Enhanced chimeric antigen receptors and their use

Abstract

To provide new CAR designs comprising a modified CD3ζ chain, and cells including genetically modified immunoresponsive cells (e.g., T cells, NK cells, or CTL cells) comprising the CARs.SOLUTION: The present disclosure provides chimeric antigen receptors (CARs) that bind to an antigen of interest. The CARs can bind to a tumor antigen or a pathogen antigen. In certain non-limiting embodiments, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a modified CD3ζ polypeptide.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 612,031, filed on 29 December 2017, which is incorporated in its entirety by reference and for which priority is claimed.

[0002] Introduction The subject matter of this disclosure provides methods and compositions for enhancing immune responses to cancer and pathogens. It relates to novel designs of chimeric antigen receptors (CARs) and engineered immune-responsive cells containing them. Engineered immune-responsive cells containing novel CARs are antigen-targeted and have extended persistence without impairment of function. [Background technology]

[0003] Chimeric antigen receptor (CAR) therapy has achieved great clinical success for hematological malignancies (PMID:23515080). It is based on synthetic receptors that possess both antigen recognition and signaling functions (PMID:20467460). The single-chain variable fragment (scFv) in CARs retains its antigen recognition specificity derived from the variable regions of the heavy and light chains of the original monoclonal antibody. On the other hand, signaling of CAR constructs is highly dependent on the signaling domain of the original immune receptor. Currently, all versions of second-generation CARs in clinical trials have two signaling capabilities / modalities, as they contain domains derived from two immune receptors, one of which is CD3ζ and the other is a co-stimulatory receptor such as CD28 or 41BB (PMID:26129802). Essentially, it combines two signals transmitted by different receptors in one synthetic receptor. Presumably, signaling in CARs is similar to that in CD3ζ and CD28 / 41BB.

[0004] CD3ζ is part of the multimeric T cell antigen receptor (TCR) complex, which binds to antigens and converts the cross-plasma binding into intracellular signals. While the TCRαβ (or TCRγδ) subunit recognizes antigens through its specific extracellular domain, CD3ζ primarily performs its signaling function within the complex through its three well-conserved immunoreceptor-activated tyrosine motifs (ITAMs) (PMID:20516133). Identified for the first time based on their sequence homology, the ITAMs consist of two consecutive YxxL / I motifs (YxxL / IX) separated by a predetermined number of amino acids. 6~8 ITAMs consist of (-YxxL / I) (PMID:2927501). ITAMs are typically found as receptors expressed in hematopoietic cells and are particularly well studied in the context of TCR signaling. TCR binding to the peptide-MHC results in the activation of the Src family kinase Lck, which phosphorylates two tyrosine residues in each of the three ITAMs in CD3ζ (PMID:25861978). Each diphosphorylated ITAM then acquires the ability to bind to the two tandem SH2 domains of the Syk family kinase, ZAP-70. This interaction brings ZAP-70 closer to Lck, resulting in phosphorylation and activation of ZAP-70 by Lck. Activated ZAP-70 further phosphorylates its downstream targets, such as the adapter proteins LAT and SLP-76. Phosphorylated LAT and SLP-76 provide a scaffold for many other proteins, including PLC-γ, Grb2 / Sos, Gads, as well as Itk, Vav, and Nck, ultimately leading to calcium mobilization, Ras / Erk activation, actin cytoskeleton rearrangement, and ultimately, activation of gene expression (PMID:20516133). Therefore, the three ITAMs in CD3ζ are major, if not the only, signaling moieties in TCR signaling.

[0005] On the other hand, CD28 contains no ITAM whatsoever. Instead, its cytoplasmic domain contains a YMNM motif that, when phosphorylated upon binding of CD28 to its ligand CD80 / CD86, can bind to the p85 subunit of PI3K and Grb2 / Gads (PMID:20534709). Furthermore, the proline-rich region of CD28 can interact with Itk, Tec, Lck, Grb2 / Vav, and filamin A (PMID:20534709). Thus, TCR signaling initiated by antigen binding via CD28 signaling initiated by CD3ζ and CD80 / 86 binding shares many common players such as Grb2, Vav, Gads, Lck, and Itk, enabling crosstalk between these two pathways. Moreover, activation of both pathways occurs in signaling complexes that assemble near the plasma membrane at immunological synapses, physically bringing signaling molecules from the two pathways into the same space. Finally, CD28-induced calcium signaling occurs at least a few seconds after the intracellular calcium increase initiated by the TCR, reflecting the temporal proximity / closeness of the two pathways (PMID:18848472). [Overview of the project] [Means for solving the problem]

[0006] This disclosure provides a chimeric antigen receptor (CAR) that binds to an antigen of interest. The CAR can bind to a tumor antigen or a pathogen antigen. In certain non-limiting embodiments, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a modified CD3ζ polypeptide.

[0007] In certain embodiments, the modified CD3ζ polypeptide lacks all or part of the immunoreceptor-activating tyrosine motif (ITAM), where ITAM is ITAM1, ITAM2, and ITAM3. In certain embodiments, the modified CD3ζ polypeptide lacks ITAM2 or part thereof. In certain embodiments, the modified CD3ζ polypeptide further lacks ITAM3 or part thereof. In certain embodiments, the modified CD3ζ polypeptide further lacks ITAM1 or part thereof. In certain embodiments, the modified CD3ζ polypeptide lacks ITAM1 or part thereof. In certain embodiments, the modified CD3ζ polypeptide further lacks ITAM3 or part thereof. In certain embodiments, the modified CD3ζ polypeptide lacks ITAM3 or part thereof. In certain embodiments, the modified CD3ζ polypeptide includes a deletion of ITAM2 or part thereof. In certain embodiments, the modified CD3ζ polypeptide further includes a deletion of ITAM3 or part thereof. In certain embodiments, the modified CD3ζ polypeptide further includes a deletion of ITAM1 or part thereof. In certain embodiments, the modified CD3ζ polypeptide includes a deletion of ITAM1 or a portion thereof. In certain embodiments, the modified CD3ζ polypeptide further includes a deletion of ITAM3 or a portion thereof. In certain embodiments, the modified CD3ζ polypeptide includes a deletion of ITAM3 or a portion thereof.

[0008] In certain embodiments, the modified CD3ζ polypeptide further lacks all or part of the basic-rich stretch (BRS) region, where the BRS region is BRS1, BRS2, ​​and BRS3. In certain embodiments, the modified CD3ζ polypeptide lacks BRS2 or part thereof. In certain embodiments, the modified CD3ζ polypeptide further lacks BRS3 or part thereof. In certain embodiments, the modified CD3ζ polypeptide further lacks BRS1 or part thereof. In certain embodiments, the modified CD3ζ polypeptide lacks BRS1 or part thereof. In certain embodiments, the modified CD3ζ polypeptide further lacks BRS3 or part thereof. In certain embodiments, the modified CD3ζ polypeptide lacks BRS3 or part thereof. In certain embodiments, the modified CD3ζ polypeptide lacks BRS1 or part thereof, BRS2 or part thereof, and BRS3 or part thereof. In certain embodiments, the modified CD3ζ polypeptide includes a deletion of BRS2 or part thereof. In certain embodiments, the modified CD3ζ polypeptide further includes a deletion of BRS3 or a portion thereof. In certain embodiments, the modified CD3ζ polypeptide further includes a deletion of BRS1 or a portion thereof. In certain embodiments, the modified CD3ζ polypeptide includes a deletion of BRS1 or a portion thereof. In certain embodiments, the modified CD3ζ polypeptide further includes a deletion of BRS3 or a portion thereof. In certain embodiments, the modified CD3ζ polypeptide includes a deletion of BRS3 or a portion thereof. In certain embodiments, the modified CD3ζ polypeptide includes a deletion of BRS1 or a portion thereof, BRS2 or a portion thereof, and BRS3 or a portion thereof. In certain embodiments, the modified CD3ζ polypeptide includes a deletion of ITAM2, ITAM3, BRS2, ​​and BRS3. In certain embodiments, the modified CD3ζ polypeptide lacks ITAM2, ITAM3, BRS2, ​​and BRS3. In certain embodiments, CAR includes the amino acid sequence described in SEQ ID NO: 45 or SEQ ID NO: 47.

[0009] In certain embodiments, the modified CD3ζ polypeptide lacks all or part of the basic residue-rich extension (BRS) region, where the BRS region is BRS1, BRS2, ​​and BRS3. In certain embodiments, the modified CD3ζ polypeptide lacks BRS2 or part thereof. In certain embodiments, the modified CD3ζ polypeptide further lacks BRS3 or part thereof. In certain embodiments, the modified CD3ζ polypeptide further lacks BRS1 or part thereof. In certain embodiments, the modified CD3ζ polypeptide lacks BRS1 or part thereof. In certain embodiments, the modified CD3ζ polypeptide further lacks BRS3 or part thereof. In certain embodiments, the modified CD3ζ polypeptide lacks BRS3 or part thereof. In certain embodiments, the modified CD3ζ polypeptide lacks BRS1 or part thereof, BRS2 or part thereof, and BRS3 or part thereof.

[0010] In certain embodiments, the modified CD3ζ polypeptide comprises a BRS variant selected from the BRS1, BRS2, ​​and BRS3 variants, the BRS variant comprising one or more loss-of-function mutations.

[0011] In certain embodiments, any of the various CARs disclosed above further comprises a hinge / spacer region containing a CD8 polypeptide, CD28 polypeptide, CD3ζ polypeptide, CD40 polypeptide, 4-1BB polypeptide, OX40 polypeptide, CD84 polypeptide, CD166 polypeptide, CD8a polypeptide, CD8b polypeptide, ICOS polypeptide, ICAM-1 polypeptide, CTLA-4 polypeptide, CD27 polypeptide, CD40 / My88 peptide, NKGD2 peptide, or a combination thereof. In certain embodiments, the hinge / spacer region comprises a CD166 polypeptide. In certain embodiments, the hinge / spacer region comprises a CD166 polypeptide having amino acids 489-527 of SEQ ID NO: 3.

[0012] In certain embodiments, the transmembrane domain of any of the various CARs disclosed above comprises a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD40 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, a CD84 polypeptide, a CD166 polypeptide, a CD8a polypeptide, a CD8b polypeptide, an ICOS polypeptide, an ICAM-1 polypeptide, a CTLA-4 polypeptide, a CD27 polypeptide, a CD40 / My88 peptide, an NKGD2 peptide, or a combination thereof. In certain embodiments, the transmembrane domain comprises a CD166 polypeptide. In certain embodiments, the transmembrane domain comprises a CD166 polypeptide having amino acids 528-527 of SEQ ID NO: 3.

[0013] In certain embodiments, the transmembrane domain and the hinge / spacer region are derived from the same molecule. In certain embodiments, the hinge / spacer region contains the CD28 polypeptide, and the transmembrane domain contains the CD28 polypeptide. In certain embodiments, the hinge / spacer region contains the CD84 polypeptide, and the transmembrane domain contains the CD84 polypeptide. In certain embodiments, the hinge / spacer region contains the CD166 polypeptide, and the transmembrane domain contains the CD166 polypeptide. In certain embodiments, the CAR contains amino acids 489-553 of SEQ ID NO: 3.

[0014] In certain embodiments, the hinge / spacer region contains a CD8a polypeptide, and the transmembrane domain contains a CD8a polypeptide. In certain embodiments, the hinge / spacer region contains a CD8b polypeptide, and the transmembrane domain contains a CD8b polypeptide.

[0015] In certain embodiments, the transmembrane domain and the hinge / spacer region are derived from different molecules. In certain embodiments, the hinge / spacer region comprises the CD28 polypeptide, and the transmembrane domain comprises the ICOS polypeptide.

[0016] In certain embodiments, the intracellular signaling domain of any of the various CARs disclosed above further comprises a co-stimulatory signaling domain. In certain embodiments, the co-stimulatory signaling domain comprises a CD28 polypeptide.

[0017] The subject matter of this disclosure also provides a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a hinge / spacer region, a transmembrane domain, and an intracellular signaling domain comprising a modified CD3ζ polypeptide, wherein the modified CD3ζ polypeptide comprises an ITAM variant comprising one or more loss-of-function mutations, and the ITAM variant is selected from the group consisting of ITAM1 variant, ITAM2 variant, and ITAM3 variant. In certain embodiments, the hinge / spacer region comprises a CD8 polypeptide, CD28 polypeptide, CD3ζ polypeptide, CD40 polypeptide, 4-1BB polypeptide, OX40 polypeptide, CD84 polypeptide, CD166 polypeptide, CD8a polypeptide, CD8b polypeptide, ICOS polypeptide, ICAM-1 polypeptide, CTLA-4 polypeptide, CD27 polypeptide, CD40 / My88 peptide, NKGD2 peptide, or a combination thereof. In certain embodiments, the ITAM2 variant has the amino acid sequence described in SEQ ID NO: 29. In certain embodiments, the ITAM3 variant has the amino acid sequence described in SEQ ID NO: 33. In certain embodiments, CAR comprises the hinge / spacer region and the transmembrane domain of the CD166 polypeptide. In certain embodiments, CAR comprises amino acids 489-553 of SEQ ID NO: 3. In certain embodiments, the modified CD3ζ polypeptide has amino acids 374-485 of SEQ ID NO: 43. In certain embodiments, CAR has the amino acid sequence described in SEQ ID NO: 43.

[0018] The subject matter of this disclosure also provides immune-responsive cells containing the CARs disclosed herein. In certain embodiments, the CARs are recombinantly expressed. In certain embodiments, the CARs are expressed from a vector. In certain embodiments, the CARs are placed at an endogenous locus of the immune-responsive cell. In certain embodiments, the endogenous locus is the TRAC locus, the TRBC locus, or the TRGC locus. In certain embodiments, the endogenous locus is the TRAC locus. In certain embodiments, the placement of the CAR disrupts or inactivates the endogenous expression of the TCR.

[0019] The subject matter of this disclosure also provides immune-responsive cells comprising two or more CARs. In certain embodiments, the immune-responsive cell comprises: a) a first CAR comprising a first extracellular antigen-binding domain, a first transmembrane domain, and a first intracellular signaling domain for binding to a first antigen; and b) a second CAR comprising a second extracellular antigen-binding domain, a second transmembrane domain, and a second intracellular signaling domain for binding to a second antigen, wherein the first CAR is one of the CARs disclosed above, or the first intracellular signaling domain comprises a modified CD3ζ polypeptide comprising one or more ITAM variants comprising one or more loss-of-function mutations, each of which is independently selected from the group consisting of ITAM1 variant, ITAM2 variant, and ITAM3 variant. In certain embodiments, the first CAR further comprises a first hinge / spacer region. In certain embodiments, the second CAR further comprises a second hinge / spacer region. In certain embodiments, the first and second hinge / spacer regions can each be independently selected from any of the hinge / spacer regions disclosed herein.

[0020] In certain embodiments, the second CAR is the CAR disclosed above. In certain embodiments, the second intracellular signaling domain of the second CAR comprises a modified CD3ζ polypeptide comprising one or more ITAM variants containing one or more loss-of-function mutations, the ITAM variants being selected from the group consisting of ITAM1 variant, ITAM2 variant, and ITAM3 variant. In certain embodiments, the second intracellular signaling domain of the second CAR comprises a modified CD3ζ polypeptide that is the same as the modified CD3ζ polypeptide contained in the first intracellular signaling domain of the first CAR. In certain embodiments, the second intracellular signaling domain of the second CAR comprises a modified CD3ζ polypeptide that is different from the modified CD3ζ polypeptide contained in the first intracellular signaling domain of the first CAR. In certain embodiments, the second intracellular signaling domain of the second CAR comprises a native CD3ζ polypeptide.

[0021] In certain embodiments, the first intracellular signaling domain of the first CAR is the same as the second intracellular signaling domain of the second CAR. In certain embodiments, the first intracellular signaling domain of the first CAR is different from the second intracellular signaling domain of the second CAR.

[0022] In a particular embodiment, the first antigen is different from the second antigen.

[0023] In certain embodiments, the first intracellular signaling domain includes or has an ITAM2 variant and an ITAM3 variant, and the second intracellular signaling domain includes or has a deletion of ITAM2 or a part thereof and a deletion of ITAM3 or a part thereof.

[0024] In certain embodiments, the first intracellular signaling domain includes or has ITAM2 variants and ITAM3 variants, and the second intracellular signaling domain includes or has ITAM1 variants and ITAM2 variants.

[0025] In certain embodiments, the cell further comprises a third CAR including a third extracellular antigen-binding domain that binds to a third antigen, a third transmembrane domain, and a third intracellular signaling domain.

[0026] In certain embodiments, the first intracellular signaling domain includes or contains the ITAM2 variant and the ITAM3 variant, the second intracellular signaling domain includes or contains deletions of ITAM2 or a portion thereof and deletions of ITAM3 or a portion thereof, and the third intracellular signaling domain includes or contains the ITAM1 variant and the ITAM2 variant.

[0027] In certain embodiments, the first intracellular signaling domain includes or has a deletion of ITAM2 or a portion thereof and a deletion of ITAM3 or a portion thereof; the second intracellular signaling domain includes or has a deletion of ITAM2 or a portion thereof and a deletion of ITAM3 or a portion thereof; and the third intracellular signaling domain includes or has an ITAM1 variant and an ITAM2 variant.

[0028] In certain embodiments, the first intracellular signaling domain includes or has a deletion of ITAM2 or a portion thereof and a deletion of ITAM3 or a portion thereof; the second intracellular signaling domain includes or has an ITAM1 variant and an ITAM2 variant; and the third intracellular signaling domain includes or has an ITAM1 variant and an ITAM2 variant.

[0029] In certain embodiments, the first intracellular signaling domain includes or contains the ITAM1 variant and the ITAM2 variant, the second intracellular signaling domain includes or contains the ITAM1 variant and the ITAM2 variant, and the third intracellular signaling domain includes or contains the ITAM1 variant and the ITAM2 variant.

