Chimeric antigen receptors targeting her2

Abstract

The present invention relates to chimeric antigen receptors targeting HER2. Chimeric transmembrane immune receptors (CARs) comprising an extracellular domain targeting HER2, a transmembrane region, a costimulatory domain and an intracellular signaling domain are described.

Description

[0001] This application is a divisional application of a patent application filed on November 4, 2016, with a priority date of November 4, 2015, application number 201680076412.0, entitled "Chimeric Antigen Receptor Targeting HER2". Background Technology

[0002] Immunotherapy based on tumor-specific T cells, including anti-tumor therapies employing engineered T cells, has been investigated. In some cases, the T cells used in such therapies do not remain active in vivo for a sufficiently long period. In other cases, the tumor specificity of T cells is relatively low, partly due to the heterogeneous nature of solid tumors and the potential for off-target effects on non-cancerous cells when targeting self-antigens. Therefore, there is a need in the art for tumor-specific cancer therapies with improved anti-tumor specificity and function.

[0003] Chimeric antigen receptors (CARs) consist of an extracellular tumor recognition / targeting domain, an extracellular linker / spacer, a transmembrane domain, and an intracellular T cell activation and co-stimulatory signaling domain. The design of the recognition / targeting region is crucial to avoiding harmful off-target effects. Most CAR tumor-targeting domains are single-chain variable fragments (scFvs) derived from antibody sequences, utilizing the specificity of antibody binding to specific antigens. Examples of CAR tumor-targeting domains derived from normal receptor ligands also exist, such as the IL-13 cytokine CAR that targets cells expressing the IL-13 receptor IL-13Rα2.

[0004] Adoptive T-cell therapy (ACT) using engineered T cells expressing CARs has demonstrated potent and durable clinical efficacy in patients with CD19+ B-cell malignancies (Priceman et al. 2015 Curr Opin Oncol; Mauset al. 2014 Blood 123: 2625-2635). Following early successes in hematologic disorders, broader applications of this approach in solid tumors are now under intensive investigation.

[0005] According to data from the National Cancer Institute's Surveillance, Epidemiology, and End Results Program (SEER), an estimated 40,000 people died from breast cancer in 2014, primarily from metastatic disease. Approximately 25-30% of breast cancer patients carry HER2 gene amplification, which confers a particularly poor prognosis. Even with the advent of newer agents, including targeted therapies, the overall mortality rate for stage IV disease has only modestly improved. For example, in randomized trials of HER2-positive breast cancer patients, the most promising combination of two HER2-targeting antibodies, trastuzumab and pertuzumab, plus docetaxel, produced a median overall survival extension of 56.5 months and a progression-free survival extension of only 6.3 months compared to trastuzumab or docetaxel alone. Summary of the Invention

[0006] This article describes a chimeric transmembrane immune receptor (chimeric antigen receptor or "CAR") comprising an extracellular domain, a transmembrane region, and an intracellular signaling domain. The extracellular domain includes a HER2-targeting scFv and optionally a spacer containing, for example, a portion of a human Fe domain. The transmembrane portion includes, for example, a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, or a CD3 transmembrane domain. The intracellular signaling domain includes a signaling domain derived from the ζ chain (CD3ξ) of the human CD3 complex and one or more co-stimulatory domains, such as a 4-1BB or CD28 co-stimulatory domain. The extracellular domain enables T cell activity to be directed to cells expressing HER2 when the CAR is expressed on the surface of T cells. Such cells include certain breast cancer cells and certain brain cancer cells. The inclusion of a co-stimulatory domain tandem with CD3ζ, such as a 4-1BB (CD137) co-stimulatory domain, in the intracellular region enables T cells to receive co-stimulatory signals. T cells, such as patient-specific autologous T cells, can be engineered to express the CAR described herein, and engineered cells can be expanded and used for ACT. Various T cell subsets can be used, including both αβ T cells and γδ T cells. Additionally, CARs can be expressed in other immune cells such as NK cells. In cases where patients are treated with immune cells expressing the CARs described herein, the cells can be autologous T cells or allogeneic T cells. In some cases, the cells used are CD4+ and CD8+ central memory T cells (T cells) comprising CD62L+, CCR7+, CD45RO+, and CD45RA-. CM The cell population, or the cells used, include both CD4+ and CD8+ T cells. CMCell populations including stem cells, stem central memory T cells, and naive T cells (i.e., T cells) CM / SCM / N (Cell population). T CM / SCM / N The cell population is CD62L+, CCR7+, and includes both CD45RA+ and CD45RO+ cells, as well as both CD4+ and CD8+ cells. Using this type of cell improves long-term cell persistence after adoptive transfer compared to using other types of patient-specific T cells.

[0007] This article describes a nucleic acid molecule encoding a CAR, which comprises: a scFv targeting HER2 (e.g., DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS; SEQ ID NO: 1) or variants thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); transmembrane domains selected from: CD4 transmembrane domain or variants thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), CD8 transmembrane domain or variants thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), CD28 transmembrane domain or variants thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), and CD3ξ transmembrane domain or variants thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); co-stimulatory domains (e.g., CD28 co-stimulatory domain or variants thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); or 4-I The BB costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution); or the CD28 costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution) and both the 4-1BB costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution); and the CD3ξ signaling domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications.

[0008] In various embodiments: the co-stimulatory domain is selected from the group consisting of: a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution), a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution), and an OX40 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications. In some embodiments, a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications is present. In some embodiments, two co-stimulatory domains are present, such as a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution) and a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution). In various embodiments, the 1-5 (e.g., 1 or 2) amino acid modifications are substitutions.

[0009] In some cases, there is a short sequence of 1-6 amino acids (e.g., GGG) between the co-stimulatory domain and the CD3ξ signaling domain and / or between the two co-stimulatory domains.

[0010] In another implementation, the CAR includes: a scFv targeting HER2; two different co-stimulatory domains selected from the group consisting of: a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, and an OX40 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications; HER2 scFv or a variant thereof having 1-2 amino acid modifications; selected from the following transmembrane domains: CD4 transmembrane domain or a variant thereof having 1-2 amino acid modifications, CD8 transmembrane domain or a variant thereof having 1-2 amino acid modifications, CD28 transmembrane domain or a variant thereof having 1-2 amino acid modifications, and CD3ξ transmembrane domain or a variant thereof having 1-2 amino acid modifications; costimulatory domains (e.g., CD28 costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution); or 4-1BB costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution); or both the CD28 costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution) and the 4-1BB costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitution); and CD3ξ signaling domain or a variant thereof having 1-2 amino acid modifications; located at HER2 The spacer region between the scFv or a variant thereof and the transmembrane domain (e.g., the spacer region contains an amino acid sequence selected from the group consisting of SEQ ID NO: 2-12 and 42 (Table 3) or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications); the spacer contains an IgG hinge region; the spacer region contains 1-150 amino acids; there is no spacer; the 4-1BB signaling domain contains the amino acid sequence of SEQ ID NO: 24, and the CD3ξ signaling domain contains the amino acid sequence of SEQ ID NO: 21 and a 3-15 amino acid linker located between the co-stimulatory domain and the CD3ξ signaling domain or a variant thereof. In some embodiments with two co-stimulatory domains, one is a 4-1BB co-stimulatory domain and the other is a co-stimulatory domain selected from the group consisting of CD28 and CD28gg. In various embodiments, the 1-5 (e.g., 1 or 2) amino acid modifications are substitutions, such as conservative substitutions.

