Q18S.
- a non-polar amino acid was replaced by a polar amino acid, so it can be considered that the properties of the protein near this site are not conserved and can be modified without affecting the function.
- the amino acid sequence 1-14 was retained and humanized, and the amino acid 15-18 was deleted.
- the rearrangement scheme of 25 amino acids at the N-terminus of the constant region of the TCR ⁇ chain (the murine sequence is DLRNVTPPKVSLFEPSKAEIANKQK), through the analysis of amino acid properties, it is found that the murine and human sequences are only homogeneous amino acid substitutions at R3K and L12V sites, while in T6F, K8E , S11A, K17E, A21S, N22H and K23T sites are all substitutions of different amino acid properties, so it can be considered that the properties of proteins near this site are not conserved, and can be modified without affecting the function.
- the amino acid sequence 1-16 was retained and humanized, and the amino acids 17, 21-25 were deleted.
- the effect of additional hinge structures on STAR was also tested.
- the scFv of M971 targeting CD22 was constructed at the N-terminus of the ⁇ chain constant region of STAR, and different hinge structures (respectively the amino acid sequence encoded by BspEI, the amino acid sequence encoded by EcoRI, the hinge region of CD8) were added in the middle. , G4S connector).
- Constructs M971(BspEI)ba, M971(CD8h)ba, M971(EcoRI)ba, M971(G4S)ba, M971ba were obtained.
- the C-terminus of the alpha chain constant region (deleting the intracellular domain) in these constructs was linked to the intracellular domain of the costimulatory molecule OX40 by a linker ( G4S)3 .
- the resulting constructs were transduced into T cells to obtain corresponding STAR-T cells.
- the specific killing of CD22-expressing Raji cells comprising the different STAR-T cells obtained was tested (effector-target ratios of 0.5:1 and 1:1 were used, respectively).
- a CD19-targeting STAR was constructed using the FMC63 scFv against CD19, wherein the FMC63 scFv was constructed at the N-terminus of the beta chain constant region, and the STAR structure was named CD19-STAR (CT).
- CT represents the murine constant region
- C cysteine modification
- T hydrophobic modification
- CD22-targeting STAR was constructed using the M971 scFv against CD22, wherein the M971 scFv was constructed at the N-terminus of the beta chain constant region, and the STAR structure was named CD22-STAR (CT).
- CD19-22 STAR CD19-STAR
- CT CD19-STAR
- CT CD22-STAR
- the human renal epithelial cell line 293T which does not express CD19 and CD22, was used as target cells.
- 293T cells are cells that overexpress luciferase-GFP by lentivirus, and on the basis of this cell, 293T-CD19/22 target cells overexpressing CD19 or CD22 are constructed by lentivirus.
- CD19-22 STAR CT basically did not kill 293T cells, but could well kill 293T target cells overexpressing CD19 or CD22.
- HA22 scFv targeting CD22 was used to replace the M971 scFv, combined with the FMC63 scFv to construct a dual-target STAR targeting both CD19 and CD22, and the CD8 hinge region was also used to connect the N of the HA22 scFv and the ⁇ chain constant region. end.
- the obtained dual-target STAR also showed strong killing ability to 293T cells overexpressing CD19 and CD22, although the killing ability and specificity were weaker than those of the M971 scFv-based dual-target STAR.
- the efficacy of the HA22 scFv-based dual-targeting STAR can be improved by optimizing the hinge region connecting the HA22 scFv and the beta chain constant region.
- CD19-22 STAR In order to further improve CD19-22 STAR, its structure was optimized, especially on the basis of CD19-22 STAR (CT), the N-terminals of the constant regions of ⁇ chain and ⁇ chain were further modified as described in Example 1 to obtain CD19 -22 STAR (NCT): the alpha chain sequence is shown in SEQ ID NO:37; the beta chain sequence is shown in SEQ ID NO:38. N in NCT stands for N-terminal modification.
- CT CD19-22 STAR
- NCT CD19-22 STAR
- CD19-22 CAR The killing ability of CD19-22 STAR (CT), CD19-22 STAR (NCT) and CD19-22 CAR as a control were then tested by lymphoma cell lines Raji cells, CD19 knockout Raji cells, CD22 knockout Raji cells .
- the CD19-22 STAR(CT) structure was further optimized by deleting the native intracellular domain at the C-terminus of the ⁇ chain (the last 2 amino acids of the C-terminus for the mouse-derived ⁇ chain constant region), and using a linker (G4S) ) 3 added OX40 costimulatory factor to obtain CD19-22 STAR-OX40 ( ⁇ chain: SEQ ID NO: 18; ⁇ chain: SEQ ID NO: 19) and tested its killing ability.