[0030] In certain embodiments, the cells are selected from a group consisting of T cells, natural killer (NK) cells, human embryonic stem cells, and pluripotent stem cells that can differentiate into lymphoid cells. In certain embodiments, the cells are T cells. In certain embodiments, the T cells are selected from a group consisting of cytotoxic T lymphocytes (CTLs), regulatory T cells, and natural killer T (NKT) cells. In certain embodiments, the immune-responsive cells are myeloid cells such as macrophages. In certain embodiments, the immune-responsive cells are autologous. In certain embodiments, the antigen is a tumor antigen. In certain embodiments, tumor antigens include CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, erb-B2, 3, 4, FBP, fetal acetylcholine receptor, folate receptor-a, GD2, GD3, HER-2, hTERT, IL-13R-a2, K-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, ERBB2, MAGEA3, p53, MART1, The antigen is selected from the group consisting of GP100, proteinase 3 (PR1), tyrosinase, survivorbin, hTERT, EphA2, NKG2D ligand, NY-ES0-1, fetal cancer antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, and CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME, CCR4, CD5, CD3, TRBC1, TRBC2, TIM-3, integrin B7, ICAM-1, CD70, Tim3, CLEC12A, and ERBB. In a particular embodiment, the antigen is CD19.

[0031] The subject matter of this disclosure further provides pharmaceutical compositions comprising an effective amount of immunoresponsive cells disclosed herein and pharmaceutically acceptable excipients. In certain embodiments, the pharmaceutical composition is for treating neoplasms.

[0032] The subject matter of this disclosure further provides a method for reducing tumor burden in a subject, comprising administering to the subject an effective amount of immune-responsive cells or a pharmaceutical composition disclosed herein. The subject matter of this disclosure also provides a method comprising administering to a subject an effective amount of immune-responsive cells or a pharmaceutical composition comprising the same, wherein the immune-responsive cells comprise a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a hinge / spacer region, a transmembrane domain, and an intracellular signaling domain comprising a modified CD3ζ polypeptide, the modified CD3ζ polypeptide comprises one or more ITAM variants comprising one or more loss-of-function mutations, and the one or more ITAM variants are each independently selected from the group consisting of ITAM1 variant, ITAM2 variant, and ITAM3 variant. In certain embodiments, the modified CD3ζ polypeptide comprises the ITAM2 variant and the ITAM3 variant. In certain embodiments, one or both of the ITAM2 variant and the ITAM3 variant comprise two loss-of-function mutations. In certain embodiments, the ITAM2 variant has the amino acid sequence described in SEQ ID NO: 29. In certain embodiments, the ITAM3 variant has the amino acid sequence described in SEQ ID NO: 33. In certain embodiments, one or more loss-of-function mutations are located at tyrosine amino acid residues. In certain embodiments, the intracellular signaling domain further comprises a co-stimulatory signaling domain. In certain embodiments, the co-stimulatory signaling domain comprises a CD28 polypeptide.

[0033] In certain embodiments, the method reduces the number of tumor cells. In certain embodiments, the method reduces the tumor size. In certain embodiments, the method eradicates the tumor in the subject.

[0034] The subject matter of this disclosure further provides methods for treating or preventing neoplasms. In certain embodiments, the method comprises administering to a subject an effective amount of an immune-responsive cell or pharmaceutical composition disclosed herein. In certain embodiments, the method comprises administering to a subject an effective amount of an immune-responsive cell or pharmaceutical composition comprising the immune-responsive cell comprising a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a hinge / spacer region, a transmembrane domain, and an intracellular signaling domain comprising a modified CD3ζ polypeptide, wherein the modified CD3ζ polypeptide comprises one or more ITAM variants comprising one or more loss-of-function mutations, and each of the one or more ITAM variants is independently selected from the group consisting of ITAM1 variant, ITAM2 variant, and ITAM3 variant.

[0035] The subject matter of this disclosure is a method for treating a subject having a recurrent neoplasm, wherein the subject receives immune-responsive cells comprising an antigen-recognizing receptor, and the antigen-recognizing receptor comprises a 4-1BB costimulatory signal. In certain embodiments, the method comprises administering to a subject an effective amount of immune-responsive cells or a pharmaceutical composition disclosed herein. In certain embodiments, the method comprises administering to a subject an effective amount of immune-responsive cells or a pharmaceutical composition comprising the same, wherein the immune-responsive cells comprise a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a hinge / spacer region, a transmembrane domain, and an intracellular signaling domain comprising a modified CD3ζ polypeptide, wherein the modified CD3ζ polypeptide comprises one or more ITAM variants comprising one or more loss-of-function mutations, and each of the one or more ITAM variants is independently selected from the group consisting of ITAM1 variant, ITAM2 variant, and ITAM3 variant. In certain embodiments, the intracellular signaling domain further comprises a costimulatory signaling domain. In certain embodiments, the costimulatory signaling domain comprises a CD28 polypeptide.

[0036] In certain embodiments, the neoplasm or tumor is selected from the group consisting of hematological cancers, B-cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, non-Hodgkin lymphoma, and ovarian cancer. In certain embodiments, the neoplasm is B-cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, or non-Hodgkin lymphoma, and the CAR binds to CD19. In certain embodiments, the neoplasm is CD19+ALL. In certain embodiments, the neoplasm comprises tumor cells having low levels of tumor-specific antigen expression.

[0037] The subject matter of this disclosure is a method for producing antigen-specific immune-responsive cells, further comprising introducing a nucleic acid sequence encoding a CAR disclosed herein into immune-responsive cells. In certain embodiments, the nucleic acid sequence is contained in a vector. In certain embodiments, the vector is a retroviral vector.

[0038] The subject matter of this disclosure further provides isolated nucleic acids (nucleotide acids) encoding CARs disclosed herein. In certain embodiments, the isolated nucleic acid is SEQ ID NO: 4 Further includes the nucleotide sequences described in 5 and SEQ ID NO: 47.

[0039] The subject matter of this disclosure further provides nucleic acid compositions comprising CARs disclosed herein. In certain embodiments, the nucleic acid sequence is contained in a vector. In certain embodiments, the vector is a retroviral vector.

[0040] The subject matter of this disclosure further provides vectors comprising nucleic acid compositions disclosed herein.