[0011] In some implementations: the nucleic acid molecule expresses a polypeptide comprising an amino acid sequence selected from SEQ ID NO: 26-41; the chimeric antigen receptor comprises an amino acid sequence selected from SEQ ID NO: 26-41.

[0012] Also disclosed is a population of human T cells transduced via a vector containing an expression cassette encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises: a HER2-targeting scFv; a transmembrane domain selected from: a CD4 transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), a CD8 transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), a CD28 transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), and a CD3ξ transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); a costimulatory domain (e.g., a CD28 costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); or a 4-1BB costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); or CD28 The co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions) and both the 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); and the CD3ξ signaling domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions). In various embodiments: the human T cell population comprises a vector expressing a chimeric antigen receptor, said chimeric antigen receptor comprising an amino acid sequence selected from any one of SEQ ID NO: 26-41 or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); the human T cell population comprises central memory T cells (T cells). CM (cells), for example, at least 20%, 30%, 40%, 50%, 60%, 70%, 80% of the cells are T cells. CM Cells, or T cell populations, comprise a combination of central memory T cells, naive T cells, and stem cell central memory cells (T cells). CM / SCM / N (cells), for example, at least 20%, 30%, 40%, 50%, 60%, 70%, 80% of the cells are T cells. CM / SCM / N Cells. In any case, the T cell population comprises both CD4+ and CD8+ cells (e.g., at least 20% of CD3+ T cells are CD4+ and at least 3% of CD3+ T cells are CD8+, and at least 70, 80, or 90% are CD4+ or CD8+; at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, or 60% of CD3+ cells are CD4+, and at least 4%, 5%, 8%, 10%, or 20% of CD3+ cells are CD8+ cells).

[0013] A method for treating cancer in patients is also described, comprising administering a population of autologous or allogeneic human T cells transduced via a vector containing an expression cassette encoding a chimeric antigen receptor (e.g., autologous or allogeneic T cells containing central memory T cells). CM T cells) or a combination of central memory T cells, naive T cells, and stem cell central memory cells (i.e., T cells are T cells that are T cells). CM / SCM / N (Cells). At least 20%, 30%, 40%, 50%, 60%, 70%, and 80% of the cells are TCM / SCM / N cells. In any case, the T cell population comprises both CD4+ and CD8+ cells (e.g., at least 20% of CD3+ T cells are CD4+ and at least 3% of CD3+ T cells are CD8+, and at least 70, 80, or 90% are CD4+ or CD8+; at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, and 60% of the CD3+ cells are CD4+, and at least 4%, 5%, 8%, 10%, and 20% of the CD3+ cells are CD8+), wherein the chimeric antigen receptor comprises an amino acid sequence selected from SEQ ID NO: 26-41 or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions). In various embodiments: the cancer is a brain cancer, for example, a HER2-expressing brain cancer metastasized from breast cancer; and transduced human T cells are prepared by a method comprising obtaining T cells from a patient, processing the T cells to isolate central memory T cells, and transducing at least a portion of the central memory cells with a viral vector containing an expression cassette encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 26 or 27 or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions). In some cases, CAR T cells are not administered directly to the brain tumor, but rather intracerebrally within the patient's brain.

[0014] It also describes: nucleic acid molecules encoding polypeptides, said polypeptides comprising an amino acid sequence that is at least 95% identical to the amino acid sequences selected from SEQ ID NO: 26-41; nucleic acid molecules encoding polypeptides comprising an amino acid sequence that is identical to the amino acid sequences selected from SEQ ID NO: 26-41 except for the presence of no more than 5 amino acid substitutions, deletions, or insertions; nucleic acid molecules encoding polypeptides comprising an amino acid sequence that is identical to the amino acid sequences selected from SEQ ID NO: 26-41 except for the presence of no more than 5 amino acid substitutions; and nucleic acid molecules encoding polypeptides comprising an amino acid sequence that is identical to the amino acid sequences selected from SEQ ID NO: 26-41 except for the presence of no more than 2 amino acid substitutions.

[0015] This disclosure also includes nucleic acid molecules encoding any of the CARs described herein (e.g., vectors containing a nucleic acid sequence encoding one of the CARs) and isolated T lymphocytes expressing any of the CARs described herein.

[0016] The CAR described herein may include a spacer region located between the HER2 binding domain (e.g., HER2 scFv) and the transmembrane domain. A variety of different spacers may be used. Some of them include at least a portion of the human Fc region, such as the hinge portion of the human Fc region or the CH3 domain or variants thereof. Table 1 below provides a list of various spacers that may be used in the CAR described herein.

[0017] Table 1: Examples of spacers

[0018]

[0019]

[0020] Some spacer regions include whole or partial immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4) hinge regions, i.e., sequences falling between the CH1 and CH2 domains of an immunoglobulin, such as the IgG4 Fc hinge or the CD8 hinge. Some spacer regions contain the immunoglobulin CH3 domain or both the CH3 and CH2 domains. Immunoglobulin-derived sequences may contain one or more amino acid modifications, such as 1, 2, 3, 4, or 5 substitutions, for example, substitutions that reduce off-target binding.

[0021] "Amino acid modification" refers to the substitution, insertion, and / or deletion of amino acids in a protein or peptide sequence. "Amino acid substitution" or "replacement" refers to the replacement of an amino acid at a specific position in a parent peptide or protein sequence with another amino acid. Substitution can be performed to change the amino acids in the resulting protein in a non-conservative manner (i.e., by changing the codon belonging to an amino acid group of a specific size or characteristic to an amino acid belonging to another group) or a conserved manner (i.e., by changing the codon belonging to an amino acid group of a specific size or characteristic to an amino acid belonging to the same group). This conserved change generally results in minimal alteration to the structure and function of the resulting protein. Here are examples of several groupings of amino acids: 1) Amino acids with nonpolar R groups: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine; 2) Amino acids with uncharged polar R groups: glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine; 3) Amino acids with charged polar R groups (negatively charged at pH 6.0): aspartic acid, glutamic acid; 4) Basic amino acids (positively charged at pH 6.0): lysine, arginine, histidine (at pH 6.0). Another grouping could be those amino acids with phenyl groups: phenylalanine, tryptophan, and tyrosine.