- G4S linker
- the target cell results are shown in Figure 6.
- the CD19-22 STAR supplemented with OX40 co-stimulatory factor has a stronger killing effect on CD19/CD22 target cells.
- NSG mice were injected with single-target and dual-target mixed target cells (90%Raji+5%Raji-CD19KO+5%Raji-CD22KO) through the tail vein for 7 days, then injected with CD19-STAR T cells, CD22-STAR T cells and CD19-22 STAR-OX40T cells in which the alpha chains of these STARs contain a deletion of the constant region intracellular region and the addition of OX40 costimulator via linker (G4S) 3 , and contain CT modifications of the alpha and beta chain constant regions.
- G4S linker
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Abstract
Description
Claims (48)
- 一种针对CD19和CD22的合成T细胞受体抗原受体(STAR),其包含α链和β链,所述α链包含第一抗原结合区和第一恒定区,所述β链包含第二抗原结合区和第二恒定区,其中所述第一抗原结合区是特异性结合CD19的抗原结合区,且所述第二抗原结合区是特异性结合CD22的抗原结合区;或者所述第一抗原结合区是特异性结合CD22的抗原结合区,且所述第二抗原结合区是特异性结合CD19的抗原结合区。
- 权利要求1的针对CD19和CD22的STAR,其中所述第一恒定区是经修饰的TCRα链恒定区。
- 权利要求2的针对CD19和CD22的STAR,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2或3的针对CD19和CD22的STAR,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2-4中任一项的针对CD19和CD22的STAR,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2-5中任一项的针对CD19和CD22的STAR,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,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2-6中任一项的针对CD19和CD22的STAR,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,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,且在第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2-6中任一项的针对CD19和CD22的STAR,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,缺失恒定区的胞内区,例如缺失第136-137位氨基酸。
- 权利要求1的针对CD19和CD22的STAR,所述经修饰的TCRα链恒定区包含SEQ ID NO:3-4和39-41之一所示氨基酸序列。
- 权利要求1-9中任一项的针对CD19和CD22的STAR,其中所述第二恒定区是经修饰的TCRβ链恒定区。
- 权利要求10的针对CD19和CD22的STAR,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10或11的针对CD19和CD22的STAR,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10-12中任一项的针对CD19和CD22的STAR,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,且在第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10-13中任一项的针对CD19和CD22的STAR,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,缺失恒定区的胞内区,例如缺失第167-172位氨基酸。
- 权利要求14的针对CD19和CD22的STAR,所述经修饰的TCRβ链恒定区包含SEQ ID NO:7-8和42-44之一所示氨基酸序列。
- 权利要求1-15中任一项的针对CD19和CD22的STAR,其中所述抗原结合区是单链抗体(如scFv)或单域抗体(如骆驼抗体),优选所述抗原结合区是单链抗体例如scFv。