[0041] The subject matter of this disclosure further provides kits comprising CARs, immune-responsive cells, pharmaceutical compositions, nucleic acid compositions, or vectors disclosed herein. In certain embodiments, the kit includes written instructions for treating and / or preventing neoplasms, pathogen infections, autoimmune disorders, or allogeneic transplants. The present invention provides, for example, the following items: (Item 1) A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a modified CD3ζ polypeptide, wherein the modified CD3ζ polypeptide lacks all or part of an immunoreceptor-activating tyrosine motif (ITAM), and the ITAM is ITAM1, ITAM2, and ITAM3. (Item 2) The CAR described in item 1, wherein the modified CD3ζ polypeptide lacks ITAM2 or a portion thereof. (Item 3) The CAR described in item 2, further lacking ITAM3 or a portion thereof. (Item 4) The CAR described in item 2, further lacking ITAM1 or a portion thereof. (Item 5) The CAR described in item 1, wherein the modified CD3ζ polypeptide lacks ITAM1 or a portion thereof. (Item 6) The CAR described in item 5, further lacking ITAM3 or a portion thereof. (Item 7) The CAR described in item 1, wherein the modified CD3ζ polypeptide lacks ITAM3 or a portion thereof. (Item 8) The CAR described in item 1, wherein the modified CD3ζ polypeptide includes a deletion of ITAM2 or a portion thereof. (Item 9) The CAR described in item 8, wherein the modified CD3ζ polypeptide further comprises a deletion of ITAM3 or a portion thereof. (Item 10) The CAR described in item 8, wherein the modified CD3ζ polypeptide further comprises a deletion of ITAM1 or a portion thereof. (Item 11) The CAR described in item 1, wherein the modified CD3ζ polypeptide comprises a deletion of ITAM1 or a portion thereof. (Item 12) The CAR described in item 11, wherein the modified CD3ζ polypeptide further comprises a deletion of ITAM3 or a portion thereof. (Item 13) The CAR described in item 1, wherein the modified CD3ζ polypeptide comprises a deletion of ITAM3 or a portion thereof. (Item 14) The CAR according to any one of items 1 to 13, wherein the modified CD3ζ polypeptide lacks all or part of the basic residue-rich elongation (BRS) region, and the BRS region is BRS1, BRS2, ​​and BRS3. (Item 15) The CAR described in item 14, wherein the modified CD3ζ polypeptide lacks BRS2 or a portion thereof. (Item 16) The CAR described in item 15, further lacking BRS3 or a portion thereof. (Item 17) The CAR described in item 16, further lacking BRS1 or a portion thereof. (Item 18) The CAR described in item 14, wherein the modified CD3ζ polypeptide lacks BRS1 or a portion thereof. (Item 19) The CAR described in item 18, further lacking BRS3 or a portion thereof. (Item 20) The CAR described in item 14, wherein the modified CD3ζ polypeptide lacks BRS3 or a portion thereof. (Item 21) The CAR according to item 14, wherein the modified CD3ζ polypeptide lacks BRS1 or a portion thereof, BRS2 or a portion thereof, and BRS3 or a portion thereof. (Item 22) The CAR described in item 1, wherein the modified CD3ζ polypeptide lacks ITAM2, ITAM3, BRS2, ​​and BRS3. (Item 23) The CAR according to item 14, wherein the modified CD3ζ polypeptide comprises a deletion of BRS2 or a portion thereof. (Item 24) The CAR according to item 23, wherein the modified CD3ζ polypeptide further comprises a deletion of BRS3 or a portion thereof. (Item 25) The CAR according to item 24, wherein the modified CD3ζ polypeptide further comprises a deletion of BRS1 or a portion thereof. (Item 26) The CAR according to item 14, wherein the modified CD3ζ polypeptide comprises a deletion of BRS1 or a portion thereof. (Item 27) The CAR according to item 26, wherein the modified CD3ζ polypeptide further comprises a deletion of BRS3 or a portion thereof. (Item 28) The CAR according to item 14, wherein the modified CD3ζ polypeptide comprises a deletion of BRS3 or a portion thereof. (Item 29) The CAR according to item 14, wherein the modified CD3ζ polypeptide includes deletions of BRS1 or a portion thereof, BRS2 or a portion thereof, and BRS3 or a portion thereof. (Item 30) The CAR described in item 1, wherein the modified CD3ζ polypeptide includes deletions of ITAM2, ITAM3, BRS2, ​​and BRS3. (Item 31) The CAR described in item 1, comprising the amino acid sequence described in SEQ ID NO: 45 or SEQ ID NO: 47. (Item 32) A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a modified CD3ζ polypeptide, wherein the modified CD3ζ polypeptide lacks all or part of a basic residue-rich extension (BRS) region, and the BRS region is BRS1, BRS2, ​​and BRS3. (Item 33) The CAR described in item 32, wherein the modified CD3ζ polypeptide lacks BRS2 or a portion thereof. (Item 34) The CAR described in item 33, further lacking BRS3 or a portion thereof. (Item 35) The CAR described in item 34, further lacking BRS1 or a portion thereof. (Item 36) The CAR described in item 35, wherein the modified CD3ζ polypeptide lacks BRS1 or a portion thereof. (Item 37) The CAR described in item 36, further lacking BRS3 or a portion thereof. (Item 38) The CAR described in item 37, wherein the modified CD3ζ polypeptide lacks BRS3 or a portion thereof. (Item 39) The CAR according to item 32, wherein the modified CD3ζ polypeptide lacks BRS1 or a portion thereof, BRS2 or a portion thereof, and BRS3 or a portion thereof. (Item 40) A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a modified CD3ζ polypeptide, wherein the modified CD3ζ polypeptide comprises a BRS variant selected from a BRS1 variant, a BRS2 variant, and a BRS3 variant, and the BRS variant comprises one or more loss-of-function mutations. (Item 41) A CAR according to any one of items 1 to 40, further comprising a hinge / spacer region containing CD8 polypeptide, CD28 polypeptide, CD3ζ polypeptide, CD4 polypeptide, 4-1BB polypeptide, OX40 polypeptide, CD166 polypeptide, CD166 polypeptide, CD8a polypeptide, CD8b polypeptide, ICOS polypeptide, ICAM-1 polypeptide, CTLA-4 polypeptide, CD27 polypeptide, CD40 / My88 peptide, NKGD2 peptide, or a combination thereof. (Item 42) The CAR according to item 41, wherein the hinge / spacer region contains the CD166 polypeptide. (Item 43) The CAR described in item 42, wherein the CD166 polypeptide has amino acids 489-527 of SEQ ID NO: 3. (Item 44) A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a hinge / spacer region, a transmembrane domain, and an intracellular signaling domain including a modified CD3ζ polypeptide, The modified CD3ζ polypeptide comprises one or more ITAM variants containing one or more loss-of-function mutations, and each of the one or more ITAM variants is independently selected from the group consisting of ITAM1 variant, ITAM2 variant, and ITAM3 variant. The hinge / spacer region comprises CD8 polypeptide, CD28 polypeptide, CD3ζ polypeptide, CD4 polypeptide, 4-1BB polypeptide, OX40 polypeptide, CD166 polypeptide, CD166 polypeptide, CD8a polypeptide, CD8b polypeptide, ICOS polypeptide, ICAM-1 polypeptide, CTLA-4 polypeptide, CD27 polypeptide, CD40 / My88 peptide, NKGD2 peptide, or a combination thereof, in a CAR. (Item 45) The method according to item 44, wherein the modified CD3ζ polypeptide comprises an ITAM2 variant and an ITAM3 variant. (Item 46) The method according to item 45, wherein the ITAM2 variant has the amino acid sequence described in SEQ ID NO: 29. (Item 47) The method according to item 45 or 46, wherein the ITAM3 variant has the amino acid sequence described in SEQ ID NO: 33. (Item 48) The CAR according to any one of items 44 to 47, wherein the modified CD3ζ polypeptide contains or consists of amino acids 374-485 of SEQ ID NO: 43. (Item 49) A CAR as described in any one of items 44 to 48, including the amino acid sequence described in SEQ ID NO: 43. (Item 50) The method according to any one of items 45 to 49, wherein one or both of the ITAM2 variant and the ITAM3 variant contain two loss-of-function mutations. (Item 51) The method according to any one of items 44 to 50, wherein the one or more loss-of-function mutations are located in a tyrosine amino acid residue. (Item 52) The CAR according to any one of items 1 to 51, wherein the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, a CD166 polypeptide, a CD166 polypeptide, a CD8a polypeptide, a CD8b polypeptide, an ICOS polypeptide, an ICAM-1 polypeptide, a CTLA-4 polypeptide, a CD27 polypeptide, a CD40 / My88 peptide, an NKGD2 peptide, or a combination thereof. (Item 53) The CAR according to item 52, wherein the transmembrane domain contains the CD166 polypeptide. (Item 54) The CAR described in item 53, wherein the CD166 polypeptide contains amino acids 528-553 of SEQ ID NO: 3. (Item 55) The CAR according to any one of items 44 to 54, wherein the transmembrane domain and the hinge / spacer region originate from the same molecule. (Item 56) The CAR according to any one of items 44 to 55, wherein the hinge / spacer region comprises a CD28 polypeptide and the transmembrane domain comprises a CD28 polypeptide. (Item 57) The CAR according to any one of items 44 to 56, wherein the hinge / spacer region comprises a CD84 polypeptide and the transmembrane domain comprises a CD84 polypeptide. (Item 58) The CAR according to any one of items 44 to 57, wherein the hinge / spacer region comprises a CD166 polypeptide and the transmembrane domain comprises a CD166 polypeptide. (Item 59) CAR as described in item 58, containing amino acids 489-553 of SEQ ID NO: 3. (Item 60) The CAR according to any one of items 44 to 59, wherein the hinge / spacer region comprises a CD8a polypeptide and the transmembrane domain comprises a CD8a polypeptide. (Item 61) The CAR according to any one of items 44 to 60, wherein the hinge / spacer region comprises a CD8b polypeptide and the transmembrane domain comprises a CD8b polypeptide. (Item 62) The CAR according to any one of items 44 to 54, wherein the transmembrane domain and the hinge / spacer region are derived from different molecules. (Item 63) The CAR according to item 62, wherein the hinge / spacer region comprises a CD28 polypeptide and the transmembrane domain comprises an ICOS polypeptide. (Item 64) The CAR according to any one of items 1 to 63, wherein the intracellular signaling domain further comprises a co-stimulatory signaling domain. (Item 65) The CAR described in item 65, wherein the aforementioned co-stimulatory signaling domain contains a CD28 polypeptide. (Item 66) Immune-responsive cells containing CARs as described in any one of items 1 through 65. (Item 67) The immune-responsive cells described in item 66, wherein the aforementioned CAR is recombinantly expressed. (Item 68) The immune-responsive cells described in item 66 or 67, wherein the CAR is expressed from the vector. (Item 69) The immune-responsive cell according to any one of items 66 to 68, wherein the CAR is located at the endogenous gene locus of the immune-responsive cell. (Item 70) The immune-responsive cells described in item 69, wherein the endogenous gene locus is the TRAC locus, the TRBC locus, or the TRGC locus. (Item 71) The immune-responsive cells described in item 69 or 70, wherein the endogenous gene locus is the TRAC gene locus. (Item 72) An immune-responsive cell according to any one of items 69 to 71, wherein the arrangement of the CAR disrupts or inactivates the endogenous expression of the TCR. (Item 73) Immune-responsive cells as described in any one of items 66 to 72, selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, myeloid cells, human embryonic stem cells, and pluripotent stem cells capable of differentiating into lymphoid cells. (Item 74) The immune-responsive cells described in any one of items 66 to 73, wherein the immune-responsive cells are of the self. (Item 75) An immune-responsive cell as described in any one of items 66 to 74, wherein the antigen is a tumor antigen. (Item 76) The aforementioned tumor antigens include CD19, MUC16, MUC1, CAlX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, erb-B2, 3, 4, FBP, fetal acetylcholine receptor, folate receptor-a, GD2, GD3, HER-2, hTERT, IL-13R-a2, K-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, ERBB2, MAGEA3, p53, MART1, GP100, and proteinase. Immune-responsive cells as described in item 75, selected from the group consisting of 3(PR1), tyrosinase, survivorbin, hTERT, EphA2, NKG2D ligand, NY-ES0-1, fetal carcinoma antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, and CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME, CCR4, CD5, CD3, TRBC1, TRBC2, TIM-3, integrin B7, ICAM-1, CD70, Tim3, CLEC12A, and ERBB. (Item 77) The immune-responsive cells described in item 77, wherein the aforementioned antigen is CD19. (Item 78) A pharmaceutical composition comprising an effective amount of immune-responsive cells as described in any one of items 66 to 77, and a pharmaceutically acceptable excipient. (Item 79) A pharmaceutical composition as described in item 78, for the treatment of neoplasms. (Item 80) A method for reducing tumor burden in a subject, comprising administering to the subject an effective amount of immune-responsive cells described in any one of items 66 to 77 or a pharmaceutical composition described in item 78 or 79. (Item 81) A method for reducing tumor burden in a subject, comprising administering to the subject an effective amount of immune-responsive cells or a pharmaceutical composition containing them, wherein the immune-responsive cells A method comprising a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a modified CD3ζ polypeptide, wherein the modified CD3ζ polypeptide comprises one or more ITAM variants comprising one or more loss-of-function mutations, and each of the one or more ITAM variants is independently selected from the group consisting of ITAM1 variant, ITAM2 variant, and ITAM3 variant. (Item 82) The method described in item 81 or 82, which reduces the number of tumor cells. (Item 83) The method described in any one of items 81 to 83 for reducing tumor size. (Item 84) The method according to any one of items 81 to 84 for eradicating the tumor in the subject. (Item 85) A method for treating or preventing a neoplasm, comprising administering to a subject an effective amount of immune-responsive cells as described in any one of items 66 to 77 or a pharmaceutical composition as described in item 78 or 79. (Item 86) A method for treating or preventing a neoplasm, comprising administering to a subject an effective amount of immune-responsive cells or a pharmaceutical composition containing them, wherein the immune-responsive cells A method comprising a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a modified CD3ζ polypeptide, wherein the modified CD3ζ polypeptide comprises one or more ITAM variants comprising one or more loss-of-function mutations, and each of the one or more ITAM variants is independently selected from the group consisting of ITAM1 variant, ITAM2 variant, and ITAM3 variant. (Item 87) The method according to any one of items 80 to 86, wherein the neoplasm or tumor is selected from the group consisting of hematological cancers, B-cell leukemias, multiple myelomas, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemias, non-Hodgkin lymphomas, and ovarian cancers. (Item 88) The method according to any one of items 80 to 87, wherein the neoplasm is a B-cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, or non-Hodgkin lymphoma, and the CAR binds to CD19. (Item 89) The method according to any one of items 80 to 87, wherein the neoplasm is a B-cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, or non-Hodgkin lymphoma, and the CAR binds to BCMA, ADGRE2, MSLN, PSMA, or a combination thereof. (Item 90) A method for treating a subject having a relapse of a neoplasm, wherein the subject has a relapse of the disease, the subject has received treatment resulting in residual tumor cells, or the subject has received immune-responsive cells comprising an antigen-recognizing receptor, the antigen-recognizing receptor comprising a 4-1BB costimulatory signal, and the method comprises administering to the subject an effective amount of immune-responsive cells according to any one of items 66 to 77 or a pharmaceutical composition according to item 78 or 79. (Item 91) A method for treating a subject having a relapse of a neoplasm, wherein the subject has a relapse of the disease, the subject has received treatment resulting in residual tumor cells, or the subject has received immune-responsive cells comprising an antigen-recognizing receptor, the antigen-recognizing receptor comprising a 4-1BB costimulatory signal, and the method comprises administering to the subject an effective amount of immune-responsive cells or a pharmaceutical composition comprising them, the immune-responsive cells A method comprising a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a modified CD3ζ polypeptide, wherein the modified CD3ζ polypeptide comprises one or more ITAM variants comprising one or more loss-of-function mutations, and each of the one or more ITAM variants is independently selected from the group consisting of ITAM1 variant, ITAM2 variant, and ITAM3 variant. (Item 92) The method according to item 90 or 91, wherein the neoplasm is selected from the group consisting of hematological cancers, B-cell leukemias, multiple myelomas, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemias, and non-Hodgkin lymphomas. (Item 93) The method according to any one of items 90 to 92, wherein the neoplasm is a B-cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, or non-Hodgkin lymphoma, and the CAR binds to CD19. (Item 94) The method according to items 90 to 93, wherein the neoplasm is CD19+ ALL. (Item 95) The method according to items 90 to 94, wherein the neoplasm comprises tumor cells having a low density of tumor-specific antigens on the surface of the tumor cells. (Item 96) Immune-responsive cells as described in any one of items 66 to 77 or a pharmaceutical composition as described in item 78 or 79, for use in reducing tumor burden, treating or preventing neoplasms, and / or treating subjects with recurrent neoplasms. (Item 97) A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain including a modified CD3ζ polypeptide, for use in reducing tumor burden, treating or preventing neoplasms, and / or treating subjects with recurrent neoplasms, wherein the modified CD3ζ polypeptide comprises one or more ITAM variants comprising one or more loss-of-function mutations, and each of the one or more ITAM variants is independently selected from the group consisting of ITAM1 variant, ITAM2 variant, and ITAM3 variant. (Item 98) A method for producing antigen-specific immune-responsive cells, comprising introducing a nucleic acid sequence encoding a CAR as described in any one of items 1 to 65 into immune-responsive cells. (Item 99) The method according to item 98, wherein the nucleic acid sequence is contained in the vector. (Item 100) The method according to item 99, wherein the vector is a retroviral vector. (Item 101) An isolated nucleic acid encoding a CAR as described in any one of items 1 through 65. (Item 102) The isolated nucleic acid described in item 101, comprising the nucleotide sequences described in SEQ ID NOs. 45 and 47. (Item 103) A nucleic acid composition containing a CAR as described in any one of items 1 to 65. (Item 104) The nucleic acid composition according to item 103, wherein the nucleic acid sequence is contained in the vector. (Item 105) The nucleic acid composition according to item 104, wherein the vector is a retroviral vector. (Item 106) A vector comprising a nucleic acid composition as described in any one of items 103 to 105. (Item 107) A kit comprising a CAR as described in any one of items 1 to 65, an immune-responsive cell as described in any one of items 66 to 77, a pharmaceutical composition as described in item 78 or 79, a nucleic acid composition as described in any one of items 103 to 105, or a vector as described in item 106. (Item 108) The kit described in item 107, further including written instructions for treating and / or preventing neoplasms, pathogen infections, autoimmune disorders, or allogeneic transplants. (Item 109) an immune-responsive cell comprising: a) a first CAR comprising a first extracellular antigen-binding domain, a first transmembrane domain, and a first intracellular signaling domain that binds to a first antigen; and b) a second CAR comprising a second extracellular antigen-binding domain, a second transmembrane domain, and a second intracellular signaling domain that binds to a second antigen, wherein the first CAR is a CAR as described in any one of items 1 to 65, or the first intracellular signaling domain comprises a modified CD3ζ polypeptide comprising one or more ITAM variants containing one or more loss-of-function mutations, and each of the one or more ITAM variants is independently selected from the group consisting of ITAM1 variant, ITAM2 variant, and ITAM3 variant. (Item 110) The immune-responsive cell as described in item 109, wherein the second CAR is a CAR as described in any one of items 1 to 65. (Item 111) The immune-responsive cell according to item 109, wherein the second intracellular signaling domain of the second CAR comprises a modified CD3ζ polypeptide comprising one or more ITAM variants containing one or more loss-of-function mutations, and each of the one or more ITAM variants is independently selected from the group consisting of ITAM1 variant, ITAM2 variant, and ITAM3 variant. (Item 112) The immune-responsive cell according to item 109, comprising a modified CD3ζ polypeptide in which the second intracellular signaling domain of the second CAR is the same as the modified CD3ζ polypeptide contained in the first intracellular signaling domain of the first CAR. (Item 113) The immune-responsive cell according to item 109, wherein the second intracellular signaling domain of the second CAR contains a modified CD3ζ polypeptide that is different from the modified CD3ζ polypeptide contained in the first intracellular signaling domain of the first CAR. (Item 114) The immune-responsive cells described in item 109, wherein the second intracellular signaling domain of the second CAR contains a native CD3ζ polypeptide. (Item 115) An immune-responsive cell as described in any one of items 109 to 113, wherein the first intracellular signaling domain of the first CAR is the same as the second intracellular signaling domain of the second CAR. (Item 116) An immune-responsive cell as described in any one of items 109 to 114, wherein the first intracellular signaling domain of the first CAR is different from the second intracellular signaling domain of the second CAR. (Item 117) An immune-responsive cell as described in any one of items 109 to 116, wherein the first antigen is different from the second antigen. (Item 118) An immune-responsive cell according to any one of items 109 to 117, wherein the first CAR further comprises a first hinge / spacer region. (Item 119) An immune-responsive cell according to any one of items 109 to 118, wherein the second CAR further comprises a second hinge / spacer region. (Item 120) An immune-responsive cell according to any one of items 109 to 119, wherein the first intracellular signaling domain comprises or has an ITAM2 variant and an ITAM3 variant, and the second intracellular signaling domain comprises or has a deletion of ITAM2 or a part thereof and a deletion of ITAM3 or a part thereof. (Item 121) An immune-responsive cell according to any one of items 109 to 119, wherein the first intracellular signaling domain comprises or has an ITAM2 variant and an ITAM3 variant, and the second intracellular signaling domain comprises or has an ITAM1 variant and an ITAM2 variant. (Item 122) An immune-responsive cell according to any one of items 109 to 121, further comprising a third CAR including a third extracellular antigen-binding domain for binding to a third antigen, a third transmembrane domain, and a third intracellular signaling domain. (Item 123) The immune-responsive cell according to item 122, wherein the first intracellular signaling domain comprises or has an ITAM2 variant and an ITAM3 variant, the second intracellular signaling domain comprises or has a deletion of ITAM2 or a portion thereof and a deletion of ITAM3 or a portion thereof, and the third intracellular signaling domain comprises or has an ITAM1 variant and an ITAM2 variant. (Item 124) The immune-responsive cell according to item 122, wherein the first intracellular signaling domain includes or has a deletion of ITAM2 or a part thereof and a deletion of ITAM3 or a part thereof, the second intracellular signaling domain includes or has a deletion of ITAM2 or a part thereof and a deletion of ITAM3 or a part thereof, and the third intracellular signaling domain includes or has an ITAM1 variant and an ITAM2 variant. (Item 125) The immune-responsive cell according to item 122, wherein the first intracellular signaling domain includes or has a deletion of ITAM2 or a part thereof and a deletion of ITAM3 or a part thereof, the second intracellular signaling domain includes or has an ITAM1 variant and an ITAM2 variant, and the third intracellular signaling domain includes or has an ITAM1 variant and an ITAM2 variant. (Item 126) The immune-responsive cell according to item 122, wherein the first intracellular signaling domain comprises or contains the ITAM1 variant and the ITAM2 variant, the second intracellular signaling domain comprises or contains the ITAM1 variant and the ITAM2 variant, and the third intracellular signaling domain comprises or contains the ITAM1 variant and the ITAM2 variant. (Item 127) A pharmaceutical composition comprising an effective amount of immune-responsive cells as described in any one of items 109 to 126, and a pharmaceutically acceptable excipient. (Item 128) Immune-responsive cells as described in any one of items 109 to 126 or a pharmaceutical composition as described in item 127, for use in reducing tumor burden, treating or preventing neoplasms, and / or treating subjects with recurrent neoplasms. (Item 129) A method for reducing tumor burden in a subject, comprising administering to the subject an effective amount of immune-responsive cells described in any one of items 109 to 126 or a pharmaceutical composition described in item 127. (Item 130) The method according to item 129, which reduces the number of tumor cells in the subject, reduces the tumor size, and / or eradicates the tumor. (Item 131) A method for treating or preventing a neoplasm, comprising administering to a subject an effective amount of immune-responsive cells as described in any one of items 109 to 126 or a pharmaceutical composition as described in item 127. (Item 132) The method according to item 131, wherein the neoplasm or tumor is selected from the group consisting of hematological cancers, B-cell leukemias, multiple myelomas, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemias, non-Hodgkin lymphomas, and ovarian cancers. (Item 133) The method according to item 131 or 132, wherein the neoplasm is a B-cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, or non-Hodgkin lymphoma, and the CAR binds to CD19. (Item 134) The method according to any one of items 131 to 133, wherein the neoplasm is a B-cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, or non-Hodgkin lymphoma, and the CAR binds to BCMA, ADGRE2, MSLN, PSMA, or a combination thereof. (Item 135) A method for treating a subject having a relapse of a neoplasm, wherein the subject has a relapse of the disease, the subject has received treatment resulting in residual tumor cells, or the subject has received immune-responsive cells comprising an antigen-recognizing receptor, the antigen-recognizing receptor comprising a 4-1BB costimulatory signal, and the method comprises administering to the subject an effective amount of immune-responsive cells according to any one of items 109 to 126 or a pharmaceutical composition according to item 127. (Item 136) The method according to item 135, wherein the neoplasm is selected from the group consisting of hematological cancer, B-cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, and non-Hodgkin lymphoma. (Item 137) The method according to item 135 or 136, wherein the neoplasm is a B-cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, or non-Hodgkin lymphoma, and the CAR binds to CD19. (Item 138) The method according to items 135 to 137, wherein the neoplasm is CD19+ ALL. (Item 139) The method according to items 135 to 138, wherein the neoplasm comprises tumor cells having a low density of tumor-specific antigens on the surface of the tumor cells.

[0042] The following detailed descriptions are provided as examples, but are not intended to limit the subject matter of this disclosure to the specific embodiments described herein and should be understood in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0043] [Figure 1A] Figure 1 shows the cytoplasmic regions of wild-type, mutant, and truncated 1928ζ(1928z)CARs. A) Cytoplasmic regions of different 1928ζ(1928z)CARs. Mutations are made in the ζ chain of the original 1928ζ CAR to construct 1928ζ CAR T cells with a specific ζ signaling domain. The three ITAM motifs in the ζ domain are called ITAM1, ITAM2, and ITAM3, from proximal to distal of the membrane. In 1XX, X2X, XX3, and X23 CARs, two tyrosine residues (Y) in each ITAM are point-mutated to two phenylalanine residues (F) relative to the ITAM in question. In D12 and D23, deletion mutations are created to remove ITAM1 and ITAM2 (D12) or ITAM2 and ITAM3 (D23). B) Cytoplasmic region of 1928z CAR with improved antitumor efficacy compared to the original 1928z structure. In 1XX, the ζ chain of the original 1928ζ CAR has point mutations in ITAM2 and ITAM3 (conversion of tyrosine (T) to phenylalanine (F)), but the amino acid basic residue-rich elongation (BRS-1, -2, -3) remains. In D12 and D23, ITAM1 and ITAM2 (D12) or ITAM2 and ITAM3 (D23) are deleted; D23 contains one BRS region (BRS-1), while D12 lacks a BRS. C) Representative flow cytometry analysis showing the expression levels of CAR and LNGFR of 1928ζ and the 1928ζ mutants shown. UT: Untransduced T cells were used as a control. [Figure 1B] Same as above. [Figure 1C] Same as above.

[0044] [Figure 2A] Figures 2A-2B show the therapeutic efficacy of T cells containing novel CAR designs or control 1928ζ(1928z)CARs. The therapeutic efficacy of 1928ζ(1928z)CAR T cells is improved by mutations in the CD3ζ chain in the CAR construct. Tumor burden (mean radiance) (n=9-10, pooled data from two independent experiments) of mice with NALM-6 treated with 0.05 × 10⁶ CAR T cells. A) In vivo response of 1928ζ mutants with unmodified CD3ζ, ITAM3(XX3), ITAM2(X2X), or a combination of ITAM2 and ITAM3(X23) at the original CAR site, and mutations in the remaining ITAM motifs. B) Tumor burden in NALM-6 mice treated with a 1928ζ mutant consisting of a single non-mutant CD3ζ sequence at a primary position (ITAM3 relative to D12 and ITAM1 relative to D23) and deletions of the remaining ζ chain domains. [Figure 2B] Same as above.

[0045] [Figure 3A-B] Figures 3A–3B show the survival of CAR T cells expressing 1928ζ(1928z) control or novel CAR constructs after treatment with the same dose. Mice with Nalm-6 were treated with 5 × 10⁴ CAR+ T cells. A) and B) Kaplan-Meier survival analysis of mice comparing the in vivo efficacy of a single dose of 1928z WT and 1928z mutants XX3, X23 and X2X (A) or 1928z mutants D12, D23 and 1XX (B). Data pooled from two independent experiments, representing n=10 mice. Control refers to untreated mice (n=3). *p<0.05 (Log-Rank-Mantel-Cox test).

[0046] [Figure 4]Figure 4 shows the counting of CAR T cells in mice with tumors. Mice with NALM-6 were treated with 5 × 10⁴ CAR T cells (n=10 / group, pooled data from two independent experiments) and euthanized 17 days after infusion; bone marrow CAR T cells and NALM-6 cells were analyzed and counted by FACS. All data are mean ± SD. *P<0.05, ***P<0.001, ****P<0.0001 (unpaired Student's t-test).

[0047] [Figure 5A-B] Figures 5A-5C show the analysis of T cell differentiation. Phenotypes of CD4+ and CD8+ CAR T cells in mouse bone marrow 10 days after CAR injection (data are mean ± SEM, and each bar represents n=5 mice). A) Percentage of CD62L / CD45RA expression on CAR T cells of the indicated 1928ζ mutant compared to 1928ζ WT. B) Percentage of central memory (CD62L+CD45RA-) and C) effector cells (CD62L-CD45RA+) in CD4+ and CD8+ CAR T cells. Data compared to 1928ζ WT. **P<0.01, ***P<0.001 (unpaired Student's t-test). [Figure 5C] Same as above.

[0048] [Figure 6] Figure 6 shows the analysis of the cell counts of central memory CD4+ CAR T cells (Tells) (CAR+CD4+CD62L+CD45RA-) and IL7R-expressing CD4+ CAR T cells in the bone marrow of mice 10 and 17 days after T cell administration (data are mean ± SEM, and each bar represents n=5 mice). **P<0.01, ***P<0.001 (unpaired Student's t-test).

[0049] [Figure 7]Figure 7 shows the analysis of T cell depletion. Mice carrying NALM-6 were treated with 5 × 10⁴ CAR T cells (n=9-10 mice / group; data pooled from two independent experiments) and euthanized 10 days after CAR infusion; bone marrow CAR T cells were analyzed by FACS. The percentage of CD4+ and CD8+ CAR T cells expressing depletion markers was quantified by FACS 10 days after CAR infusion. Data are mean ± SEM, and each point represents one mouse. *P<0.05, **P<0.01, ***P<0.001 (unpaired Student's t-test).