[0022] In some embodiments, the spacer is derived from IgG1, IgG2, IgG3, or IgG4 and comprises one or more amino acid residues substituted with amino acid residues different from those present in the unmodified spacer. The one or more substituted amino acid residues are selected from, but not limited to, one or more amino acid residues at positions 220, 226, 228, 229, 230, 233, 234, 235, 234, 237, 238, 239, 243, 247, 267, 268, 280, 290, 292, 297, 298, 299, 300, 305, 309, 218, 326, 330, 331, 332, 333, 334, 336, and 339, or combinations thereof. In this numbering system, as described in more detail below, the first amino acid in the IgG4 (L235E, N297Q) spacer in Table 1 is 219, and the first amino acid in the IgG4 (HL-CH3) spacer in Table 1 is also 219, as are the first amino acids in the IgG hinge sequence and the IgG4 hinge joint (HL) sequence in Table 1.

[0023] In some embodiments, the modified spacer is derived from IgG1, IgG2, IgG3, or IgG4 and comprises, but is not limited to, substitutions for one or more of the following amino acid residues: C220S, C226S, S228P, C229S, P230S, E233P, V234A, L234V, L234F, L234A, L235A, L235E, G236A, G237A, P238S, S239D, F243L, P247I, S267E, H268Q, S280H, K2 90S, K290E, K290N, R292P, N297A, N297Q, S298A, S298G, S298D, S298V, T299A, Y300L, V305I, V309L, E318A, K326A, K326W, K326E, L328F, A330L, A330S, A331S, P331S, I332E, E333A, E333S, E333S, K334A, A339D, A339Q, P396L, or combinations thereof.

[0024] In some embodiments, the modified spacer is derived from the IgG4 region and comprises one or more amino acid residues substituted with amino acid residues different from those present in the unmodified region. The one or more substituted amino acid residues are selected from, but not limited to, one or more amino acid residues or combinations thereof at positions 220, 226, 228, 229, 230, 233, 234, 235, 234, 237, 238, 239, 243, 247, 267, 268, 280, 290, 292, 297, 298, 299, 300, 305, 309, 218, 326, 330, 331, 332, 333, 334, 336, and 339.

[0025] In some embodiments, the modified spacer is derived from the IgG4 region and comprises, but is not limited to, one or more of the following amino acid residues substituted: 220S, 226S, 228P, 229S, 230S, 233P, 234A, 234V, 234F, 234A, 235A, 235E, 236A, 237A, 238S, 239D, 243L, 247I, 267E, 268Q, 280H, 290S, 290E, 290N, 292P 297A, 297Q, 298A, 298G, 298D, 298V, 299A, 300L, 305I, 309L, 318A, 326A, 326W, 326E, 328F, 330L, 330S, 331S, 331S, 332E, 333A, 333S, 333S, 334A, 339D, 339Q, 396L or combinations thereof, wherein the amino acid in the unmodified spacer is replaced by the amino acid identified above at the indicated position.

[0026] For the amino acid positions in the immunoglobulins discussed in this article, they were numbered according to the EU index or the EU numbering system (Kabat et al. 1991 Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda, vector incorporated in full by reference). The EU index in the EU index or Kabat or EU numbering system refers to the number of EU antibodies (Edelman et al. 1969 Proc Natl Acad Sci USA 63:78-85).

[0027] Various transmembrane domains can be used. Table 2 contains examples of suitable transmembrane domains. In the presence of spacer domains, the transmembrane domain is located at the carboxyl terminus of the spacer domain.

[0028] Table 2: Examples of transmembrane domains

[0029]

[0030] Many of the CARs described herein contain one or more (e.g., two) co-stimulatory domains. These co-stimulatory domains are located between the transmembrane domain and the CD3ζ signaling domain. Table 3 includes examples of suitable co-stimulatory domains and sequences of the CD3ζ signaling domains.

[0031] Table 3: Examples of CD3ζ domain and co-stimulatory domain

[0032]

[0033] The present invention further relates to the following embodiments:

[0034] 1. A nucleic acid molecule encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises: a HER2 targeting sequence; a transmembrane domain selected from the following: a CD4 transmembrane domain or a variant thereof having a modification of 1-5 amino acids, a CD8 transmembrane domain or a variant thereof having a modification of 1-5 amino acids, a CD28 transmembrane domain or a variant thereof having a modification of 1-5 amino acids, and a CD3ξ transmembrane domain or a variant thereof having a modification of 1-5 amino acids; a costimulatory domain selected from the following: a CD28 costimulatory domain or a variant thereof having a modification of 1-5 amino acids, and a 4-1BB costimulatory domain or a variant thereof having a modification of 1-5 amino acids; and a CD3ξ signaling domain or a variant thereof having a modification of 1-5 amino acids.

[0035] 2. The nucleic acid molecule of implementation scheme 1, wherein the HER2 targeting domain is HER2 scFv.

[0036] 3. The nucleic acid molecule of embodiment 1, wherein the HER2 scFv comprises an amino acid sequence:

[0037] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGSTSGGGSGGGSGGGSSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS or variants thereof having a sequence of 1 to 5 amino acids.

[0038] 4. The nucleic acid molecule of embodiment 1, wherein the chimeric antigen receptor comprises: a HER2 targeting sequence; a transmembrane domain selected from the following: a CD4 transmembrane domain or a variant thereof having a 1-2 amino acid modification, a CD8 transmembrane domain or a variant thereof having a 1-2 amino acid modification, a CD28 transmembrane domain or a variant thereof having a 1-2 amino acid modification, and a CD3ξ transmembrane domain or a variant thereof having a 1-2 amino acid modification; a costimulatory domain selected from the following: a CD28 costimulatory domain or a variant thereof having a 1-2 amino acid modification, and a 4-1BB costimulatory domain or a variant thereof having a 1-2 amino acid modification; and a CD3ξ signaling domain or a variant thereof having a 1-2 amino acid modification.

[0039] 5. The nucleic acid molecule of embodiment 1, comprising a spacer region located between the HER2 targeting domain and the transmembrane domain.

[0040] 6. The nucleic acid molecule of embodiment 5, wherein the spacer region comprises 5-300 amino acids.

[0041] 7. The nucleic acid molecule of embodiment 5, wherein the spacer region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2-12 or variants thereof having 1-5 amino acid modifications.