- 权利要求16的针对CD19和CD22的STAR,其中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,例如,所述接头是(G4S)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1-17中任一项的针对CD19和CD22的STAR,其中所述特异性结合CD19的抗原结合区包含重链可变区和轻链可变区,所述重链可变区包含SEQ ID NO:25所示的VH CDR1、SEQ ID NO:26所示的VH CDR2、和SEQ ID NO:27所示的VH CDR3,所述轻链可变区包含SEQ ID NO:28所示的VL CDR1、SEQ ID NO:29所示的VL CDR2、和SEQ ID NO:30所示的VL CDR3,例如,所述特异性结合CD19的抗原结合区包含SEQ ID NO:9所示的重链可变区以及SEQ ID NO:10所示的轻链可变区。
- 权利要求1-18中任一项的针对CD19和CD22的STAR,其中所述特异性结合CD22的抗原结合区包含重链可变区和轻链可变区,其中i)所述重链可变区包含SEQ ID NO:31所示的VH CDR1、SEQ ID NO:32所示的VH CDR2、和SEQ ID NO:33所示的VH CDR3,所述轻链可变区包含SEQ ID NO:34所示 的VL CDR1、SEQ ID NO:35所示的VL CDR2、和SEQ ID NO:36所示的VL CDR3,ii)所述重链可变区包含SEQ ID NO:58所示的VH CDR1、SEQ ID NO:59所示的VH CDR2、和SEQ ID NO:60所示的VH CDR3,所述轻链可变区包含SEQ ID NO:55所示的VL CDR1、SEQ ID NO:56所示的VL CDR2、和SEQ ID NO:57所示的VL CDR3;iii)所述重链可变区包含SEQ ID NO:65所示的VH CDR1、SEQ ID NO:66所示的VH CDR2、和SEQ ID NO:67所示的VH CDR3,所述轻链可变区包含SEQ ID NO:61所示的VL CDR1、SEQ ID NO:62所示的VL CDR2、和SEQ ID NO:63所示的VL CDR3;iv)所述重链可变区包含SEQ ID NO:73所示的VH CDR1、SEQ ID NO:74所示的VH CDR2、和SEQ ID NO:75所示的VH CDR3,所述轻链可变区包含SEQ ID NO:69所示的VL CDR1、SEQ ID NO:70所示的VL CDR2、和SEQ ID NO:71所示的VL CDR3;或v)所述重链可变区包含SEQ ID NO:81所示的VH CDR1、SEQ ID NO:82所示的VH CDR2、和SEQ ID NO:83所示的VH CDR3,所述轻链可变区包含SEQ ID NO:77所示的VL CDR1、SEQ ID NO:78所示的VL CDR2、和SEQ ID NO:79所示的VL CDR3,例如,所述特异性结合CD22的抗原结合区包含a)SEQ ID NO:11所示的重链可变区,以及SEQ ID NO:12所示的轻链可变区;b)SEQ ID NO:46所示的重链可变区,以及SEQ ID NO:45所示的轻链可变区;c)SEQ ID NO:68所示的重链可变区,以及SEQ ID NO:64所示的轻链可变区;d)SEQ ID NO:76所示的重链可变区,以及SEQ ID NO72所示的轻链可变区;e)SEQ ID NO:84所示的重链可变区,以及SEQ ID NO80所示的轻链可变区。
- 权利要求1-19中任一项的针对CD19和CD22的STAR,其中所述α链或β链的抗原结合区直接或间接融合至所述恒定区的N末端。
- 权利要求1-20中任一项的针对CD19和CD22的STAR,其中所述α链或β链的抗原结合区通过接头融合至所述恒定区的N末端。
- 权利要求21的针对CD19和CD22的STAR,其中所述α链或β链,优选β链的抗原结合区通过CD8铰链区融合至所述恒定区的N末端,例如,所述CD8铰链区包含SEQ ID NO:13所示氨基酸序列。
- 权利要求1-22中任一项的针对CD19和CD22的STAR,其中所述α链和/或β链在其C末端连接有至少一个外源胞内功能结构域。
- 权利要求23的针对CD19和CD22的STAR,其中所述外源胞内功能结构域直接或通过接头连接至所述α链和/或β链的恒定区的C末端,优选地,所述外源胞内功能结构域通过接头连接至胞内区缺失的所述α链和/或β链的恒定区的C末端,优选地,所述接头是(G4S)n接头,其中n代表1-10的整数,优选地,n是3。
- 权利要求23或24的针对CD19和CD22的STAR,其中所述外源胞内功能结构域是共刺激分子的胞内结构域,优选OX40的胞内结构域,例如,所述OX40的胞内结 构域包含SEQ ID NO:14的氨基酸序列。
- 权利要求1-25中任一项的针对CD19和CD22的STAR,其中所述α链包含特异性结合CD19的抗原结合区以及经修饰的TCRα链恒定区,其中特异性结合CD19的抗原结合区包含通过接头(G4S) 3连接的SEQ ID NO:9所示的重链可变区以及SEQ ID NO:10所示的轻链可变区,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1-25中任一项的针对CD19和CD22的STAR,其中所述α链包含特异性结合CD19的抗原结合区以及经修饰的TCRα链恒定区,其中特异性结合CD19的抗原结合区包含通过接头(G4S) 3连接的SEQ ID NO:9所示的重链可变区以及SEQ ID NO:10所示的轻链可变区,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,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,且在第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1-25中任一项的针对CD19和CD22的STAR,其中所述α链包含特异性结合CD19的抗原结合区、经修饰的TCRα链恒定区以及OX40的胞内结构域,其中特异性结合CD19的抗原结合区包含通过接头(G4S) 3连接的SEQ ID NO:9所示的重链可变区以及SEQ ID NO:10所示的轻链可变区,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,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,且在第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,第136-137位氨基酸被缺失。