[0050] [Figure 8] Figure 8 shows in vitro functional studies of CAR T cell efficacy. A) Cytotoxic activity determined by a 4-hour 51Cr release assay using EL4-CD19 as the target (n=2 independent experiments performed in 3 series, data are mean ± SEM). B) and C) Cytotoxic activity using an 18-hour bioluminescence assay with firefly luciferase (FFL)-expressing NALM-6 as the target. Data are mean ± SEM (n=5 independent experiments performed in 3 series). Experiments were performed one week after the enlargement of effector cells in irradiated 3T3-CD19.

[0051] [Figure 9A] Figure 9 shows the in vitro cytokine profile of CAR T cells. Percentages of CD4+ and CD8+ CAR T cells with positive expression of single-positive (A) and double-positive Th1 cytokines, determined by intracellular cytokine staining 18 hours after the second stimulation with 3T3-CD19 (data are mean ± SEM, compared to 1928ζ WT, paired Student's t-test, n=3-5 independent experiments). *P<0.05, **P<0.01. [Figure 9B] Same as above.

[0052] [Figure 10-1]Figure 10 shows schematic diagrams of 1XX CARs with different hinge (H) and transmembrane domain (TM) regions. Flow cytometry profiles show CAR and LNGFR expression using goat IgG anti-mouse IgG(F(ab')2) fragments and anti-LNGFR, respectively. A 1928z-LNGFR CAR containing the CD28 / CD28 H / TM region is used as a control. [Figure 10-2] Same as above. [Figure 10-3] Same as above.

[0053] [Figure 11] Figure 11 shows the cumulative CAR T cell count when CAR T cells were stimulated weekly, starting from 10⁶ cells / ml. Arrows indicate the time of stimulation. Data are mean ± SEM. n=3.

[0054] [Figure 12] Figure 12 shows in vitro cytotoxicity of CAR T cells. Cytotoxic activity of CAR T cells was measured using a 4-hour 51Cr release assay at the end of the third stimulation (D21). T cells and EL-4-CD19+ target cells were used in different effector:target ratios (E:T). Data are mean ± SEM. Data are representative of four independent experiments.

[0055] [Figure 13] Figure 13 shows the in vivo antitumor efficacy of different H / TM CAR T cells using NOD.Cg PrkdcscidIl2rgtm1Wjl / SzJ(NSG) mice. In the upper panel, tumor burden is tracked by weekly quantification of bioluminescence signals. CAR constructs are shown for each treatment. In the lower panel, Kaplan-Meier analysis of tumor-free survival of mice from the same experiment. The log-rank (Mantel-Cox) test is used for survival comparisons. n=7 mice / group.

[0056] [Figure 14]Figure 14 shows exemplary deimmunization strategies for novel CAR constructs. J1 and J2: No modification from the WT junction. J3: Substitution of Ser(S), the last amino acid of CD28, with Lys(K). J: Junction; X: Point mutation.

[0057] [Figure 15] Figure 15 shows various survival curves for mice treated with CAR T cells. NSG mice were injected into the tail vein with 5 × 10⁵ FFLuc-GFP NALM6 cells (pre-B ALL cell line), and 4 days later, 2 × 10⁵ 19BBz CAR T cells were injected. Ten days after the initial T cell injection (a non-effective dose that only slowed tumor progression), the mice were again injected with either 19BBz CAR T cells or 1928z (5 × 10⁵ cells / mouse). Arrows indicate CAR T cell injection times. In the upper panel, tumor burden is tracked by weekly quantification of bioluminescence signals. In the lower panel, Kaplan-Meier analysis of tumor-free survival in mice from the same experiment. The log-rank (Mantel-Cox) test is used for survival comparison. n=7 mice / group.

[0058] [Figure 16A]Figures 16A–16E show that 1928ζ iTAM differentially modulates the efficacy of CAR T cells. A) Cytoplasmic regions of wild-type and mutant 1928ζ CARs. The ζ chain of the 1928ζ CAR is mutated in one or two of its three ζ signaling domains, and these are referred to as ITAM1, ITAM2, and ITAM3 from proximal to distal membrane. In 1XX, X2X, XX3, and X23 CARs, two tyrosine (Y) molecules in each ITAM are point-mutated to two phenylalanine (F) molecules relative to the indicated ITAM. B) Flow cytometry analysis showing the expression levels of CAR and LNGFR for the constructs shown in A). Data are representative of at least five independent experiments with similar results. Non-transduced T (UT) cells were used as controls. C)–E) Mice with Nalm6 were treated with 5 × 10⁴ CAR+ T cells. C) Mouse tumor burden (mean radiance) is shown, comparing the in vivo efficacy of wild-type 1928ζ, 1XX, X2X, XX3, and X23 (n=10 mice / group, results pooled from two independent experiments). The control refers to untreated mice (n=6). D) CAR T cell count in mouse bone marrow 17 days after injection (results pooled from two independent experiments, n=10 mice / group). E) Phenotype of CAR T cells in mouse bone marrow 10 days after CAR injection, demonstrated by percentages of TCM and TEFF cells. Representative results from two independent experiments are shown (n=5 mice / group). All data are mean ± sem. In D) and E), p-values ​​were determined by a two-sided Mann-Whitney U test. [Figure 16B] Same as above. [Figure 16C] Same as above. [Figure 16D] Same as above. [Figure 16E] Same as above.

[0059] [Figure 17A-B]Figures 17A-17E show that the iTAM position within the 1928ζ CAR determines its antitumor efficacy. A) Cytoplasmic domain of the 1928ζ CAR with CD3ζ chain deletion. In D12, the deletion mutation removes ITAM1 and ITAM2, while in D23, ITAM2 and ITAM3 are removed. B) Flow cytometry analysis showing CAR and LNGFR expression levels for the constructs indicated. Data are representative of at least five independent experiments with similar results. UT, non-transduced T cells were used as a control. C)-E) Mice with Nalm6 were treated with 5 × 10⁴ CAR+ T cells. C) Tumor load (mean radiance) of mice treated with wild-type 1928ζ, D12, or D23 (wild-type 1928ζ and D23: n=10; D12: n=9, pooled data from two independent experiments). D) Number of CAR T cells in mouse bone marrow 17 days after injection (results pooled from two independent experiments, n=10 mice / group). E) Phenotype of CAR T cells in mouse bone marrow 10 days after CAR injection, as demonstrated by percentages of TCM and TEFF cells (data pooled from two independent experiments, n=10 mice / group). All data are mean ± sem. For D) and E), p-values ​​were determined by a two-sided Mann-Whitney U test. [Figure 17C] Same as above. [Figure 17D] Same as above. [Figure 17E] Same as above.

[0060] [Figure 18A-D]Figures 18A–18H show that TRAC-1XX enhances T cell efficacy by reducing T cell depletion and by inducing the development of long-lived memory T cells with effective recall responses. A)–D) Mice with Nalm6 were treated with 1 × 10⁵ or 5 × 10⁵ TRAC-CAR T cells. A) Kaplan-Meier survival analysis of mice treated with 1 × 10⁵ TRAC-1XX or TRAC-XX3 compared with TRAC-1928ζ (TRAC-1928ζ and TRAC-XX3: n=5 mice / group; TRAC-1XX: n=7). Control refers to untreated mice (n=3). P-values ​​were determined by a one-sided log-rank-Mantel-Cox test. B) Kaplan-Meier survival analysis of mice treated with 5 × 10⁵ cells (n=5 mice / group) or 1 × 10⁵ cells (TRAC-1928ζ: n=10 mice, TRAC-1XX: n=5 mice) of TRAC-CAR T cells. P-values ​​were determined by a one-sided log-rank-Mantel-Cox test. C) and D) Cell count (C) and expression of the depletion markers PD1+TIM3+LAG3+ on bone marrow CAR T cells (D) were determined for TRAC-1XX and TRAC-XX3 and compared with TRAC-1928ζ. Data are shown as mean ± sem, and each symbol refers to an individual mouse (n=5 mice / group). P-values ​​were determined by a two-sided Mann-Whitney U test. E)~H) Mice possessing Nalm6 were treated with 5 × 10⁵ TRAC-edited naive T cells, and Nalm6 cells (n=5 mice / group) were rechallenged as indicated by the arrows. No further tumor rechallenge was performed in the control group (TRAC-1928ζ: n=6 mice; TRAC-1XX: n=7 mice). E) Tumor burden (mean radiance) of mice comparing the in vivo efficacy of TRAC-1928ζ and TRAC-1XX after tumor rechallenge with no further rechallenge. All data are mean ± sem. A two-sided, unpaired Student's t-test was used for statistical analysis of tumor burden at 59 days post-CAR administration (TRAC-1928ζ: n=4; TRAC-1XX: n=5).F) and G) Number of total CAR T cells (F), TCM, TEFF, and IL7R+ CAR T cells (G) in the bone marrow of treated mice 63 days after CAR administration (with rechallenge: TRAC-1928ζ: n=4 mice, TRAC-1XX: n=5 mice; without rechallenge: n=5 mice / group). All data are mean ± sem; a two-sided, unpaired Student's t-test was used for statistical analysis. H) Expression of the depletion markers PD1+TIM3+LAG3+ on bone marrow CAR T cells. All data are mean ± sem. P-values ​​were determined by a two-sided, unpaired Student's t-test. [Figure 18E-F] Same as above. [Figure 18G-H] Same as above.

[0061] [Figure 19A-1]Figures 19A–19H show that the CD3ζ ITAM mutation in 1928ζ CAR establishes a distinctive transcriptional signature. Gene expression profiles of CD8+ TRAC-1928ζ, TRAC-1XX, and TRAC-XX3 CAR T cells (first selected naive T cells) 24 hours after stimulation with CD19+ target cells. A) Normalized enrichment scores of significantly upregulated or downregulated gene sets in comparisons of 1928ζ and 1XX and 1928ζ and XX3 (n=3 repeats / group) as determined by GSEA using the MSigDB C7 gene ontology set. For all pathways, the false discovery rate (FDR) q ≤ 0.02. GSE datasets are shown in parentheses. B) Differentially expressed genes (FDRq<0.05) (left) (n=6 repeats / group) between selected effector and naive / memory T cells, and heatmaps demonstrating the expression profiles of the same genes for CAR T cells (n=3 repeats / group). TF, transcription factor. C) Normalized enrichment scores (FDRq≦0.03) (n=3 repeats / group) of gene sets significantly enriched in relation to phenotypic and functional T cell characteristics, comparing TRAC-1928ζ and TRAC-XX3, TRAC-1928ζ and TRAC-1XX, and TRAC-1XX and TRAC-XX3, identified by GSEA analysis. JAK-STAT, Janus kinase signaling activator. D) Effects of defined CD3z ITAM mutations in 1928ζ CAR T cells on effector and memory-related T cell attributes. 1XX CAR T cells exhibit balanced effector and memory traits. [Figure 19A-2] Same as above. [Figure 19B-1] Same as above. [Figure 19B-2] Same as above. [Figure 19C] Same as above. [Figure 19D] Same as above.

[0062] [Figure 20A-C]Figures 20A–20E show the effects of the ITAM mutant 1928ζ CAR on in vitro T cell function, T cell differentiation, and in vivo antitumor activity. A) Cytotoxic activity determined by a 4-hour 51Cr release assay one week after the enlargement of effector cells on irradiated 3T3-CD19 (data are shown as mean values ​​from n=2 independent experiments performed in 3 series). B) Cumulative cell count of the indicated CAR T cells after weekly stimulation with CD19+ target cells (n=3 independent experiments). All data are mean ± sem. P-values ​​were calculated by two-sided paired Student's t-test. C) Mice with NALM6 were treated with 5 × 10⁴ CAR+ T cells. Kaplan-Meier survival analysis comparing the in vivo efficacy of wild-type 1928ζ or the indicated 1928ζ mutant (n=10 mice, pooled data from two independent experiments). The control group (Ctl) refers to untreated mice (n=6). P-values ​​were determined by a one-sided log-rank-Mantel-Cox test. D) Phenotype of CAR T cells demonstrated by the percentage of central memory (CD62L+CD45RA-) and effector memory (CD62L-CD45RA-) CD4+ CAR T cells 48 hours after the second stimulation with CD19+ target cells. A two-sided paired Student's t-test was performed, and the data represent the mean ± sem of n=4 independent experiments. E) Mice with NALM6 were treated with 5 × 10⁴ CAR T cells and euthanized 10 days after injection; bone marrow CAR T cells were analyzed by FACS. Representative flow cytometry analysis of the CAR T cell phenotype as indicated, gated for CAR+CD4+ T cells and determined by CD62L / CD45RA expression. Representative of 5 mice / group in at least n=2 independent experiments with similar results. [Figure 20D] Same as above. [Figure 20E] Same as above.

[0063] [Figure 21A-B]Figures 21A–21C show an analysis of effector function in the 1928ζ mutant compared to wild-type 1928ζ. A) Cytotoxic activity of the 1928ζ mutant compared to wild-type 1928ζ using an 18-hour bioluminescence assay with FFL-expressing NALM6 cells as a target. Experiments were performed one week after the enlargement of effector cells on CD19+ target cells. Data are mean ± sem (n=4 independent experiments performed in triplicates). P<0.05 (21:P=0.0273, 2-1:P=0.0387, 2-2:P=0.0125), **P=0.0018, calculated by a two-sided paired Student's t-test on the mean of the triplicates. B) and C) Granzyme B (GrB) expression on CD8+ CAR T cells (independent experiments of n=4) (B), and cytokine secretion from CD4+ and CD8+ CAR T cells after a second stimulation by CD19-expressing target cells (C). All data are mean ± sem (IFNγ and IL2, n=4; TNFα, n=5, independent experiments). Unstimulated wild-type 1928ζ cells were used as a control. Significant differences compared to 1928ζ were determined by two-sided paired Student's t-tests. [Figure 21C] Same as above.

[0064] [Figure 22]Figures 22A-22D show the effect of ITAM location within 1928ζ CAR on T cell function and therapeutic efficacy. A) Cytotoxic activity determined by a 4-hour 51Cr release assay one week after enlargement of effector cells on irradiated 3T3-CD19 (data are mean values ​​from n=2 independent experiments performed in 3 series). B) Cumulative cell count of notated CAR T cells stimulated weekly with CD19+ target cells (n=3 independent experiments). All data are mean ± sem; P-values ​​were calculated by two-sided paired Student's t-test. C) Cytotoxic activity of D12 and D23 compared to wild-type 1928ζ, determined by an 18-hour bioluminescence assay using FFL-expressing NALM6 cells as a target. Experiments were performed one week after enlargement of effector cells on CD19+ target cells. Data are mean ± sem (n=4 independent experiments performed in 3 series). P-values ​​were calculated by a two-sided paired Student's t-test on the mean of triplicates, and no significant difference was found between D12 / D23 and wild-type 1928ζ for any E / T ratio (P>0.05). D) Mice with NALM6 were treated with 5 × 10⁴ CAR T cells. Kaplan-Meier survival analysis of mice treated with wild-type 1928ζ or the 1928ζ mutant (n=10 mice / group). Controls refer to untreated mice (n=6). P-values ​​were calculated by a one-sided log-rank-Mantel-Cox test.

[0065] [Figure 23A] Figures 23A-23B show the effect of iTAM location within 1928ζ CAR cells on effector function in vitro. A) Granzyme B (GrB) expression on CD8+ CAR T cells (n=5 independent experiments). B) Cytokine secretion from CD4+ and CD8+ CAR T cells after second stimulation by CD19-expressing target cells. Unstimulated wild-type 1928ζ cells were used as a control. All data are mean ± sem (IFNγ and IL2, n=4; TNFα, n=5 independent experiments). Each individual symbol represents one sample. Significant differences compared to 1928ζ were determined by two-sided paired Student's t-test. [Figure 23B] Same as above.

[0066] [Figure 24A-B] Figures 24A–24F show T cell differentiation and effector function of the TRAC-coded 1928ζ mutant. Mice possessing NALM6 were treated with 1 × 10⁵ CAR T cells and euthanized 17 days after injection. CAR T cells from the bone marrow and spleen were analyzed and counted by FACS. A) Histogram and flow cytometry analysis of CAR expression 4 days after integration of the CAR gene into the TRAC locus. Representative of four independent experiments with similar results. B) Cell count of CD4+ and CD8+ CAR T cells. C) Percentage of CD8+ TCM (CD62L+CD45RA-) and flow cytometry analysis of CD62L / CD45RA expression on bone marrow CD8+ CAR T cells (representative of n=5 mice / group from one independent experiment). D) Ratio of CAR+IL7R+ to tumor cells in mouse bone marrow and exemplary flow cytometry analysis of IL7R+ CAR T cells. E) Counting of CAR T cells in mouse spleens. In B), C), D), and E), all data are mean ± sem, and two-sided Mann-Whitney analysis was performed. n=5 mice / group. F) Cytotoxic activity of TRAC-1XX, TRAC-XX3, and wild-type TRAC-1928ζ (18-hour bioluminescence assay with NALM6 expressing FFL as a target). Experiments were performed 4 days after transduction, and 1 and 3 weeks after weekly CD19 antigen stimulation for amplification. The symbols indicate the mean of 3 consecutive values ​​(one representative donor). [Figure 24C-D] Same as above. [Figure 24E-F] Same as above.

[0067] [Figure 25A]Figures 25A–25G show in vivo T cell depletion of TRAC-1928ζ mutants compared to wild-type TRAC-1928ζ. A) Mice with NALM6 were treated with 1 × 10⁵ CAR T cells and euthanized 17 days after injection. FACS analysis of depletion marker expression on CAR+ T cells, representative of n=5 mice / group in one independent experiment. B)–G) Mice with NALM6 were treated with 1 × 10⁵ TRAC-edited naive T cells. TRAC-1928ζ and TRAC-1XX cells from bone marrow and spleen were exposed to ex vivo stimulation with NALM6 or PMA / ionomycin (Iono) 16 days (B and C) and 36 days (E and G) after CAR administration. Cytokine and granzyme B (GrB) / CD107a expression on CAR T cells, demonstrated by percentage expression and flow cytometry analysis, representative of n=3 mice in two independent experiments (B) and n=3 replicates (G). Expression of the exhaustion marker PD1+LAG3+ on CAR T cells after 10 hours of co-culture with NALM6 (D) and cytotoxic activity of TRAC-1XX (day 36) (F). All data are mean ± sem, n=3 mice / group. [Figure 25B] Same as above. [Figure 25C] Same as above. [Figure 25D-E] Same as above. [Figure 25F] Same as above. [Figure 25G-1] Same as above. [Figure 25G-2] Same as above.