[0042] 8. The nucleic acid molecule of embodiment 6, wherein the spacer comprises an IgG hinge region.

[0043] 9. The nucleic acid molecule of embodiment 6, wherein the spacer comprises 10-50 amino acids.

[0044] 10. The nucleic acid molecule of embodiment 1, wherein the co-stimulatory domain is a 4-1BB co-stimulatory domain comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof having 1-5 amino acid modifications.

[0045] 11. The nucleic acid molecule of embodiment 1, wherein the CD3ξ signal transduction domain comprises the amino acid sequence of SEQ ID NO: 7.

[0046] 12. The nucleic acid molecule of embodiment 1, wherein a linker of 3-15 amino acids is located between the co-stimulatory domain or a variant thereof and the CD3ξ signaling domain or a variant thereof.

[0047] 13. The nucleic acid molecule of embodiment 1, wherein the nucleic acid molecule expresses a polypeptide comprising an amino acid sequence selected from SEQ ID NO: 26 and 27 or a variant thereof having 1-5 amino acid modifications.

[0048] 14. The nucleic acid molecule of embodiment 1, wherein the chimeric antigen receptor comprises a 4-1BB co-stimulatory domain and a spacer region, said spacer region comprising an amino acid sequence of any one of SEQ ID NO: 2-12 or a variant thereof having a modification of 1-5 amino acids.

[0049] 15. The nucleic acid molecule of embodiment 1, wherein the chimeric antigen receptor comprises an amino acid sequence selected from SEQ ID NO: 26 and 27.

[0050] 16. The nucleic acid molecule of implementation scheme 1, wherein the 1-5 amino acid modification is a modification of 1 or 2 amino acids.

[0051] 17. The nucleic acid molecule of implementation scheme 1, wherein the 1-5 amino acid modifications are 1-5 amino acid substitutions.

[0052] 18. A population of human T cells transduced via a vector, said vector comprising an expression cassette encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises: a HER2 targeting sequence; a transmembrane domain selected from the following: a CD4 transmembrane domain or a variant thereof having a 1-5 amino acid modification, a CD8 transmembrane domain or a variant thereof having a 1-5 amino acid modification, a CD28 transmembrane domain or a variant thereof having a 1-5 amino acid modification, and a CD3ξ transmembrane domain or a variant thereof having a 1-5 amino acid modification; a costimulatory domain selected from the following: a CD28 costimulatory domain or a variant thereof having a 1-5 amino acid modification, and a 4-1BB costimulatory domain or a variant thereof having a 1-5 amino acid modification; and a CD3ξ signaling domain or a variant thereof having a 1-5 amino acid modification.

[0053] 19. A population of human T cells comprising a vector expressing a chimeric antigen receptor, said chimeric antigen receptor comprising an amino acid sequence selected from SEQ ID NO: 26 and 27 or a variant thereof having a modification of 1-5 amino acids.

[0054] 20. The human T cell population of embodiment 19, wherein the T cells comprise a population of central memory T cells.

[0055] 21. A method for treating HER2-expressing brain cancer in patients, comprising administering an autologous or allogeneic population of human T cells transduced by a vector comprising an expression cassette encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an amino acid sequence selected from SEQ ID NO: 26 and 27 or a variant thereof having a modification of 1-5 amino acids.

[0056] 22. The method of implementation scheme 21, wherein the human T cell population comprises CD62L+ memory T cells.

[0057] 23. The method of implementation scheme 21, wherein the cancer is a breast-to-brain metastasis.

[0058] 24. The method of embodiment 21, wherein transduced human T cells are prepared by means of a method comprising obtaining T cells from the patient, processing the T cells to isolate central memory T cells, and transducing at least a portion of the central memory cells with a viral vector comprising an expression cassette encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an amino acid sequence selected from SEQ ID NO: 26 and 27 or a variant thereof having a modification of 1-5 amino acids.

[0059] 25. The method of implementation scheme 21, wherein the T cells are administered intratumorally.

[0060] 26. The method of implementation scheme 21, wherein the T cells are administered indoors.

[0061] 27. The method of implementation scheme 21, wherein the T cells are administered in a room near the tumor.

[0062] 28. A nucleic acid molecule encoding a polypeptide, said polypeptide comprising at least 95% identical amino acid sequences to those selected from SEQ ID NO: 26 and 27.

[0063] 29. T cells expressing a polypeptide, said polypeptide comprising the same amino acid sequence as that selected from SEQ ID NO: 26 and 27 or variants thereof having 1-5 amino acid modifications.

[0064] 30. The nucleic acid molecule of embodiment 1, wherein the nucleic acid molecule encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NO: 26-41.

[0065] 31. A nucleic acid molecule of any one of embodiments 1-4, wherein the chimeric antigen receptor comprises a spacer region located between the HER2 targeting domain and the transmembrane domain.

[0066] 32. A nucleic acid molecule according to the embodiments, wherein the spacer region comprises an amino acid sequence of any one of SEQ ID NO: 9, 7, 3 and 2 or an amino acid sequence of SEQ ID NO: 3 and the following SEQ ID NO: 2, or is composed of an amino acid sequence of any one of SEQ ID NO: 9, 7, 3 and 2 or an amino acid sequence of SEQ ID NO: 3 and the following SEQ ID NO: 2. Attached Figure Description

[0067] Figure 1 The amino acid sequence of Her2scFv-IgG4 (L235E, N297Q)CD28tm-CD28gg-Zeta-T2A-CD19t is depicted. Each domain is listed sequentially below the sequence and indicated by alternating underscores and ununderscores. The mature CAR sequence (SEQ ID NO: 26) does not include the GMCSFRa signal peptide, the T2A skipping sequence, or a truncated CD19.

[0068] Figure 2 The amino acid sequence of Her2scFv-IgG4(L235E, N297Q)CD8tm-41BB-Zeta-T2A-CD19t is depicted. Each domain is listed sequentially below the sequence and indicated by alternating underscores and ununderscores. The mature CAR sequence (SEQ ID NO: 27) does not include the GMCSFRa signal peptide, the T2A skipping sequence, or a truncated CD19.

[0069] Figures 3A-3DData on HER2-specific CAR constructs and CAR T-cell expansion were described.

[0070] Figures 4A-4D Describe the in vitro characterization of HER2-CAR T cells against breast cancer cell lines.

[0071] Figures 5A-5F The results of in vitro tumor activity studies on HER2-CAR T cells were described.

[0072] Figure 6A-6I The results of an in vivo study on the antitumor efficacy of HER2-CAR T cells delivered locally within tumors were described.

[0073] Figures 7A-7D The results of a study on local delivery of HER2-CAR T cells in a human orthotopic BBM xenograft model are described.