- 权利要求1的针对CD19和CD22的STAR,其中所述α链包含SEQ ID NO:16或18或37所示的氨基酸序列。
- 权利要求1-27中任一项的针对CD19和CD22的STAR,其中所述β链包含特异性结合CD22的抗原结合区以及经修饰的TCRβ链恒定区,其中特异性结合CD22的抗原结合区包含通过接头(G4S) 3连接的SEQ ID NO:11所示的重链可变区以及SEQ ID NO:12所示的轻链可变区,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1-27中任一项的针对CD19和CD22的STAR,其中所述β链包含特异性结合CD22的抗原结合区以及经修饰的TCRβ链恒定区,其中特异性结合CD22的抗原结合区包含通过接头(G4S) 3连接的SEQ ID NO:11所示的重链可变区以及SEQ ID NO:12所示的轻链可变区,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,其中所述特异性结合CD22的抗原结合区通过CD8铰链区连接至所述经修饰的TCRβ链恒定区。
- 权利要求1-27中任一项的针对CD19和CD22的STAR,其中所述β链包含特异性结合CD22的抗原结合区以及经修饰的TCRβ链恒定区,其中特异性结合CD22的抗原结合区包含通过接头(G4S) 3连接的SEQ ID NO:11所示的重链可变区以及SEQ ID NO:12所示的轻链可变区,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,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,且在第56位的氨基酸例如丝氨酸S被突变为半胱氨酸C,其中所述特异性结合CD22的抗原结合区通过CD8铰链区连接至所述经修饰的TCRβ链恒定区。
- 权利要求1-27中任一项的针对CD19和CD22的STAR,其中所述β链包含特异性结合CD22的抗原结合区、经修饰的TCRβ链恒定区以及OX40的胞内结构域,其中特异性结合CD22的抗原结合区是抗体m971的scFv,所述m971的scFv包含通过接头(G4S) 3连接的SEQ ID NO:11所示的重链可变区以及SEQ ID NO:12所示的轻链可变区,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,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,且在第56位的氨基酸例如丝氨酸S被突变为半胱氨酸C,第167-172位氨基酸被缺失,其中所述特异性结合CD22的抗原结合区通过CD8铰链区连接至所述经修饰的TCRβ链恒定区。
- 权利要求1的针对CD19和CD22的STAR,所述β链包含SEQ ID NO:17或19或38所示的氨基酸序列。
- 权利要求1的针对CD19和CD22的STAR,其中所述α链包含SEQ ID NO:16所示的氨基酸序列,且所述β链包含SEQ ID NO:17所示的氨基酸序列。
- 权利要求1的针对CD19和CD22的STAR,其中所述α链包含SEQ ID NO:18所示的氨基酸序列,且所述β链包含SEQ ID NO:19所示的氨基酸序列。
- 权利要求1的针对CD19和CD22的STAR,其中所述α链包含SEQ ID NO:37所示的氨基酸序列,且所述β链包含SEQ ID NO:38所示的氨基酸序列。
- 一种分离的多核苷酸,其包含编码权利要求1-37中任一项的针对CD19和CD22的STAR的α链和/或β链的核苷酸序列。
- 权利要求38的多核苷酸,所述多核苷酸包含在同一读码框内的i)编码所述α链的核苷酸序列、ii)编码所述β链的核苷酸序列和iii)位于i)和ii)之间的编码自裂解肽的核苷酸序列。
- 权利要求39的多核苷酸,其中所述自裂解肽是2A多肽,例如P2A多肽。
- 一种表达载体,其包含与调控序列可操作连接的权利要求38-40中任一项的多核苷酸。
- 权利要求41的表达载体,其是病毒载体,例如慢病毒载体。
- 一种制备治疗性T细胞的方法,包括在T细胞中表达权利要求1-37中任一项的针对CD19和CD22的STAR。
- 权利要求41的方法,所述方法包括将权利要求38-40中任一项的多核苷酸或权利要求41或42的表达载体导入T细胞。
- 一种治疗性T细胞,其包含权利要求1-37中任一项的针对CD19和CD22的STAR,或其通过权利要求43或44的方法获得。
- 一种药物组合物,其包含权利要求45的治疗性T细胞,和药物可接受的载体。
- 权利要求45的治疗性T细胞或权利要求46的药物组合物在制备用于在对象中治疗疾病的药物中的用途,例如所述疾病是CD19和/或CD22相关癌症。
- 权利要求47的用途,其中所述癌症可以是B细胞恶性肿瘤,例如慢性或急性白血病(包括急性髓细胞样白血病、慢性髓细胞样白血病、急性淋巴细胞性白血病、慢性淋巴细胞性白血病)、淋巴细胞性淋巴瘤、非霍奇金淋巴瘤、以及所述癌症的组合。
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