[0068] [Figure 26A-B]Figures 26A–26G show T cell memory formation in TRAC-1XX compared to wild-type TRAC-1928ζ. Mice with NALM6 were treated with 1 × 10⁵ or 5 × 10⁵ TRAC-edited naive T cells. A)–C) CARs were isolated from bone marrow and spleen 16 and 36 days after administration of 1 × 10⁵ TRAC-1928ζ and TRAC-1XX. A)–B) Cell counts of CAR T cells (A), central memory (TCM: CD62L+CD45RA-), effector (TEFF: CD62L-CD45RA+), and IL7R-expressing bone marrow CAR T cells (B). All data are mean ± sem. n = 3 mice / group. C) Representative flow cytometry analysis of CD62L / CD45RA expression on TRAC-1928ζ and TRAC-1XX bone marrow CAR T cells at day 36 in one independent experiment (n=3 mice / group). D)-G) Mice with NALM6 were treated with 5 × 10⁵ TRAC-edited naive T cells and either NALM6 cells were rechallenged (n=5 mice / group) or no further rechallenge of the tumor was performed (TRAC-1928ζ, n=6 mice; TRAC-1XX, n=7 mice). D)-E) Cell counts of total CAR T cells (D), TCM, TEFF and IL7R+ CAR T cells (E) in the spleen of treated mice 63 days after CAR administration (with rechallenge: TRAC-1928ζ, n=4 mice; TRAC-1XX, n=5 mice; without rechallenge: n=5 mice / group). All data are mean ± sem; two-tailed, unpaired Student's t-tests were used for statistical analysis. F) FACS analysis of IL7R, CD62L, and CD45RA expression on TRAC-1928ζ and TRAC-1XX CAR T cells 63 days after CAR infusion (representing at least n=3 mice / group in one independent experiment). G) Expression of the exhaustion markers PD1+TIM3+LAG3+ on spleen-derived CAR T cells (with rechallenge: TRAC-1928ζ, n=4 mice; TRAC-1XX: n=5 mice, without rechallenge, n=5 mice / group).All data are presented as mean ± s.e.m.; P values were determined by two-tailed unpaired Student's t-test. [Figure 26C] Same as above. [Figure 26D-E] Same as above. [Figure 26F-1] Same as above. [Figure 26F-2] Same as above. [Figure 26G] Same as above.

[0069] [Figure 27A] Figures 27A - 27C show the transcriptional profiles of the TRAC - coding 1928ζ variant and sorted control T cells. A) Principal component analysis (PCA) of the full transcriptional profiles of CD8+ TRAC - 1XX, TRAC - XX3, and TRAC - 1928ζ (left) and sorted control T cell subsets (right): TN, TSCM, and TEFF after stimulation with CD19 - target cells. Experiments were performed technically in triplicate for each CAR construct and six replicates for each control subset. B) Representative GSEA enrichment plots (GSE10239) demonstrate the down - regulation of memory genes compared to effector - related genes and naive genes compared to effector - related genes in the comparison of 1928ζ with 1XX and in the comparison of 1928ζ with XX3 (n = 3 mice / group). C) Heatmap of 900 differentially expressed genes in the CD8 T cell subsets described by Gattinoni et al. (left) compared to the differential gene expression of sorted control T cell subsets (TEFF, TSCM, and TN). [Figure 27B] Same as above. [Figure 27C] Same as above.

[0070] [Figure 28A-1]Figures 28A–28D show the effects of the CD3ζ ITAM mutation in TRAC-1928ζ on T cell differentiation state and effector profiles. A) GSEA of the top 200 gene signatures upregulated in exhausted CD8 T cells compared to naive or memory CD8 T cells derived from GSE41867 demonstrates enrichment of the exhausted signature in comparisons of TRAC-1928ζ with TRAC-1XX or TRAC-XX3, and with selected control TEFF with TN and TSCM. Experiments were performed in technical triplicates for each CAR construct and in 6 replicates for each control subset. B) Gene ontology analysis (n=3) demonstrating significantly enriched gene sets related to inflammation, cytokine, and chemokine signaling in comparisons of 1928ζ with XX3, 1XX with XX3, and 1928ζ with 1XX. CAR genes were incorporated into the TRAC locus of naive T cells, and transcriptional analysis was performed after stimulation with CD19+ target cells. Results are shown in order of significance as -log10 (corrected P-value). P-values ​​were determined by one-sided Fisher's exact test, and multiple hypothesis tests were corrected using the Benjamin-Hochberg method. C) Heatmap of differentially expressed genes selected among CAR constructs related to inflammation, cytokines, and chemokine activity. D) Flow cytometry analysis of T cell differentiation status in CD8+ CAR T cells after stimulation with CD19 antigen (representative of n=2 independent experiments with similar results). [Figure 28A-2] Same as above. [Figure 28A-3] Same as above. [Figure 28B] Same as above. [Figure 28C] Same as above. [Figure 28D] Same as above.

[0071] [Figure 29A]Figures 29A-29B show the gate setting strategy for analyzing CAR T cells obtained from the bone marrow of treated mice. A)-B) Representative flow cytometry analysis of TRAC-1928ζ(A) ​​compared with TRAC-1XX(B) 17 days after CAR injection. The gate setting configuration was based on FMO control. [Figure 29B] Same as above. [Modes for carrying out the invention]

[0072] The subject matter of this disclosure provides novel CAR designs comprising modified CD3ζ chains, and cells comprising genetically modified immune-responsive cells (e.g., T cells, NK cells, or CTL cells) comprising said CARs. The subject matter of this disclosure also provides methods of using such cells to induce and / or enhance an immune response to a target antigen, and / or to treat and / or prevent neoplasms or other diseases / disorders for which an increased antigen-specific immune response is desirable. The subject matter of this disclosure is at least in part based on the finding that immune-responsive cells comprising the CARs of this disclosure showed improved therapeutic efficacy (e.g., reduced cell waste) compared with control cells comprising conventional CARs.

[0073] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. The following references provide general definitions of many terms used in the subject matter of this disclosure: Singletonetal, Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, unless otherwise specified, the following terms have the meanings of the following:

[0074] As used herein, the terms “about” or “approximately” mean within an acceptable margin of error for a particular value as determined by those skilled in the art, and will depend in part on how that value is measured or determined, i.e., on the limits of the measurement system. For example, “about” may mean within three standard deviations or more than three standard deviations per practice in the art. Alternatively, “about” may mean a range of up to 20% of a given value, for example, up to 10%, up to 5%, or up to 1%. Or, particularly with respect to biological systems or processes, the term may mean within one order of magnitude of a value, for example, up to five times or up to two times.

[0075] "Activating immune-responsive cells" means inducing signaling or protein changes within cells that lead to the initiation of an immune response. For example, when CD3 chains cluster in response to ligand binding and immunoreceptor-suppressive tyrosine motifs (ITAMs), a signaling cascade is produced. In certain embodiments, when an endogenous TCR or exogenous CAR binds to an antigen, immunological synapse formation occurs near the bound receptor (e.g., CD4 or CD8, CD3γ / δ / ε / ζ, etc.), involving the clustering of many molecules. This clustering of membrane-bound signaling molecules leads to phosphorylation of the ITAM motif contained within the CD3 chain. This phosphorylation then initiates the T cell activation pathway, ultimately activating transcription factors such as NF-κB and AP-1. These transcription factors induce overall gene expression in T cells, increasing IL-2 production for the proliferation and expression of master regulatory T cell proteins, and initiating a T cell-mediated immune response.

[0076] "Stimulating immune-responsive cells" refers to signals that elicit a robust and sustained immune response. In various embodiments, this occurs after the activation of immune cells (e.g., T cells) or is simultaneously mediated via receptors including CD28, CD137(4-1BB), OX40, CD40, CD27, CD40 / My88, NKGD2, and ICOS, but is not limited to these. Receiving multiple stimulating signals may be crucial for initiating a robust and prolonged T-cell-mediated immune response. T cells can be rapidly inhibited and become unresponsive to antigens. While the effects of these co-stimulatory signals may vary, they generally result in increased gene expression to produce long-surviving, proliferating, and anti-apoptotic T cells that respond robustly to antigens for complete and sustained eradication.

[0077] As used herein, the term "antigen recognition receptor" refers to a receptor that can activate an immune or immunoreactive cell (e.g., a T cell) in response to its binding to an antigen. Non-limiting examples of antigen recognition receptors include natural or endogenous T cell receptors ("TCRs"), and chimeric antigen receptors ("CARs").

[0078] As used herein, the term "antibody" means not only intact antibody molecules but also fragments of antibody molecules that retain immunogen-binding ability. Such fragments are also well known in the art and are commonly used both in vitro and in vivo. Thus, as used herein, the term "antibody" means not only intact immunoglobulin molecules but also well-known active fragments F(ab')2, and Fab. Fragments F(ab')2 and Fab that lack the Fc fragment of the intact antibody may disappear more rapidly from the circulation and may have low non-specific tissue binding of the intact antibody (Wahletal., J. Nucl. Med. 24:316-325 (1983)). As used herein the term "antibody" includes whole native antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab', single-chain V region fragments (scFv), fusion polypeptides, and unconventional antibodies. In certain embodiments, an antibody is a glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as V H and a heavy chain constant (C H ) region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as V L and a light chain constant C L region. The light chain constant region consists of one domain, C L . The V H and V L regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) interspersed with regions called more conserved framework regions (FRs). Each V H and V LIt consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0079] As used herein, “CDR” is defined as the amino acid sequence of the complementarity-determining region of an antibody, which is the hypervariable region of the immunoglobulin heavy and light chains. See, for example, Kabatetal., Sequences of Proteins of Immunological Interest, 4th U.S. Department of Health and Human Services, National Institutes of Health (1987). Generally, an antibody contains three heavy chains and three light chain CDRs or CDR regions within the variable region. The CDRs provide the majority of the contact residues for the antibody's binding to an antigen or epitope. In certain embodiments, the CDR region is described using the Kabat system (Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).

[0080] As used herein, the terms “single-chain variable fragment” or “scFv” mean V H ::V L A heavy chain of immunoglobulin covalently linked to form a heterodimer (V H ) and light chain (V L It is a fusion protein of the variable region of ). H and V L They are either directly concatenated or VH The N-terminus of V L Connect to the C terminal of, or V H The C-terminus of V L The N-terminus of the protein is linked by a peptide-encoding linker (e.g., 10, 15, 20, or 25 amino acids). The linker is typically glycine-rich for flexibility and serine or threonine-rich for solubility. Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies are used to test for V, as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). H and V L To express from nucleic acids containing a sequence that codes for This is possible. See also U.S. Patents No. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Application Publications No. 20050196754 and 20050196754. The inhibitory antagonist scFv has been described (see, for example, Zhaoetal., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J CachexiaSarcopeniaMuscle 2012 August 12; Shieh et al., J Imunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife eta., J Clin Invst 2006 116(8):2252-61; Brockset al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 2(10:31-40)). The agonist scFv is described (see, for example, Peter et al., J Bioi Chern 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho etal., BioChim Biophys Acta 2003 1638(3):257-66).

[0081] As used herein, the term “affinity” means a measure of binding strength. Affinity may depend on the closeness of stereochemical fit between the antibody binding site and the antigenic determinant, the size of the contact area between them, and / or the distribution of charged and hydrophobic groups. As used herein, the term “affinity” also includes “avidity,” which refers to the strength of antigen-antibody binding after the formation of a reversible complex. Methods for calculating the affinity of an antibody to an antigen are known in the art and are not limited to, but include various antigen-binding experiments, such as functional assays (e.g., flow cytometry assays).

[0082] As used herein, the term “chimeric antigen receptor” or “CAR” refers to a molecule comprising an extracellular antigen-binding domain fused to an intracellular signaling domain capable of activating or stimulating immune-responsive cells, and a transmembrane domain. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an scFv. The scFv can be derived from fusing the variable heavy and light chain regions of an antibody. Alternatively, or further, the scFv may be derived from Fab' (obtained, for example, from a Fab library, instead of being derived from an antibody). In certain embodiments, the scFv is fused to the transmembrane domain and then to the intracellular signaling domain. In certain embodiments, the CAR is selected to have high binding affinity or avidity to the antigen.

[0083] As used herein, the term "nucleic acid molecule" includes any nucleic acid molecule encoding a polypeptide of interest or a fragment thereof. Such nucleic acid molecules need not be 100% identical or the same as the endogenous nucleic acid sequence, but may exhibit substantial identity. Polynucleotides having "substantial identity" or "substantial homology" to an endogenous sequence are typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. "Hybridize" means to form pairs between complementary polynucleotide sequences (e.g., the genes described herein), or portions thereof, under various stringency conditions. (See, e.g., Wahl, G.M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

[0084] For example, stringent salt concentrations will typically be less than about 750 mM NaCl and less than about 75 mM trisodium citrate, such as less than about 500 mM NaCl and less than about 50 mM trisodium citrate, or less than about 250 mM NaCl and less than about 25 mM trisodium citrate. Low stringency hybridization is obtained in the absence of organic solvents such as formamide, while high stringency hybridization is obtained in the presence of at least about 35% formamide, such as at least about 50% formamide. Stringent temperature conditions will typically include a temperature of at least about 30°C, at least about 37°C, or at least about 42°C. Additional varying parameters such as hybridization time, concentration of detergents such as sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA are well known to those of skill in the art. By combining these various conditions as appropriate, various levels of stringency are achieved. In certain embodiments, hybridization is performed at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In certain embodiments, hybridization is performed at 500 mM The hybridization is carried out at 37°C in NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA). In certain embodiments, hybridization is carried out at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations of these conditions will be readily apparent to those skilled in the art.

[0085] For many applications, the stringency will also change during the washing step after hybridization. Washing stringency conditions can be defined by salt concentration and temperature. As mentioned above, washing stringency can be increased by decreasing the salt concentration or increasing the temperature. For example, a stringent salt concentration for the washing step may be less than about 30 mM NaCl and less than 3 mM trisodium citrate, e.g., less than about 15 mM NaCl and less than 1.5 mM trisodium citrate. Stringent temperature conditions for the washing step will typically include temperatures of at least about 25°C, at least about 42°C, or at least about 68°C. In certain embodiments, the washing step is performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In certain embodiments, the washing step is performed at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In certain embodiments, the washing step is carried out at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations of these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, by Benton and Davis (Science 196:180, 1977); Grunstein and Rogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York. It is.

[0086] "Substantially identical" or "substantially homologous" means a polypeptide or nucleic acid molecule that exhibits at least about 50% homology or identity with a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). In certain embodiments, such a sequence is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% homology or identity with the amino acid or nucleic acid sequence used for comparison.

[0087] Sequence identity can be verified using sequence analysis software (e.g., Sequence Analysis). This can be measured by using the Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705 (BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine substitutions. An exemplary method for determining the degree of identity is to use the BLAST program with probability scores from e-3 to e-100 indicating closely related sequences.

[0088] "Analog" refers to a structurally related polypeptide or nucleic acid molecule that has the function of a reference polypeptide or nucleic acid molecule.

[0089] As used herein, the term "ligand" refers to a molecule that binds to a receptor. In certain embodiments, a ligand binds to a receptor on another cell, enabling intercellular recognition and / or interaction.

[0090] As used herein, the terms “constitutive expression” or “to be constitutively expressed” mean expression or being expressed under any physiological conditions.

[0091] "Disease" means any condition, disease, or disorder that damages or interferes with the normal function of a cell, tissue, or organ, such as neoplasms and pathogenic infections of cells.

[0092] "Effective dose" means an amount sufficient to have a therapeutic effect. In certain embodiments, the "effective dose" is an amount sufficient to stop, improve, or inhibit the continued proliferation, growth, or metastasis (e.g., invasion or migration) of a neoplasm.

[0093] "Forcing tolerance" means preventing the activation of autoreactive or immune-responsive cells that target the transplanted organ or tissue.

[0094] "Endogenous" refers to nucleic acid molecules or polypeptides that are normally expressed in cells or tissues.

[0095] "Exogenous" means a nucleic acid molecule or polypeptide that is not endogenously present in the cell. Therefore, the term "exogenous" encompasses any recombinant nucleic acid molecule or polypeptide expressed in a cell, including foreign, heterogeneous, overexpressed nucleic acid molecules and polypeptides. "Exogenous" nucleic acid means a nucleic acid that is not present in natural wild-type cells; for example, an exogenous nucleic acid may differ from its endogenous counterpart by sequence, location / situation, or both. For clarity, an exogenous nucleic acid may have the same or different sequences compared to its natural endogenous counterpart; it may be introduced into the cell itself or its precursor cells by genetic engineering and, if necessary, ligated to an alternative regulatory sequence, such as a non-natural promoter or secretory sequence.

[0096] "Heterogeneous nucleic acid molecules or polypeptides" means nucleic acid molecules (e.g., cDNA, DNA, or RNA molecules) or polypeptides that are not normally present in cells or samples obtained from cells. These nucleic acids may originate from another organism, or they may be, for example, mRNA molecules that are not normally expressed in cells or samples.

[0097] "Immune-responsive cells" refer to cells that function in the immune response, or their progenitor cells or offspring.

[0098] "Modulating" means changing something in a positive or negative direction. Examples of modulation include changes of approximately 1%, 2%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0099] "Increase" means a positive change of at least approximately 5%. The change may be approximately 5%, 10%, 25%, 30%, 50%, 75%, 100%, or higher.

[0100] "Decrease" means a negative change of at least about 5%. The change may be about 5%, 10%, 25%, 30%, 50%, 75%, or even 100%.

[0101] "Isolated cells" refers to cells that have been separated from the molecules and / or cellular components that naturally accompany them.

[0102] The terms “isolated,” “purified,” or “biologically pure” refer to material that contains, to the extent that it alters the components normally associated with it as found in its natural state. “Isolating” indicates the degree of separation from the original source or its surroundings. “Purifying” indicates a higher degree of separation than isolation. A “purified” or “biologically pure” protein is free from other material to such an extent that impurities do not substantially affect the protein’s biological properties or cause other harmful consequences. That is, a nucleic acid or peptide is purified if it is substantially free from cellular material, viral material, or culture medium if produced by recombinant DNA technology, or from chemical precursors or other chemicals if chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, e.g., polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term “purified” may also indicate that the nucleic acid or protein produces essentially one band in the electrophoretic gel. For proteins that can be modified, e.g., phosphorylated or glycosylated, different modifications may produce different isolated proteins that can be purified separately.