[0074] Figures 8A-8E The results of an in-situ delivery study of HER2-CAR T cells are described.

[0075] Figure 9-14 Another CAR targeting HER2 is described.

[0076] Figures 15A-15C The study results characterizing some additional CARs with various spacers are described. IgG3 (EQ) in Figure 2 DeltaCh2 in Figure 11 In the middle; CD8h in Figure 9 In; HL in Figure 10 In; L in Figure 14 middle.

[0077] Figure 16 The AC results show the results of an examination of CD107a and INFγ produced when TCMs expressing various CAARs were exposed to cells that did not express HER2 (MDA-MB-468), low HER2 (231BR), low HER2 (231BRHER2LO), or high HER2 (231BRHER2HI).

[0078] Figures 17A-17D Displayed for inspection Figure 2 (HER2(EQ)BBζ) or Figure 14 Results of a study on PD-1 production and tumor cell killing in various cell lines of (HER2(L)BB ζ) CAR.

[0079] Figures 18A-18BThe results show the effects of examining CD107a and INFγ produced when TCMs expressing various CAARs were exposed to cells that did not express HER2 (MDA-MB-468), had low HER2 (231BR), low HER2 (231BRHER2LO), or high HER2 (231BRHER2HI). Detailed Implementation

[0080] The following describes the structure, construction, and characterization of various chimeric antigen receptors that target HER2 and can be used to treat HER2-expressing breast cancer and breast-to-brain metastases. Importantly, the CARs described herein can be used in ACTs to treat HER2-expressing tumors in the brain via intratumoral or intratumoral delivery.

[0081] Chimeric antigens (CARs) are recombinant biomolecules containing at least an extracellular recognition domain, a transmembrane domain, and an intracellular signaling domain. Therefore, the term "antigen" is not limited to molecules that bind antibodies, but refers to any molecule capable of specifically binding a target. For example, a CAR may include a ligand that specifically binds to a cell surface receptor. The extracellular recognition domain (also called the extracellular domain or simply referred to by the recognition element it contains) contains a recognition element that specifically binds to molecules present on the cell surface of a target cell. The transmembrane domain anchors the CAR to the membrane. The intracellular signaling domain contains a signaling domain derived from the ζ chain of the human CD3 complex and optionally includes one or more co-stimulatory signaling domains. CARs can both bind antigens and transduce T cell activation, independent of MHC restriction. Therefore, CARs are "universal" immune receptors that can treat patient populations with antigen-positive tumors, regardless of their HLA genotype. Adoptive immunotherapy using T lymphocytes expressing tumor-specific CARs can be a powerful therapeutic strategy for treating cancer.

[0082] In some cases, the CAR described herein can be generated using a vector in which the CAR read frame is followed by a T2A ribosomal skipping sequence and a truncated CD19 (CD19t) lacking a cytoplasmic signaling tail (truncated at amino acid 323). In this arrangement, co-expression of CD19t provides an inert, non-immunogenic surface marker that allows for accurate measurement of genetically modified cells and enables positive selection of genetically modified cells, as well as efficient cell tracking and / or imaging of therapeutic T cells in vivo after adoptive transfer. Co-expression of CD19t provides a marker that functions as a suicide switch for in vivo immunological targeting of transduced cells using clinically available antibodies and / or immunotoxins to selectively deplete therapeutic cells.

[0083] The CAR described herein can be generated by any means known in the art, although recombinant DNA technology is preferred. Where convenient, nucleic acids encoding several regions of the chimeric receptor can be prepared and assembled into a complete coding sequence using standard molecular cloning techniques known in the art (genomic library screening, PCR, primer-assisted ligation, site-directed mutagenesis, etc.). The resulting coding regions are preferably inserted into an expression vector and used to transform a suitable expression host cell line, preferably a T lymphocyte line, and most preferably selected from in vivo T lymphocyte lines.

[0084] Various T cell subgroups isolated from patients can be transduced using vectors for CAR expression, including unselected PBMCs or enriched CD3 T cells or enriched CD3 or memory T cell subgroups. Central memory T cells are a useful T cell subgroup. Central memory T cells can be isolated from peripheral blood mononuclear cells (PBMCs) by immunomagnetically selecting cells expressing the desired receptor using devices such as CliniMACS®, enriched CD45RO+ / CD62L+ cells. Cells rich in central memory T cells can be activated with anti-CD3 / CD28 and transduced with SIN lentiviral vectors, such as those guiding CAR expression and truncated human CD19 (CD19t), which is a non-immunogenic surface marker for both in vivo detection and potential in vitro selection. Activated / genetically modified central memory T cells can be expanded in vitro with IL-2 / IL-15 and then cryopreserved.

[0085] Example 1: Structures of Two HER2-CARs

[0086] One CAR containing the HER2 scFv described in this article is called Her2scFv-IgG4(L235E, N297Q)-CD28tm-CD28gg-Zeta-T2A-CD19t. This CAR includes several important features, including: a HER2-targeting scFv; IgG4 Fc regions mutated at two sites (L235E; N297Q) within the CH2 region to reduce Fc receptor (FcR) binding; and a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3ζ activation domain. Figure 1 The amino acid sequence of this CAR is presented, including the truncated CD19 sequence for monitoring CAR expression and the T2A ribosomal jumping sequence that allows CAR generation without fusing the truncated CD19 sequence. Figure 2As shown, the immature CAR includes: the GMCSFR signal peptide, HER2 scFv, IgG4 which acts as a spacer, the CD8 transmembrane region, a 4-1BB co-stimulatory domain containing LL to GG sequence alterations, a triple Gly sequence, and a CD3 Zeta-stimulatory domain. The transcript also encodes the T2A ribosomal sequence and a truncated CD19 sequence, which are not part of the CAR protein sequence. The mature CAR is the same as the immature CAR, but lacks the GMCSFR signal peptide.

[0087] Example 2: Construction and structure of epHIV7 for expressing HER2-specific CAR T cells

[0088] The epHIV7 vector is a vector that can be used to express a HER2-specific CAR. It is derived from the pHIV7 vector. Importantly, this vector uses the human EFI promoter to drive CAR expression. The 5' and 3' sequences of the vector are both derived from pv653RSN, as previously derived from the HXBc2 provirus. The polypurine tract DNA flap sequence (cPPT) is derived from the HIV-1 strain pNL4-3 from the NIH AIDS Repository. The prairie posttranscriptional regulatory element (WPRE) sequence has been previously described.