[0103] As used herein, the term "antigen-binding domain" refers to a domain that can specifically bind to a particular antigenic determinant or set of antigenic determinants present on a cell.

[0104] As used herein, “linker” should mean a functional group (e.g., a chemical or polypeptide) that covalently links two or more polypeptides or nucleic acids so that they are linked to one another. As used herein, “peptide linker” means a functional group (e.g., a peptide linker) that couples two proteins together (e.g., a peptide linker). H and VL This refers to one or more amino acids used to couple domains. In certain embodiments, the linker includes the sequence described in GGGGSGGGGSGGGGS (SEQ ID NO: 66).

[0105] "Neoplasm" refers to a disease characterized by the pathological proliferation of cells or tissues and their subsequent migration or invasion into other tissues or organs. Neoplasmic proliferation is typically uncontrolled, progressive, and occurs under conditions that do not induce or cause the cessation of replication of normal cells. Neoplasms can affect a variety of cell types, tissues, or organs, including, but are not limited to, organs selected from the group consisting of the bladder, bone, brain, breast, cartilage, glia, esophagus, fallopian tubes, gallbladder, heart, intestines, kidneys, liver, lungs, lymph nodes, nerve tissue, ovaries, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urogenital tract, ureters, urethra, uterus, and vagina, or their tissues or cell types. Neoplasms include cancers such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells).

[0106] A "receptor" refers to a polypeptide, or a part thereof, located on the cell membrane that selectively binds to one or more ligands.

[0107] "Recognizing" means selectively binding to a target. T cells that recognize tumors can express receptors (e.g., TCRs or CARs) that bind to tumor antigens.

[0108] "Reference" or "control" refers to the standard of comparison. For example, the level of scFv-antigen binding in cells expressing both CAR and scFv can be compared to the level of scFv-antigen binding in corresponding cells expressing only CAR.

[0109] "Secreted" refers to polypeptides that are released from a cell via secretory pathways through the endoplasmic reticulum and Golgi apparatus, and as vesicles that transiently fuse at the cell plasma membrane, releasing proteins to the outside of the cell.

[0110] A "signal sequence" or "leader sequence" refers to a peptide sequence (e.g., 5, 10, 15, 20, 25, or 30 amino acids) located at the N-terminus of a newly synthesized protein that directs its entry into the secretory pathway. Exemplary leader sequences include, but are not limited to, IL-2 signal sequences: MYRMQLLSCIALSLALVTNS (SEQ ID NO: 67) (human), MYSMQLASCVTLTLVLLVNS (SEQ ID NO: 68) (mouse); kappa leader sequences: METPAQLLFLLLLWLPDTTG (SEQ ID NO: 69) (human), METDTLLLWVLLLWVPGSTG (SEQ ID NO: 70) (mouse); CD8 leader sequence: MALPVTALLLPLALLLHAARP (SEQ ID NO: 71) (human); truncated human CD8 signal peptide: MALPVTALLLPLALLLHA (SEQ ID NO: 72) (human); albumin signal sequence: MKWVTFISLLFSSAYS (SEQ ID NO: 73) (human); and prolactin signal sequence: MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS (SEQ ID NO: 74) (human). In certain embodiments, the leader sequence may be an IgG signal peptide or a GM-CSF signal peptide. "Soluble" means a polypeptide that can freely diffuse in an aqueous environment (for example, one that is not bound to a membrane).

[0111] "Specifically binding" means a polypeptide or fragment thereof that recognizes and binds to a target biomolecule (e.g., a polypeptide), but substantially does not recognize or bind to other molecules in a sample, e.g., a biological sample naturally containing the polypeptide of this disclosure.

[0112] As used herein, the term “tumor antigen” refers to an antigen (e.g., polypeptide) that is uniquely or differentially expressed on tumor cells compared to normal or non-IS neoplastic cells. In certain embodiments, the tumor antigen includes any polypeptide expressed by a tumor that can activate or induce an immune response via CAR (e.g., CD19, MUC-16) or suppress an immune response via receptor-ligand binding (e.g., CD47, PD-L1 / L2, B7.1 / 2).

[0113] The terms "comprises" and "comprising" are intended to have a broad meaning in U.S. patent law, and may also mean "includes" and "including."

[0114] As used herein, “treatment” refers to a clinical intervention in an attempt to alter the course of a disease in an individual or cell being treated, and may be performed for preventive purposes or during the course of a clinicopathological condition. The therapeutic effects of treatment include, but are not limited to, prevention of disease onset or recurrence, reduction of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of the rate of disease progression, improvement or mitigation of the disease state, and remission or improvement of prognosis. By preventing the progression of disease or disability, treatment can not only prevent the exacerbation caused by the disability in a subject who is affected or diagnosed with the disability or suspected to have the disability, but also prevent the onset of the disability or symptoms of the disability in a subject who is at risk of disability or suspected to have the disability.

[0115] In this specification, “individual” or “subject” refers to a human or a non-human animal, such as a vertebrate, including mammals. Mammals include, but are not limited to, humans, primates, livestock, sport animals, rodents, and pets. Non-exclusive examples of non-human animal subjects include rodents such as mice, rats, hamsters, and guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cattle, horses, and non-human primates such as apes and monkeys. The term “immunely vulnerable” as used herein refers to a subject that is immunocompromised. Such a subject is highly vulnerable to opportunistic infections, which are infections caused by organisms that do not normally cause disease in individuals with a healthy immune system but can affect individuals with a poorly functioning or suppressed immune system.

[0116] Other aspects of the subject matter of this disclosure are described in the following disclosures and are within the scope of the subject matter of this disclosure.

[0117] 2. Chimeric antigen receptor This disclosure provides a chimeric antigen receptor (CAR) that binds to an antigen of interest. The CAR can bind to a tumor antigen or a pathogen antigen.

[0118] 2.1. Antigen In certain embodiments, the CAR binds to a tumor antigen. Any tumor antigen (antigenic peptide) can be used in the tumor-related embodiments described herein. Sources of antigens include, but are not limited to, oncoproteins. The antigen can be expressed as a peptide, or as part of a intact protein. The intact protein or part thereof may be naturally occurring or mutagenic.Non-limiting examples of tumor antigens include carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, CD123, CD44V6, antigens of cells infected with cytomegalovirus (CMV) (e.g., cell surface antigens), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), and epithelial cell adhesion Molecules (EpCAM), receptor tyrosine protein kinases erb-B2, 3, 4 (erb-B2, 3, 4), folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-α, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ light chain, kinase insertion domain receptor (KDR), Lewis Y (LeY), L1 cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), mucin 16 (MUC16), mucin 1 (MUC1), mesothelin (MSLN), ERBB2, MAGEA3, p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivorbin, hTERT, EphA2, NKG2D ligand, cancer testicular antigen NY-ES0-1, cancer fetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen ( Examples include PSMA, ROR1, tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), and Wilms oncoprotein (WT-1), BCMA, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME, CCR4, CD5, CD3, TRBC1, TRBC2, TIM-3, integrin B7, ICAM-1, CD70, Tim3, CLEC12A, and ERBB.

[0119] In certain embodiments, the CAR binds to the CD19 polypeptide. In certain embodiments, the CAR binds to the human CD19 polypeptide. In certain embodiments, the human CD19 polypeptide contains the amino acid sequence described in SEQ ID NO: 75.

[0120] [ka]

[0121] In a particular embodiment, CAR binds to the extracellular domain of the CD19 protein.

[0122] In certain embodiments, CARs bind to pathogenic antigens for use, for example, in the treatment and / or prevention of pathogenic infections or other infectious diseases in immunocompromised subjects. Non-limiting examples of pathogens include viruses, bacteria, fungi, parasites, and protozoa that can cause disease.

[0123] Non-exclusive examples of viruses include Retroviridae (e.g., human immunodeficiency viruses such as HIV-1 (also known as HDTV-III, LAVE, or HTLV-III / LAV) or HIV-III, and other isolated strains such as HIV-LP); Picornaviridae (e.g., poliovirus, hepatitis A virus; enterovirus, human coxsackievirus, rhinovirus, echovirus); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flavi idae (e.g., dengue virus, encephalitis virus, yellow fever virus), Coronaviridae (e.g., coronavirus), Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus), Filoviridae (e.g., Ebola virus), Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, polynuclear respiratory virus), Orthomyxoviridae (e.g., influenza virus), Bunyaviridae (e.g., Hantan virus, Bunya virus, Fre Boviruses and Nairo (Naira) viruses, Arenaviridae (hemorrhagic fever viruses) Reoviruses, orbiviruses (for example, reoviruses, orbiviruses) Examples include virulence factors for hepatitis delta (and rotavirus), Birnaviridae, Hepadnaviridae (hepatitis B virus), Parvoviridae (parvovirus), Papovaviridae (papillomavirus, polyomavirus), Adenoviridae (most adenoviruses), Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpesvirus), Poxviridae (smallpox virus, vaccinia virus, poxvirus), and Iridoviridae (e.g., African swine fever virus), as well as unclassified viruses (e.g., virulence factors for hepatitis delta (considered to be a deficient satellite of hepatitis B virus), virulence factors for non-A, non-B hepatitis (Class 1 = internally transmitted, Class 2 = parenterally transmitted (i.e., hepatitis C), Norwalk and related viruses, and astroviruses)).

[0124] Non-specific examples of bacteria include Pasteurella, Staphylococcus, Streptococcus, Escherichia coli, Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria, though not limited to these, include Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), and Streptococcus coccus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus antracis, Corynebacterium diphtheriae, Corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia and Actinomyces israelli.

[0125] In certain embodiments, the pathogen antigen is a viral antigen present in cytomegalovirus (CMV), a viral antigen present in Epstein-Barr virus (EBV), a viral antigen present in human immunodeficiency virus (HIV), or a viral antigen present in influenza virus.

[0126] 2.2. Chimeric antigen receptors (CARs) CARs are engineered receptors that transplant or confer desired specificity to immune effector cells. Using CARs, the specificity of monoclonal antibodies can be transplanted to T cells, along with the transfer of their coding sequences facilitated by retroviral vectors.

[0127] Three generations of CARs exist. "First-generation" CARs typically consist of a transmembrane domain fused to a cytoplasmic / intracellular signaling domain and an extracellular antigen-binding domain (e.g., scFv). "First-generation" CARs provide de novo antigen recognition and, independently of HLA-mediated antigen presentation, transmit CD4 ζ-chain signaling domains within a single fusion molecule. + and CD8 + It can induce activation of both T cells. "Second-generation" CARs provide further signaling to T cells by adding intracellular signaling domains derived from various co-stimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40, CD27, CD40 / My88, and NKGD2) to the cytoplasmic tail of the CAR. "Second-generation" CARs include those that provide both co-stimulation (e.g., CD28 or 4-1BB) and activation (CD3ζ). "Third-generation" CARs include those that provide multiple co-stimulations (e.g., CD28 and 4-1BB) and activation (CD3ζ). In certain embodiments, the CAR is a second-generation CAR. In certain embodiments, the CAR includes an extracellular antigen-binding domain that binds to an antigen, a transmembrane domain, and an intracellular signaling domain, the intracellular signaling domain including a co-stimulatory signaling domain. In certain embodiments, the CAR further includes a hinge / spacer region.

[0128] In certain non-limiting embodiments, the extracellular antigen-binding domain of the CAR (embodied, for example, as scFv or its analogue) is approximately 2 × 10⁻⁶ -7 M or a dissociation constant less than (K) d ) binds to the antigen. In a particular embodiment, K d It is approximately 2 x 10 -7 M or less, approximately 1 x 10 -7 M or smaller, approximately 9 x 10 -8 M or less, approximately 1 x 10 -8 M or smaller, approximately 9 x 10 -9 M or less, approximately 5 x 10 -9 M or less, approximately 4 x 10 -9M or less, approximately 3 x 10 -9 M or less, approximately 2 x 10 -9 M or less, or approximately 1 × 10 -9 M or less. In certain non-limiting embodiments, K d It is approximately 3 x 10 -9 M or less. In certain non-limiting embodiments, K d It is approximately 1 x 10 -9 M ~ approx. 3×10 -7 M is M. In certain non-limiting embodiments, K d It is approximately 1.5 × 10 -9 M ~ approx. 3×10 -7 M is M. In certain non-limiting embodiments, K d It is approximately 1.5 × 10 -9 M ~ approx. 2.7×10 -7 M is M. In certain non-limiting embodiments, K d It is approximately 1 x 10 -4 M ~ approx. 1×10 -6 M is M. In certain non-limiting embodiments, K d It is approximately 1 x 10 -13 M ~ approx. 1×10 -15 It is M.

[0129] The binding of the extracellular antigen-binding domain (e.g., in scFv or its analogues) can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot assay. Each of these assays typically detects the presence of a specific target protein-antibody complex by using a reagent (e.g., antibody, or scFv) that is specific to the complex of interest. For example, scFv can be radiolabeled and used in radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on RadioligandAssayTechniques, The Endocrine Society, March, 1986, incorporated herein by reference). Radioisotopes can be detected by means such as the use of a gamma counter or scintillation counter, or by autoradiography. In certain embodiments, the extracellular antigen-binding domain of CAR is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalama1), cyan fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet).

[0130] 2.2.1. Extracellular antigen-binding domain of CAR In certain embodiments, the extracellular antigen-binding domain specifically binds to the antigen. In certain embodiments, the extracellular antigen-binding domain is scFv. In certain embodiments, scFv is human scFv. In certain embodiments, scFv is humanized scFv. In certain embodiments, scFv is mouse scFv. In certain embodiments, the extracellular antigen-binding domain is Fab, which is crosslinked as needed. In certain embodiments, the extracellular antigen-binding domain is F(ab)2. In certain embodiments, any of the above molecules may be contained in a fusion protein with heterologous sequences forming the extracellular antigen-binding domain. In certain embodiments, scFv is identified by screening an scFv phage library containing an antigen-Fc fusion protein. scFv can be derived from mice carrying human VL and / or VH genes. Alternatively, scFv can be substituted with camelid heavy chains (e.g., VHH derived from camels, llamas, etc.) or partial native ligands for cell surface receptors. In certain embodiments, the antigen is a tumor antigen, e.g., one disclosed herein. In certain embodiments, the antigen is a pathogenic antigen, for example, one disclosed herein.

[0131] In certain embodiments, the extracellular antigen-binding domain is a mouse scFv. In certain embodiments, the extracellular antigen-binding domain is a mouse scFv that binds to a human CD19 polypeptide. In certain embodiments, the extracellular antigen-binding domain is a mouse scFv that contains the amino acid sequence of SEQ ID NO: 84 and specifically binds to a human CD19 polypeptide (e.g., a human CD19 polypeptide containing the amino acid sequence described in SEQ ID NO: 75). In certain embodiments, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 84 is described in SEQ ID NO: 85. In certain embodiments, the mouse scFv has a heavy chain variable region (V) containing the amino acid sequence described in SEQ ID NO: 82. H ) includes. In a particular embodiment, mouse scFv has a light chain variable region (V) containing the amino acid sequence described in SEQ ID NO: 83. L) includes. In a particular embodiment, the mouse scFv may, if necessary, V H and V L (iii) a linker sequence, for example, a linker peptide, together with the amino acid sequence described in SEQ ID NO: 82, V H and V containing the amino acid sequence described in SEQ ID NO: 83 L This includes. In certain embodiments, the linker contains amino acids having the sequence described in SEQ ID NO: 66. In certain embodiments, the extracellular antigen-binding domain contains an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous to SEQ ID NO: 82. H For example, the extracellular antigen-binding domain contains an amino acid sequence that is approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to SEQ ID NO: 82. H Includes. In certain embodiments, the extracellular antigen-binding domain includes the amino acid sequence described in SEQ ID NO: 82. H Includes. In certain embodiments, the extracellular antigen-binding domain includes an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least 90%, or at least about 95%) homologous to SEQ ID NO: 83. L For example, the extracellular antigen-binding domain contains an amino acid sequence that is approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to SEQ ID NO: 83. L Includes. In certain embodiments, the extracellular antigen-binding domain includes the amino acid sequence described in SEQ ID NO: 83. L In certain embodiments, the extracellular antigen-binding domain includes an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least 90%, or at least about 95%) homologous or identical to SEQ ID NO: 82. HAnd V containing an amino acid sequence that is at least approximately 80% (e.g., at least approximately 85%, at least 90%, or at least approximately 95%) homologous or identical to sequence number 83. L This includes. In a particular embodiment, the extracellular antigen-binding domain is V containing the amino acid sequence described in SEQ ID NO: 82. H V containing the amino acid sequence described in Sequence ID No. 83 L This includes the following. In a particular embodiment, the extracellular antigen-binding domain is V containing the amino acid sequence described in SEQ ID NO: 76. H CDR1 or its conservative modification, V containing the amino acid sequence described in SEQ ID NO: 77 H CDR2 or its conservative modifications, and V containing the amino acid sequence described in SEQ ID NO: 78 H Includes CDR3 or a conservative modification thereof. In certain embodiments, the extracellular antigen-binding domain includes the amino acid sequence described in SEQ ID NO: 76. H V containing the amino acid sequence described in CDR1, SEQ ID NO: 77 H V containing the amino acid sequence described in CDR2 and SEQ ID NO: 78 H Contains CDR3. In certain embodiments, the extracellular antigen-binding domain contains the amino acid sequence described in SEQ ID NO: 79. L CDR1 or its conservative modification, V containing the amino acid sequence described in SEQ ID NO: 80 L V containing CDR2 or its conservative modifications, and the amino acid sequence described in SEQ ID NO: 81 L Includes CDR3 or a conservative modification thereof. In certain embodiments, the extracellular antigen-binding domain includes the amino acid sequence described in SEQ ID NO: 79. L V containing the amino acid sequence described in CDR1, SEQ ID NO: 80 L V containing the amino acid sequence described in CDR2 and SEQ ID NO: 81 L Contains CDR3. In certain embodiments, the extracellular antigen-binding domain contains the amino acid sequence described in SEQ ID NO: 76. H CDR1 or its conservative modification, V containing the amino acid sequence described in SEQ ID NO: 77 H CDR2 or its conservative modification, V containing the amino acid sequence described in SEQ ID NO: 78 HCDR3 or a conservative modification thereof, V comprising the amino acid sequence set forth in SEQ ID NO: 79 L CDR1 or a conservative modification thereof, V comprising the amino acid sequence set forth in SEQ ID NO: 80 L CDR2 or a conservative modification thereof, and V comprising the amino acid sequence set forth in SEQ ID NO: 81 L CDR3 or a conservative modification thereof. In certain embodiments, the extracellular antigen-binding domain comprises V comprising an amino acid having the sequence set forth in SEQ ID NO: 76 H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO: 77 H CDR2, V comprising the amino acid sequence set forth in SEQ ID NO: 78 H CDR3, V comprising the amino acid sequence set forth in SEQ ID NO: 79 L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO: 80 L CDR2, and V comprising the amino acid sequence set forth in SEQ ID NO: 81 L CDR3

Table 1

[0132] As used herein, the term “conservative sequence modification” refers to an amino acid modification that does not significantly affect or alter the binding properties of the CAR of this disclosure, including the amino acid sequence (e.g., the extracellular antigen-binding domain of the CAR). Conservative modifications may include amino acid substitutions, additions, and deletions. Modifications can be introduced into the human scFv of the CAR of this disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified into groups according to their physicochemical properties, such as charge and polarity. A conservative amino acid substitution is one in which an amino acid residue is replaced by an amino acid within the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, and histidine; negatively charged amino acids include aspartic acid and glutamic acid; and neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Furthermore, amino acids can be classified by polarity: polar amino acids include arginine (basic polarity), asparagine, aspartic acid (acidic polarity), glutamic acid (acidic polarity), glutamine, histidine (basic polarity), lysine (basic polarity), serine, threonine, and tyrosine; nonpolar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acids in the CDR region can be replaced with other amino acid residues from the same group, and the modified antibody can be tested for retained function (i.e., the function described in (c) to (l) above) using the functional assay described herein. In certain embodiments, one or fewer, two or fewer, three or fewer, four or five or fewer residues in a particular sequence or CDR region are modified.

[0133] V having at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homology or identity to a specific sequence (e.g., SEQ ID NO: 82 and SEQ ID NO: 83) H and / or V L The amino acid sequence may contain substitutions (e.g., conservative substitutions), insertions, or deletions compared to the specific sequence, but retains the ability to bind to a target antigen (e.g., CD19). In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in the specific sequence (e.g., SEQ ID NO: 82 and SEQ ID NO: 83). In certain embodiments, the substitutions, insertions, or deletions are present in regions outside the CDRs of the extracellular antigen-binding domain (e.g., in the FRs). In certain embodiments, the extracellular antigen-binding domain is a V selected from the group consisting of SEQ ID NO: 82 and SEQ ID NO: 83, including post-translational modifications of the sequences (SEQ ID NO: 82 and SEQ ID NO: 83). H and / or V L including the sequence.

[0134] As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap (i.e., % homology = number of identical positions / total number of positions × 100). Comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm.

[0135] The homology percentage between two amino acid sequences can be determined using the E. Meyers and W. Miller algorithm (Comput.Appl.Biosci., 4:11-17 (1988)) incorporated into the ALIGN program (version 2.0), using a PAM120 residue weighting table, 12 gap length penalties, and 4 gap penalties. Furthermore, the homology percentage between two amino acid sequences can be determined using either the Blossum 62 matrix or the PAM250 matrix, and the Needleman and Wunsch algorithm (J.Mol.Biol.48:444-453 (1970)), using either a Blossum 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. It can then be decided.

[0136] Furthermore, or alternatively, the amino acid sequences of the subject matter of this disclosure can be further used, for example, as “query sequences” for performing searches against public databases to identify relevant sequences. Such searches can be performed using the XBLAST program (version 2.0) described in Altschul, et al. (1990) J. Mol. Biol. 215:403-10. This can be done. A BLAST protein search can be performed using the XBLAST program, score=50, and word length=3 to obtain amino acid sequences homologous to specific sequences disclosed herein (e.g., the heavy and light chain variable region sequences of scFv m903, m904, m905, m906, and m900). To obtain a gapped alignment for comparison, Gapped BLAST can be used with Altschuletal., (1997) Nucleic Acids Res. It can be used as described in 25(17):3389-3402. BLAST and Gappe When using BLAST programs, you can use the default parameters for each program (e.g., XBLAST and NBLAST).

[0137] 2.2.2. Transmembrane domain of CAR In certain non-limiting embodiments, the transmembrane domain of a CAR includes a hydrophobic alpha-helix spanning at least a portion of the membrane. Different transmembrane domains result in different receptor stabilities. After antigen recognition, receptor clusters and signals are transmitted to the cell. According to the subject of this disclosure, the transmembrane domain of a CAR may include native or modified transmembrane domains of CD8 polypeptide, CD28 polypeptide, CD3ζ polypeptide, CD40 polypeptide, 4-1BB polypeptide, OX40 polypeptide, CD84 polypeptide, CD166 polypeptide, CD8a polypeptide, CD8b polypeptide, ICOS polypeptide, ICAM-1 polypeptide, CTLA-4 polypeptide, CD27 polypeptide, CD40 / My88 peptide, NKGD2 peptide, synthetic polypeptides (not based on proteins associated with immune responses), or combinations thereof.

[0138] CD8 In certain embodiments, the transmembrane domain comprises a CD8 polypeptide. In certain embodiments, the CD8 polypeptide comprises or has an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to a sequence or fragment thereof having NCBI reference number: NP_001139345.1 (SEQ ID NO: 86) provided below (homology in this specification can be determined using standard software such as BLAST or FASTA), and / or may optionally contain up to one, two, or three conserved amino acid substitutions. In certain embodiments, the CD8 polypeptide comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 86, having at least 20, or at least 30, or at least 40, or at least 50, and up to 235 amino acid lengths. Alternatively, or further, in various non-limiting embodiments, the CD8 polypeptide comprises or has the amino acid sequence of amino acids 1-235, 1-50, 50-100, 100-150, 150-200, or 200-235 of SEQ ID NO: 86. In certain embodiments, the CAR of the present disclosure comprises a transmembrane domain comprising a CD8 polypeptide comprising or having the amino acid sequence of amino acids 137-209 of SEQ ID NO: 86.

[0139] [ka]

[0140] In certain embodiments, the CD8 polypeptide contains or has an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to a sequence or fragment thereof having NCBI reference number: AAA92533.1 (Sequence ID 87) provided below (homology in this specification can be determined using standard software such as BLAST or FASTA), and / or may contain up to one, two, or three conservative amino acid substitutions as appropriate. In certain embodiments, the CD8 polypeptide contains or has an amino acid sequence that is at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 100, or at least about 200, and a contiguous portion of Sequence ID 87 having a maximum amino acid length of 247 amino acids. Alternatively, or further, in various non-limiting embodiments, the CD8 polypeptide comprises or has the amino acid sequence of amino acids 1-247, 1-50, 50-100, 100-150, 150-200, 151-219, or 200-247 of SEQ ID NO: 87. In certain embodiments, the CAR of the present disclosure comprises a transmembrane domain comprising a CD8 polypeptide comprising or having the amino acid sequence of amino acids 151-219 of SEQ ID NO: 87. [ka]

[0141] In a particular embodiment, the CD8 polypeptide comprises or has the amino acid sequence described in SEQ ID NO: 88, provided below: STTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICY[Sequence ID] 88]

[0142] According to the subject matter of this disclosure, “CD8 nucleic acid molecule” refers to a polynucleotide that encodes a CD8 polypeptide.

[0143] In a particular embodiment, a CD8 nucleic acid molecule encoding a CD8 polypeptide having the amino acid sequence described in SEQ ID NO: 88 comprises or has a nucleic acid having the sequence described in SEQ ID NO: 89, which is provided below.

[0144] [ka]

[0145] CD28 In certain embodiments, the transmembrane domain of the CAR of the Disclosure comprises a CD28 polypeptide. The CD28 polypeptide may have an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the sequence or fragment thereof having NCBI reference number: P10747 or NP_006130 (SEQ ID NO: 90), and / or may optionally contain up to one, two, or three conservative amino acid substitutions. In certain non-limiting embodiments, the CD28 polypeptide comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 90, having at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acid lengths. Alternatively, or further, in various non-limiting embodiments, the CD28 polypeptide comprises or has the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 114-220, 150-200, or 200-220 of SEQ ID NO: 90. In certain embodiments, the CD28 polypeptide contained in the transmembrane domain of the CAR of the present disclosure comprises or has the amino acid sequence of amino acids 153-179 of SEQ ID NO: 90. Sequence ID 90 is provided below: [ka]

[0146] According to the subject matter of this disclosure, “CD28 nucleic acid molecule” refers to a polynucleotide encoding a CD28 polypeptide. In certain embodiments, a CD28 nucleic acid molecule encoding a CD28 polypeptide having amino acids 153–179 of SEQ ID NO: 90 comprises or has a nucleic acid having the sequence described in SEQ ID NO: 91, which is provided below. [ka]

[0147] In certain embodiments, the intracellular signaling domain of CAR includes a human CD28 transmembrane domain. The human CD28 transmembrane domain contains, or may contain, an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to SEQ ID NO: 92 or a fragment thereof, and / or may optionally contain up to one, two, or three conserved amino acid substitutions. SEQ ID NO: 92 is provided below:

[0148] [ka]

[0149] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 92 is described in SEQ ID NO: 93, provided below. [ka]

[0150] CD84 In certain embodiments, the transmembrane domain of the CAR of the Disclosure comprises a native or modified transmembrane domain of a CD84 polypeptide. The CD84 polypeptide may have an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the sequence or fragment thereof having NCBI reference number: NP_001171808.1 (Sequence ID 1), and / or may optionally include up to one, two, or three conservative amino acid substitutions. In certain non-limiting embodiments, the CD84 polypeptide comprises or has an amino acid sequence that is a contiguous portion of Sequence ID 1, having a length of at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids. Alternatively, or further, in various non-limiting embodiments, the CD84 polypeptide comprises or has the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 114-220, 150-200, or 200-220 of SEQ ID NO: 1. In certain embodiments, the CD84 polypeptide contained in the transmembrane domain of the CAR of the present disclosure comprises or has the amino acid sequence of amino acids 226-250 of SEQ ID NO: 1. Sequence ID 1 is provided below: [ka]

[0151] According to the subject matter of this disclosure, “CD84 nucleic acid molecule” refers to a polynucleotide encoding a CD84 polypeptide. In certain embodiments, a CD84 nucleic acid molecule encoding a CD84 polypeptide having amino acids 226-250 of SEQ ID NO: 1 comprises or has a nucleic acid having the sequence described in SEQ ID NO: 2, which is provided below. [ka]

[0152] CD166 In certain embodiments, the transmembrane domain of the CAR of the Disclosure comprises a native or modified transmembrane domain of the CD166 polypeptide. The CD166 polypeptide may have an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the sequence or fragment thereof having NCBI reference number: NP_001618.2 (Sequence ID 3), and / or may optionally include up to one, two, or three conservative amino acid substitutions. In certain non-limiting embodiments, the CD166 polypeptide comprises or has an amino acid sequence that is a contiguous portion of Sequence ID 3, having a length of at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids. Alternatively, or further, in various non-limiting embodiments, the CD166 polypeptide comprises or has the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 114-220, 150-200, or 200-220 of SEQ ID NO: 3. In certain embodiments, the CD166 polypeptide contained in the transmembrane domain of the CAR of the Disclosure comprises or has the amino acid sequence of amino acids 528-553 of SEQ ID NO: 3. In certain embodiments, the CD166 polypeptide contained in the transmembrane domain of the CAR of the Disclosure comprises or has the amino acid sequence of amino acids 528-549 of SEQ ID NO: 3. Sequence ID 3 is provided below: [ka]

[0153] According to the subject matter of this disclosure, “CD166 nucleic acid molecule” refers to a polynucleotide encoding the CD166 polypeptide. In certain embodiments, a CD166 nucleic acid molecule encoding the CD166 polypeptide having amino acids 528-553 of SEQ ID NO: 3 comprises or has a nucleic acid having the sequence described in SEQ ID NO: 4, which is provided below. [ka]

[0154] CD8a In certain embodiments, the transmembrane domain of the CAR of the Disclosure comprises a native or modified transmembrane domain of a CD8a polypeptide. The CD8a polypeptide may have an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the sequence or fragment thereof having NCBI reference number: NP_001139345.1 (SEQ ID NO: 5), and / or may optionally include up to one, two, or three conservative amino acid substitutions. In certain non-limiting embodiments, the CD8a polypeptide comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 5, having at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acid lengths. Alternatively, or further, in various non-limiting embodiments, the CD8a polypeptide comprises or has the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 114-220, 150-200, or 200-220 of SEQ ID NO: 5. In certain embodiments, the CD8a polypeptide contained in the transmembrane domain of the CAR of the present disclosure comprises or has the amino acid sequence of amino acids 183-207 of SEQ ID NO: 5. Sequence ID 5 is provided below: [ka]

[0155] According to the subject matter of this disclosure, “CD8a nucleic acid molecule” refers to a polynucleotide encoding a CD8a polypeptide. In certain embodiments, a CD8a nucleic acid molecule encoding a CD8a polypeptide having amino acids 183-207 of SEQ ID NO: 5 comprises or has a nucleic acid having the sequence described in SEQ ID NO: 6, which is provided below. [ka]

[0156] CD8b In certain embodiments, the transmembrane domain of the CAR of the Disclosure comprises a native or modified transmembrane domain of a CD8b polypeptide. The CD8b polypeptide may have an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the sequence or fragment thereof having NCBI reference number: NP_742099.1 (Sequence ID 7), and / or may optionally include up to one, two, or three conservative amino acid substitutions. In certain non-limiting embodiments, the CD8b polypeptide comprises or has an amino acid sequence that is a contiguous portion of Sequence ID 7, having a length of at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids. Alternatively, or further, in various non-limiting embodiments, the CD8b polypeptide comprises or has the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 114-220, 150-200, or 200-220 of SEQ ID NO: 7. In certain embodiments, the CD8b polypeptide contained in the transmembrane domain of the CAR of the present disclosure comprises or has the amino acid sequence of amino acids 171-195 of SEQ ID NO: 7. Sequence ID 7 is provided below: [ka]

[0157] According to the subject matter of this disclosure, “CD8b nucleic acid molecule” refers to a polynucleotide encoding a CD8b polypeptide. In certain embodiments, a CD8b nucleic acid molecule encoding a CD8b polypeptide having amino acids 171-195 of SEQ ID NO: 7 comprises or has a nucleic acid having the sequence described in SEQ ID NO: 8, which is provided below. [ka]

[0158] ICOS In certain embodiments, the transmembrane domain of the CAR of the Disclosure comprises a native or modified transmembrane domain of the ICOS polypeptide. The ICOS polypeptide may have an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the sequence or fragment thereof having NCBI reference number: NP_036224.1 (SEQ ID NO: 9), and / or may optionally include up to one, two, or three conservative amino acid substitutions. In certain non-limiting embodiments, the ICOS polypeptide comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 9, having at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acid lengths. Alternatively, or further, in various non-limiting embodiments, the ICOS polypeptide comprises or has the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 114-220, 150-200, or 200-220 of SEQ ID NO: 9. In certain embodiments, the ICOS polypeptide contained in the transmembrane domain of the CAR of the present disclosure comprises or has the amino acid sequence of amino acids 141-165 of SEQ ID NO: 9. Sequence ID 9 is provided below: [ka]

[0159] According to the subject matter of this disclosure, “ICOS nucleic acid molecule” refers to a polynucleotide encoding the ICOS polypeptide. In certain embodiments, an ICOS nucleic acid molecule encoding the ICOS polypeptide having amino acids 141-165 of SEQ ID NO: 9 comprises or has a nucleic acid having the sequence described in SEQ ID NO: 10, which is provided below. [ka]

[0160] CTLA-4 In certain embodiments, the transmembrane domain of the CAR of the Disclosure comprises a native or modified transmembrane domain of a CTLA-4 polypeptide. The CTLA-4 polypeptide may have an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the sequence or fragment thereof having NCBI reference number: NP_005205.2 (SEQ ID NO: 11), and / or may optionally include up to one, two, or three conservative amino acid substitutions. In certain non-limiting embodiments, the CTLA-4 polypeptide comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 11, having at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acid lengths. Alternatively, or further, in various non-limiting embodiments, the CTLA-4 polypeptide comprises or has the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 114-220, 150-200, or 200-220 of SEQ ID NO: 11. In certain embodiments, the CTLA-4 polypeptide contained in the transmembrane domain of the CAR of the present disclosure comprises or has the amino acid sequence of amino acids 162-186 of SEQ ID NO: 11. Sequence ID 11 is provided below: [ka]

[0161] According to the subject matter of this disclosure, “CTLA-4 nucleic acid molecule” refers to a polynucleotide encoding a CTLA-4 polypeptide. In certain embodiments, a CTLA-4 nucleic acid molecule encoding a CTLA-4 polypeptide having amino acids 162-186 of SEQ ID NO: 11 comprises or has a nucleic acid having the sequence described in SEQ ID NO: 12, which is provided below. [ka]

[0162] ICAM-1 In certain embodiments, the transmembrane domain of the CAR of the Disclosure comprises a native or modified transmembrane domain of the ICAM-1 polypeptide. The ICAM-1 polypeptide may have an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the sequence or fragment thereof having NCBI reference number: NP_000192.2 (SEQ ID NO: 13), and / or may optionally include up to one, two, or three conserved amino acid substitutions. In certain non-limiting embodiments, the ICAM-1 polypeptide comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 13, having at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acid lengths. Alternatively, or further, in various non-limiting embodiments, the ICAM-1 polypeptide comprises or has the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 114-220, 150-200, or 200-220 of SEQ ID NO: 13. In certain embodiments, the ICAM-1 polypeptide contained in the transmembrane domain of the CAR of the present disclosure comprises or has the amino acid sequence of amino acids 481-507 of SEQ ID NO: 13. Sequence ID 13 is provided below: [ka]

[0163] According to the subject matter of this disclosure, “ICAM-1 nucleic acid molecule” refers to a polynucleotide encoding the ICAM-1 polypeptide. In certain embodiments, an ICAM-1 nucleic acid molecule encoding the ICAM-1 polypeptide having amino acids 481-507 of SEQ ID NO: 13 comprises or has a nucleic acid having the sequence described in SEQ ID NO: 14, which is provided below. [ka]

[0164] 2.2.3. Hinge / Spacer Area In certain non-limiting embodiments, the CAR may also include a hinge / spacer region that links an extracellular antigen-binding domain to a transmembrane domain. The hinge / spacer region may be flexible enough to orient the antigen-binding domain in different orientations to facilitate antigen recognition. In certain non-limiting embodiments, the hinge / spacer region of the CAR may include a native or modified hinge region of a CD8 polypeptide, CD28 polypeptide, CD3ζ polypeptide, CD40 polypeptide, 4-1BB polypeptide, OX40 polypeptide, CD84 polypeptide, CD166 polypeptide, CD8a polypeptide, CD8b polypeptide, ICOS polypeptide, ICAM-1 polypeptide, CTLA-4 polypeptide, CD27 polypeptide, CD40 / My88 peptide, NKGD2 peptide, synthetic polypeptide (not based on proteins associated with immune responses), or a combination thereof. The hinge / spacer region may be a hinge region derived from IgG1, or a portion of the CH2CH3 region and CD3 of an immunoglobulin, a portion of a CD28 polypeptide (e.g., a portion of SEQ ID NO: 90), a portion of a CD8 polypeptide (e.g., a portion of SEQ ID NO: 86 or a portion of SEQ ID NO: 87), any of the above variants that are at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% homologous or identical thereto, or a synthetic spacer sequence.