[0089] pHIV7 was constructed as follows. In short, the pv653RSN containing a 653bp sequence from gag-pol, plus 5' and 3' long terminal repeats (LTRs) and the intermediate SL3-neomycin phosphotransferase gene (Neo), was subcloned into pBluescript: In step 1, p5'HIV-I 5I was prepared from the sequence from the 5'LTR to the rev-response element (RRE), then the 5'LTR was modified by removing the sequence upstream of the TATA box and first ligated to the CMV enhancer, then ligated to the SV40 origin of replication (p5'HIV-2). In step 2, after cloning the 3'LTR into pBluescript to prepare p3'HIV-1, a 400-bp deletion was performed in the 3'LTR enhancer / promoter to remove the cis-regulatory element in HIV U3 and form p3'HIV-2. In step 3, fragments isolated from p5'HIV-3 and p3'HIV-2 were ligated to prepare pHIV-3. In step 4, p3'HIV-2 was further modified by removing additional upstream HIV sequences to generate p3'HIV-3, and a 600-bp BamHI-SalI fragment containing WPRE was added to p3'HIV-3 to prepare p3'HIV-4. In step 5, the size of the pHIV-3 RRE was reduced by PCR and ligated to the 5' fragment (not shown) from pHIV-3 and to p3'HIV-4 to prepare pHIV-6. In step 6, a 190-bp BglII-BamHI fragment containing the cPPT DNA flap sequence from HIV-1 pNL4-3(55) was amplified from pNL4-3 and placed between the RRE and WPRE sequences in pHIV-6 to prepare pHIV-7. Using this parental plasmid pHIV7-GFP (GFP, green fluorescent protein), the parental vector was packaged using a 4-plasmid system.

[0090] The packaging signal psi ψ is required for the efficient packaging of the viral genome into the vector. RRE and WPRE enhance RNA transcript transport and transgene expression. Valve sequences combined with WPRE have been shown to enhance the transduction efficiency of lentiviral vectors in mammalian cells.

[0091] The auxiliary functions required to generate the viral vector were divided into three separate plasmids to reduce the possibility of generating replicative lentiviruses through recombination: 1) pCgp encodes the gag / pol protein required for viral vector assembly; 2) pCMV-Rev2 encodes the Rev protein, which acts on the RRE sequence to help transport the viral genome efficiently; and 3) pCMV-G encodes the vesicular stomatitis virus (VSV) glycoprotein required for the infectivity of the viral vector.

[0092] Minimal DNA sequence homology exists between the pHIV7-encoded vector genome and the helper plasmid. Homologous regions include a packaging signal region of approximately 600 nucleotides located in the gag / pol sequence of the pCgp helper plasmid; the CMV promoter sequences in all three helper plasmids; and the RRE sequence in the pCgp helper plasmid. It is highly unlikely that replicative recombinant viruses could be generated due to homology in these regions, as it requires multiple recombination events. Furthermore, any resulting recombinants would lack the functional LTR and tat sequences required for lentiviral replication.

[0093] The CMV promoter was replaced by the EF1α-HTLV promoter (EF1p), and the new plasmid was named epHIV7. EF1p has a length of 563 bp and epHIV7 was introduced using NruI and NheI after the CMV promoter was removed.

[0094] The lentiviral genome has been removed from this system, eliminating the gag / pol and rev, which are essential for the pathogenicity of the wild-type virus and for productive infection of the target cells. Furthermore, the vector construct does not contain the complete 3'LTR promoter, so the expressed and reverse-transcribed proviral DNA genome obtained in the target cells will have an inactive LTR. Due to this design, no HIV-1-derived sequences will be transcribed from the provirus, and only therapeutic sequences will be expressed from their respective promoters. The removal of LTR promoter activity in the SIN vector significantly reduces the likelihood of unintentional activation of the host gene.

[0095] Example 3: Preparation of a vector for transducing patient T cells

[0096] Vectors for transducing patient T cells can be prepared as follows. For each plasmid expressing a CAR, express the CAR and optionally a marker, such as truncated CD19; 2) pCgp; 3) pCMV-G; and 4) pCMV-Rev2), to generate a seed bank, which is used to inoculate a fermenter to produce a sufficient quantity of plasmid DNA. The identity, sterility, and endotoxin content of the plasmid DNA are tested before use to generate lentiviral vectors.

[0097] In short, cells are expanded from 293T working cells (WCBs) that have been tested and confirmed to be sterile and free from viral contamination. A vial of 293T cells from the 293T WCBs is thawed. The cells are cultured and expanded until a sufficient number of cells are available for plating into appropriate numbers of 10-layer cell factories (CFs) for vector production and cell culture maintenance. Single-train cell cultures can be used for production.

[0098] Lentiviral vectors were generated in sub-batches of up to 10 CFs. Two sub-batches could be generated within the same week, resulting in approximately 20 L of lentiviral supernatant per week. Materials from all sub-batches were combined during downstream processing to produce a single batch of product. 293T cells were seeded in CFs in 293T medium (DMEM containing 10% FBS). The factory was placed in a 37°C incubator and laid horizontally to achieve uniform cell distribution across all layers of the CF. After two days, cells were transfected using the CaPO4 method with the four lentiviral plasmids described above, involving a mixture of Tris:EDTA, 2M CaCh, 2X HBS, and the four DNA plasmids. On day 3 post-transfection, the supernatant containing the secreted lentiviral vector was collected, purified, and concentrated. After removing the supernatant from the CFs, the final produced cells were collected from each CF. Cells from each factory were digested with trypsin and collected by centrifugation. The cells were resuspended in freezing medium and cryopreserved. These cells were later used for replication-capable lentivirus (RCL) assays.

[0099] To purify and prepare the vector, the crude supernatant was clarified by membrane filtration to remove cell debris. Host cell DNA and residual plasmid DNA were degraded by endonuclease digestion (Benzonase®). Cell debris was clarified from the viral supernatant using a 0.45 μm filter. The clarified supernatant was collected into a pre-weighed container containing Benzonase® (final concentration 50 U / mL). Endonuclease digestion of residual plasmid DNA and host genomic DNA was performed at 37°C for 6 hours. Initial tangential flow ultrafiltration (TFF) of the endonuclease-treated supernatant removed residual low molecular weight components from the crude supernatant, concentrating the virus approximately 20-fold. The clarified endonuclease-treated viral supernatant was then maintained at the designed flow rate for approximately 4,000 seconds at the maximum flow rate. -1 A flow rate with an even lower shear rate was circulated through a hollow fiber cartridge with an NMWCO of 500 kD. Perfiltration of the nuclease-treated supernatant was initiated during the concentration process to maintain cartridge performance. An 80% permeate exchange rate was established using 4% lactose in PBS as the perfiltration buffer. The viral supernatant was brought to the target volume, representing a 20-fold concentration of the crude supernatant, and perfiltration was continued for another 4 exchange volumes, achieving a 100% permeate exchange rate.

[0100] Viral products were further concentrated using high-speed centrifugation. Lentiviral fragments from each sub-batch were precipitated at 6000 RPM (6,088 RCF) for 16–20 hours at 6°C using a Sorvall RC-26 plus centrifuge. Viral clumps from each sub-batch were then reconstituted in 50 mL of PBS with 4% lactose. The reconstituted clumps in this buffer represent the final formulation of the viral preparation. The entire vector concentration process resulted in approximately a 200-fold volume reduction. After all sub-batch concentrations were completed, the material was placed at -80°C while samples from each sub-batch were tested for sterility. Once sample sterility was confirmed, the sub-batch was rapidly thawed at 37°C with frequent stirring. The material was then pooled and manually aliquoted in a Class II A / B3 biosafety cabinet. A 1 mL packing configuration of concentrated lentivirus was used in sterile USP Class 6, externally threaded O-ring cryovials.

[0101] To ensure the purity of the lentiviral vector preparation, it was tested for residual host DNA contamination and transfer of residual host and plasmid DNA. In other tests, vector identity was assessed by RT-PCR to ensure the presence of the correct vector.

[0102] Example 4: Preparation of T cells suitable for use in ACT

[0103] If you want to use T cm To express CAR, suitable patient cells can be prepared as follows: First, T lymphocytes are obtained from the patient via leukopheresis, and then a suitable subset of allogeneic or autologous T cells (e.g., central memory T cells) is added. CM Genetic alterations are made to express CAR, which is then administered back to the patient through any clinically acceptable means to achieve anti-cancer treatment.

[0104] A suitable T can be generated as follows: CM Blood components obtained from consenting study participants were treated with Ficoll, washed, and incubated overnight. They were then processed using GMP-grade anti-CD14, anti-CD25, and anti-CD45RA reagents (Miltenyi Biotec) and CliniMACS. TM The separation device reduces the populations of monocytes, regulatory T cells, and naïve T cells within the cells. After reduction, the cells are processed in CliniMACS. TM The separation device uses DREG56-Biotin (COH clinical grade) and anti-biotin microbeads (MiltenyiBiotec) targeting CD62L+ T. CM Negative fraction cells enriched by cell accumulation.

[0105] After enrichment, T was prepared in complete X-Vivo15 with 50 IU / mL IL-2 and 0.5 ng / mL IL-15. CM Cells were transferred to Teflon cell culture bags and stimulated with Dynal ClinEx™ Vivo CD3 / CD28 beads. Cells were transduced with a lentiviral vector expressing the desired CAR for up to five days post-stimulation, at a multiplicity of infection (MOI) of 1.0 to 0.3. Cultures were maintained for up to 42 days (keeping cell density at 3 x 10⁻⁶ cells / year) with complete X-Vivo15 and IL-2 and IL-15 cytokines as needed for cell expansion. 5 and 2x10 6 Live cells / mL, and cytokines are supplemented every Monday, Wednesday, and Friday during culture. Cells typically expand to approximately 10⁻¹¹ within 21 days under these conditions. 9 Cells were collected at the end of the culture period, washed twice, and prepared in clinical-grade cryopreservation medium (Cryostore CS5, BioLife Solutions).

[0106] On the day of T-cell infusion, the cryopreserved and released product was thawed, washed, and prepared for re-infusion. The cryopreserved vials containing the released cell product were removed from liquid nitrogen storage, thawed, cooled, and washed with PBS / 2% human serum albumin (HSA) washing buffer. After centrifugation, the supernatant was removed, and the cells were resuspended in preservative-free physiological saline (PFNS) / 2% HSA infusion diluent. Samples were then used for quality control testing.

[0107] Example 5: Expression of CARs targeting HER2

[0108] Figure 3A yes Figure 1 and Figure 2 The diagram illustrates two HER2-specific CAR constructs. In HER2(EQ)28ξ, scFv is tethered to the membrane via a modified IgG4 Fc linker (dual mutant, L235E; N297Q), containing a CD28 transmembrane domain, an intracellular CD28 costimulatory domain, and a cytolytic CD3ζ domain. A T2A skipping sequence separates the CAR from the truncated CD19 (CD19t) protein used for cell tracking. HER2(EQ)BBξ is similar, except that the costimulatory domain is 4-1BB instead of CD28 and the transmembrane domain is the CD8 transmembrane domain instead of the CD28 transmembrane domain. Human central memory (TCM) cells were transfected with lentiviral vectors expressing HER2(EQ)28ξ or HER2(EQ)BBξ. Figure 3B Representative FACS data depicting human TCM surface phenotypes were obtained. Figure 3CThe results of CD19 and protein L expression assays in TCM cells transfected with lentiviral vectors expressing HER2(EQ)28ξ or HER2(EQ)BBξ were presented. These results show that transfection efficiencies, as assessed by CD19 expression, are similar for both CARs. However, protein L expression was lower for HER2(EQ)BBξ than for HER2(EQ)28ξ, suggesting that the stability of the HER2(EQ)BBξ CAR is lower than that of the HER2(EQ)BBξ CAR. Cell expansion analysis ( Figure 3D The results showed that neither of these two CARs interfered with T cell expansion.

[0109] Example 6: In vitro characterization of HER2-CAR T cells against various breast cancer cell lines

[0110] HER2(EQ)28 and HER2(EQ)BBξ were characterized using a variety of breast cancer cell lines, including HER2-negative lines (LCL lymphoma, MDA-MB-468, U87 glioma), low HER2 expression lines (MDA-MB-361, 231BR), and high HER2 expression lines (SKBR3, BT474, BBM1). Figure 4A HER2 expression levels for each of these lineages were characterized. Flow cytometry (CAR+ T cell gated) was used to characterize CD107a degranulation and IFNγ production in CAR T cells co-cultured for 5 hours with MDA-MB-361 tumor cells (low HER2 expression) or BBM1 tumor cells (high HER2 expression), including untransduced, HER2(EQ)28ξ, or HER2(EQ)BBξ cells. The results of this analysis were presented in [the study / analysis]. Figure 4B Presented in [the document / article]. Similar studies were conducted on other breast cancer cell lines, and the results are summarized in [the document / article / article]. Figure 4C The production of IFNγ by HER2-CAR T cells after 24 hours of culture with recombinant HER2 protein or tumor targets was measured by ELISA, and the results of this analysis showed... Figure 4D middle.

[0111] Example 7: In vitro antitumor activity

[0112] Flow cytometry was used to evaluate tumor cell killing after co-culturing HER2(EQ)28ξ or HER2(EQ)BBξ CAR T cells with tumor targets for 72 hours. The results of this analysis were presented in [the following text is missing from the original] Figure 5A Presented in [the table]. PD-1 and LAG-3 induction in total CAR T cells after co-culturing with HER2-negative MDA-MB-468 or HER2-positive BBM1 cells for 72 hours were measured, and the results of this analysis are presented in [the table]. Figure 5BIn CD8+ CAR T cells, PD-1 was induced after co-culturing with tumor targets of HER2-negative (LCL lymphoma, MDA-MB-468, U87 glioma), low HER2 expression (MDA-MB-361, 231BR), or high HER2 expression (SKBR3, BT474, BBM1) for 72 hours. The results of this analysis... Figure 5C Presented in [the text]. These studies suggest that HER2(EQ)BBξ induces lower PD-1 induction than HER2(EQ)28ξ. Tumor cell killing was measured in both HER2(EQ)28ξ and HER2(EQ)BBξ CAR T cells with effector:tumor (E:T) ratios ranging from 0.25:1 to 2:1. The results of this analysis are presented in [the text]. Figure 5D The results showed that both HER2(EQ)28ξ and HER2(EQ)BBξ were effective in killing tumor cells in vitro. CFSE proliferation of HER2-CART cells after co-culturing with MDA-MB-468 or BBMI cells for 72 hours was measured by flow cytometry. Figure 5E The study showed that HER2(EQ)BBξ CAR T cells proliferated more than HER2(EQ)28ξ CAR T cells.

[0113] Example 8: In vivo antitumor activity

[0114] Evaluation of the activity of intratumorally delivered HER2 CAR T cells in a patient-derived breast-to-brain metastasis model. Figures 6A-6C This is H&E staining of the tumor. Mice were treated by directly injecting either a mimic (untransduced) or HER2(EQ)BBξ CAR T cells into the tumor. Figure 6D-6F The optical imaging results of the tumor were described, while Figure 6G-6I These are Kaplan-Meier survival curves for mice treated locally on days 3, 8, or 14 after tumor injection. These studies demonstrate that HER2(EQ)BBξ CAR T cells possess potent in vivo antitumor efficacy when directly injected into tumors.

[0115] To evaluate the antitumor efficacy in a human breast-to-brain xenograft model, BBM1 cells (0.2M) or BT474 (0.15M) were intracranially injected into NSG mice. On day 8 post-tumor injection, HER2(EQ)28ξ, HER2(EQ)BBξ, or mimic (untransduced) T cells (0.5M) were injected intratumorally. BBMI (BBM1 cells) were monitored using luciferase-based optical imaging. Figure 7A ) and BT474 ( Figure 7B ). Figure 7C and Figure 7DThe Kaplan-Meier curve is presented in the image.

[0116] Orthotopic xenograft models derived from human patients with breast-to-brain metastases are also used to evaluate HER2(EQ)28ξ or HER2(EQ)BBξ CAR T cells. Figure 8A The tumor implantation site is shown after stereotactic injection of BBM1 cells (0.2M) and intraventricular T cell delivery. Tumor staining is depicted on... Figure 8B On day 14 after tumor injection, HER2(EQ)28ξ, HER2(EQ)BBξ, or mimic (untransduced) T cells (0.5 M) were injected intratumorally. Figure 8D The average flux for each treatment group is presented, while Figure 8E The Kaplan Meier survival curves for each treatment group are presented.

[0117] Example 9: Another CAR targeting HER2

[0118] Figure 9-14 The amino acid sequences of various CARs with different linkers were depicted. Specifically, the CARs differ in the sequence and length of the portion between the scFv targeting HER2 and the transmembrane domain. The transmembrane domain is CD8, CD28, or CD28gg. The co-stimulatory domain is 4-1BB or CD28. All have a CD3ζ stimulatory domain. In each case, a T2A skipping sequence separates the CAR from the truncated CD19 (CD19t) protein used for cell tracking.

[0119] Figure 15A Various HER2 CARs are schematically depicted; they are identical except for the sequence and length of the portion between the HER2 scFv and the CD8 transmembrane domain. All include a 4-1BB co-stimulatory domain, followed by a CD3ζ stimulatory domain. Figure 15B The text describes the transfection of T cells with a lentiviral vector expressing the CAR shown. CM The results of CD19 and protein L expression assays were obtained. These results show that the transfection efficiency, as assessed by CD19 expression, is similar for both CARs. However, protein L expression was lower for HER2(EQ)BBξ than for HER2(EQ)28ξ, suggesting that the stability of the HER2(EQ)BBξ CAR is less than that of the HER2(EQ)BBξ CAR. Analysis of cell expansion (…) Figure 15C The results showed that CAR did not interfere with T cell expansion. Figure 16-1 8 shows further research results, which demonstrate CARs with very short spacers ( Figure 14 CARs expressing high levels of HER2 exhibit relative selectivity. Such CARs could be used to treat cancers expressing HER2, where it is desirable to have remaining cells expressing lower levels of HER2 than cancer cells.

Claims

1. A chimeric antigen receptor comprising an amino acid sequence selected from SEQ ID NO: 34-41.

2. The chimeric antigen receptor of claim 1, wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO:

34.

3. The chimeric antigen receptor of claim 1, wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO:

35.

4. The chimeric antigen receptor of claim 1, wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO:

36.

5. The chimeric antigen receptor of claim 1, wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO:

37.

6. The chimeric antigen receptor of claim 1, wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO:

38.

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

39.

8. The chimeric antigen receptor of claim 1, wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO:

40.

9. The chimeric antigen receptor of claim 1, wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO:

41.

10. A polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 28-33.

11. The polypeptide of claim 10, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

28.

12. The polypeptide of claim 10, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

29.

13. The polypeptide of claim 10, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

30.

14. The polypeptide of claim 10, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

31.

15. The polypeptide of claim 10, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

32.

16. The polypeptide of claim 10, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

33.

17. A population of human T cells expressing a polypeptide or chimeric antigen receptor selected from the amino acid sequence of SEQ ID NO: 34-41.

18. The human T cell population of claim 17, wherein the T cells comprise a central memory T cell population.

19. An autologous or allogeneic human T cell population expressing a polypeptide or chimeric antigen receptor for the treatment of HER2-expressing cancer in a patient, wherein the autologous or allogeneic human T cell population is administered to the patient, and wherein the polypeptide or the chimeric antigen receptor is an amino acid sequence selected from SEQ ID NO: 28-41.

20. The autologous or allogeneic human T cell population of claim 19, wherein the human T cell population comprises CD62L+ memory T cells.

21. The autologous or allogeneic human T-cell population of claim 19, wherein the cancer is breast cancer, brain cancer, or breast-to-brain metastatic cancer.

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