[0165] CD28 In certain embodiments, the hinge / spacer region of the CAR of the Disclosure comprises a native or modified hinge region of the CD28 polypeptide described herein. In certain embodiments, the CD28 polypeptide contained in the hinge / spacer region of the CAR of the Disclosure comprises or has the amino acid sequence of amino acids 114-152 of SEQ ID NO: 90. In certain embodiments, the CD28 nucleic acid molecule encoding the CD28 polypeptide having amino acids 114-152 of SEQ ID NO: 90 comprises or has the nucleic acid having the sequence described in SEQ ID NO: 15, which is provided below. IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP[Sequence ID 15]

[0166] CD84 In certain embodiments, the hinge / spacer region of the CAR of the Disclosure comprises a native or modified hinge region of the CD84 polypeptide described herein. In certain embodiments, the CD84 polypeptide contained in the hinge / spacer region of the CAR of the Disclosure comprises or has the amino acid sequence of amino acids 187-225 of SEQ ID NO: 1. In certain embodiments, the CD84 nucleic acid molecule encoding the CD84 polypeptide having amino acids 187-225 of SEQ ID NO: 1 comprises or has the nucleic acid having the sequence described in SEQ ID NO: 16, provided below. [ka]

[0167] CD166 In certain embodiments, the hinge / spacer region of the CAR of the Disclosure comprises a native or modified hinge region of the CD166 polypeptide described herein. In certain embodiments, the CD166 polypeptide contained in the hinge / spacer region of the CAR of the Disclosure comprises or has amino acids 489-527 of SEQ ID NO: 3. In certain embodiments, the CD166 nucleic acid molecule encoding the CD166 polypeptide having amino acids 489-527 of SEQ ID NO: 3 comprises or has a nucleic acid having the sequence described in SEQ ID NO: 17, which is provided below. [ka]

[0168] In certain embodiments, the CD166 polypeptide contained in the hinge / spacer region of the CAR of the present disclosure contains or has amino acids 484-527 of SEQ ID NO: 3. In certain embodiments, the CD166 polypeptide contained in the hinge / spacer region of the CAR of the present disclosure contains or has amino acids 506-527 of SEQ ID NO: 3. In certain embodiments, the CD166 polypeptide contained in the hinge / spacer region of the CAR of the present disclosure contains or has amino acids 517-527 of SEQ ID NO: 3. In certain embodiments, the CD166 polypeptide contained in the hinge / spacer region of the CAR of the present disclosure contains or has the amino acid sequence described in SEQ ID NO: 109 or SEQ ID NO: 110. [ka]

[0169] In certain embodiments, the CD166 polypeptide contained in the hinge / spacer region and transmembrane domain of the CAR of the present disclosure contains or has the amino acid sequence described in SEQ ID NOs: 111, 112, 113, 114, 115, 116, or 117. [ka]

[0170] CD8a In certain embodiments, the hinge / spacer region of the CAR of the Disclosure comprises a native or modified hinge region of the CD8a polypeptide described herein. In certain embodiments, the CD8a polypeptide contained in the hinge / spacer region of the CAR of the Disclosure comprises or has amino acids 137-182 of SEQ ID NO: 5. In certain embodiments, the CD8a nucleic acid molecule encoding the CD8a polypeptide having amino acids 137-182 of SEQ ID NO: 5 comprises or has a nucleic acid having the sequence described in SEQ ID NO: 18, provided below. [ka]

[0171] CD8b In certain embodiments, the hinge / spacer region of the CAR of the Disclosure comprises a native or modified hinge region of the CD8b polypeptide described herein. In certain embodiments, the CD8b polypeptide contained in the hinge / spacer region of the CAR of the Disclosure comprises or has amino acids 132-170 of SEQ ID NO: 7. In certain embodiments, the CD8b nucleic acid molecule encoding the CD8b polypeptide having amino acids 132-170 of SEQ ID NO: 7 comprises or has a nucleic acid having the sequence described in SEQ ID NO: 19, which is provided below. [ka]

[0172] ICOS In certain embodiments, the hinge / spacer region of the CAR of the Disclosure comprises a native or modified hinge region of the ICOS polypeptide described herein. In certain embodiments, the ICOS polypeptide contained in the hinge / spacer region of the CAR of the Disclosure comprises or has amino acids 102-140 of SEQ ID NO: 9. In certain embodiments, the ICOS nucleic acid molecule encoding the ICOS polypeptide having amino acids 102-140 of SEQ ID NO: 9 comprises or has a nucleic acid having the sequence described in SEQ ID NO: 20, which is provided below. [ka]

[0173] CTLA-4 In certain embodiments, the hinge / spacer region of the CAR of the Disclosure comprises a native or modified hinge region of a CTLA-4 polypeptide described herein. In certain embodiments, the CTLA-4 polypeptide contained in the hinge / spacer region of the CAR of the Disclosure comprises or has amino acids 123-161 of SEQ ID NO: 11. In certain embodiments, the CTLA-4 nucleic acid molecule encoding the CTLA-4 polypeptide having amino acids 123-161 of SEQ ID NO: 11 comprises or has a nucleic acid having the sequence described in SEQ ID NO: 21, which is provided below. [ka]

[0174] ICAM-1 In certain embodiments, the hinge / spacer region of the CAR of the Disclosure comprises a native or modified hinge region of the ICAM-1 polypeptide described herein. In certain embodiments, the ICAM-1 polypeptide contained in the hinge / spacer region of the CAR of the Disclosure comprises or has amino acids 442-480 of SEQ ID NO: 13. In certain embodiments, the ICAM-1 nucleic acid molecule encoding the ICAM-1 polypeptide having amino acids 442-480 of SEQ ID NO: 13 comprises or has a nucleic acid having the sequence described in SEQ ID NO: 22, which is provided below. [ka]

[0175] In certain embodiments, the CAR of the Disclosure includes a hinge / spacer region. In certain embodiments, the hinge / spacer region is located between the extracellular antigen-binding domain and the transmembrane domain. In certain embodiments, the hinge / spacer region includes CD8 polypeptide, CD28 polypeptide, CD3ζ polypeptide, CD4 polypeptide, 4-1BB polypeptide, OX40 polypeptide, CD166 polypeptide, CD8a polypeptide, CD8b polypeptide, ICOS polypeptide, ICAM-1 polypeptide, CTLA-4 polypeptide, CD27 polypeptide, CD40 / My88 peptide, NKGD2 peptide, synthetic polypeptide (not based on proteins associated with immune responses), or a combination thereof. In certain embodiments, the transmembrane domain includes CD8 polypeptide, CD28 polypeptide, CD3ζ polypeptide, CD4 polypeptide, 4-1BB polypeptide, OX40 polypeptide, CD166 polypeptide, CD8a polypeptide, CD8b polypeptide, ICOS polypeptide, ICAM-1 polypeptide, CTLA-4 polypeptide, CD27 polypeptide, CD40 / My88 peptide, NKGD2 peptide, synthetic polypeptide (not based on proteins associated with immune responses), or a combination thereof.

[0176] In certain embodiments, the transmembrane domain and the hinge / spacer region are derived from the same molecule. In certain embodiments, the transmembrane domain and the hinge / spacer region are derived from different molecules. In certain embodiments, the hinge / spacer region of CAR contains the CD28 polypeptide, and the transmembrane domain of CAR contains the CD28 polypeptide. In certain embodiments, the hinge / spacer region of CAR contains the CD28 polypeptide, and the transmembrane domain of CAR contains the CD28 polypeptide. In certain embodiments, the hinge / spacer region of CAR contains the CD84 polypeptide, and the transmembrane domain of CAR contains the CD84 polypeptide. In certain embodiments, the hinge / spacer region of CAR contains the CD166 polypeptide, and the transmembrane domain of CAR contains the CD166 polypeptide. In certain embodiments, the hinge / spacer region of CAR contains the CD8a polypeptide, and the transmembrane domain of CAR contains the CD8a polypeptide. In certain embodiments, the hinge / spacer region of CAR contains the CD8b polypeptide, and the transmembrane domain of CAR contains the CD8b polypeptide. In certain embodiments, the hinge / spacer region of the CAR contains the CD28 polypeptide, and the transmembrane domain of the CAR contains the ICOS polypeptide.

[0177] 2.2.4. Intracellular signaling domain of CAR In certain non-limiting embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide that can activate or stimulate cells (e.g., lymphoid cells, e.g., T cells). Wild-type ("native") CD3ζ comprises three immunoreceptor-activated tyrosine motifs ("ITAM") (e.g., ITAM1, ITAM2, and ITAM3), three basic residue-rich elongation (BRS) regions (BRS1, BRS2, ​​and BRS3), and transmits an activation signal to cells (e.g., lymphoid cells, e.g., T cells) after antigen binding. The intracellular signaling domain of the native CD3ζ chain is the primary signaling molecule from the endogenous TCR. When used in the embodiments herein, CD3ζ is modified CD3ζ rather than native CD3ζ. In certain embodiments, the modified CD3ζ polypeptide contains or has an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homologous or identical to the sequence or fragment having NCBI reference number: NP_932170 (Sequence ID 94). In certain non-limiting embodiments, the modified CD3ζ polypeptide contains or has an amino acid sequence that is a contiguous portion of Sequence ID 94 having at least 20, or at least 30, or at least 40, or at least 50, or at least 100, or at least 110, or at least 113, and at most 163 amino acids in length. Alternatively, or further, in various non-limiting embodiments, the modified CD3ζ polypeptide comprises or has the amino acid sequence of amino acids 1-50, 50-100, 100-150, 50-164, 55-164, or 150-164 of SEQ ID NO: 94. In certain embodiments, the modified CD3ζ polypeptide comprises or has the amino acid sequence of amino acids 52-164 of SEQ ID NO: 94. Sequence ID 94 is provided below: [ka]

[0178] In certain embodiments, the intracellular signaling domain of CAR comprises a modified human CD3ζ polypeptide. The modified human CD3ζ polypeptide comprises, or may comprise, an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homologous or identical to SEQ ID NO: 95 or a fragment thereof, and / or may optionally comprise up to one, two, or three conserved amino acid substitutions. SEQ ID NO: 95 is provided below: [ka]

[0179] An example nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 95 is described in SEQ ID NO: 96, provided below.

[0180] [ka]

[0181] Immune receptor-activated tyrosine motif (ITAM) In certain non-limiting embodiments, the intracellular signaling domain of CAR comprises a modified CD3ζ polypeptide containing one, two, or three ITAMs. In certain embodiments, the modified CD3ζ polypeptide comprises a natural ITAM1 having the amino acid sequence described in SEQ ID NO: 23. QNQLYNELNLGRREEYDVLDKR[Sequence ID 23]

[0182] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 23 is described in SEQ ID NO: 24, provided below. [ka]

[0183] In certain embodiments, the modified CD3ζ polypeptide comprises an ITAM1 variant containing one or more loss-of-function mutations. In certain embodiments, the modified CD3ζ polypeptide has an ITAM1 variant containing two loss-of-function mutations. In certain embodiments, the loss-of-function mutations comprise mutations in tyrosine residues in ITAM1. In certain embodiments, the ITAM1 variant consisting of two loss-of-function mutations comprises the amino acid sequence described in Sequence ID No. 25 provided below. QNQLFNELNLGRREEFDVLDKR[Sequence ID 25]

[0184] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 25 is described in SEQ ID NO: 26, provided below. [ka]

[0185] In certain embodiments, the modified CD3ζ polypeptide comprises natural ITAM2 having the amino acid sequence described in Sequence ID No. 27 provided below. QEGLYNELQKDKMAEAYSEIGMK[Sequence ID 27]

[0186] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 27 is described in SEQ ID NO: 28, provided below. [ka]

[0187] In certain embodiments, the modified CD3ζ polypeptide comprises an ITAM2 variant containing one or more loss-of-function mutations. In certain embodiments, the modified CD3ζ polypeptide has an ITAM2 variant containing two loss-of-function mutations. In certain embodiments, the loss-of-function mutations comprise mutations in tyrosine residues in ITAM2. In certain embodiments, the ITAM2 variant consisting of two loss-of-function mutations comprises the amino acid sequence described in Sequence ID No. 29 provided below. QEGLFNELQKDKMAEAFSEIGMK[Sequence ID 29]

[0188] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 29 is described in SEQ ID NO: 30, provided below. [ka]

[0189] In certain embodiments, the modified CD3ζ polypeptide comprises natural ITAM3 having the amino acid sequence described in SEQ ID NO: 31 provided below. HDGLYQGLSTATKDTYDALHMQ[Sequence ID 31]

[0190] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 131 is described in SEQ ID NO: 32, provided below. cacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcag[SEQ ID NO: 32]

[0191] In certain embodiments, the modified CD3ζ polypeptide comprises an ITAM3 variant containing one or more loss-of-function mutations. In certain embodiments, the modified CD3ζ polypeptide has an ITAM3 variant containing two loss-of-function mutations. In certain embodiments, the loss-of-function mutations comprise mutations in tyrosine residues in ITAM3. In certain embodiments, the ITAM3 variant consisting of two loss-of-function mutations comprises the amino acid sequence described in Sequence ID No. 33, provided below. HDGLFQGLSTATKDTFDALHMQ[Sequence ID 33]

[0192] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 33 is described in SEQ ID NO: 34, provided below. [ka]

[0193] In certain embodiments, the intracellular signaling domain of CAR comprises an ITAM1 variant containing one or more loss-of-function mutations, an ITAM2 variant containing one or more loss-of-function mutations, an ITAM3 variant containing one or more loss-of-function mutations, or a combination thereof, or essentially consisting thereof, or a modified CD3ζ polypeptide consisting thereof. In certain embodiments, the intracellular signaling domain of CAR comprises a modified CD3ζ polypeptide comprising an ITAM2 variant containing one or more (e.g., two) loss-of-function mutations and an ITAM3 variant containing one or more (e.g., two) loss-of-function mutations. In certain embodiments, the intracellular signaling domain of CAR comprises a modified CD3ζ polypeptide comprising native ITAM1, an ITAM2 variant containing two loss-of-function mutations, and an ITAM3 variant containing two loss-of-function mutations. In certain embodiments, the intracellular signaling domain of CAR comprises a modified CD3ζ polypeptide (e.g., a construct named "1XX") which includes a native ITAM1 having the amino acid sequence described in SEQ ID NO: 23, an ITAM2 variant having the amino acid sequence described in SEQ ID NO: 29, and an ITAM3 variant having the amino acid sequence described in SEQ ID NO: 33.

[0194] In certain embodiments, the intracellular signaling domain of CAR comprises a modified CD3ζ polypeptide comprising an ITAM1 variant containing one or more (e.g., two) loss-of-function mutations and an ITAM3 variant containing one or more (e.g., two) loss-of-function mutations. In certain embodiments, the intracellular signaling domain of CAR comprises a modified CD3ζ polypeptide comprising an ITAM1 variant containing two loss-of-function mutations, native ITAM2, and an ITAM3 variant containing two loss-of-function mutations. In certain embodiments, the intracellular signaling domain of CAR comprises a modified CD3ζ polypeptide comprising an ITAM1 variant having the amino acid sequence described in SEQ ID NO: 25, native ITAM2 having the amino acid sequence described in SEQ ID NO: 27, and an ITAM3 variant having the amino acid sequence described in SEQ ID NO: 33 (e.g., a construct named "X2X").

[0195] In certain embodiments, the intracellular signaling domain of CAR comprises a modified CD3ζ polypeptide comprising an ITAM1 variant containing one or more (e.g., two) loss-of-function mutations and an ITAM2 variant containing one or more (e.g., two) loss-of-function mutations. In certain embodiments, the intracellular signaling domain of CAR comprises a modified CD3ζ polypeptide comprising an ITAM1 variant containing two loss-of-function mutations, an ITAM2 variant containing two loss-of-function mutations, and native ITAM3. In certain embodiments, the intracellular signaling domain of CAR comprises a modified CD3ζ polypeptide comprising an ITAM1 variant having the amino acid sequence described in SEQ ID NO: 25, an ITAM2 variant having the amino acid sequence described in SEQ ID NO: 29, and native ITAM3 having the amino acid sequence described in SEQ ID NO: 31 (e.g., a construct named "XX3").

[0196] In certain embodiments, the intracellular signaling domain of CAR comprises a modified CD3ζ polypeptide comprising an ITAM1 variant containing one or more (e.g., two) loss-of-function mutations. In certain embodiments, the intracellular signaling domain of CAR comprises a modified CD3ζ polypeptide comprising an ITAM1 variant containing two loss-of-function mutations, native ITAM2, and native ITAM3. In certain embodiments, the intracellular signaling domain of CAR comprises a modified CD3ζ polypeptide comprising an ITAM1 variant having the amino acid sequence described in SEQ ID NO: 27, native ITAM2 having the amino acid sequence described in SEQ ID NO: 29, and native ITAM3 having the amino acid sequence described in SEQ ID NO: 31 (e.g., a construct named "X23").

[0197] In certain embodiments, the intracellular signaling domain of CAR comprises a modified CD3ζ polypeptide comprising native ITAM1, native ITAM2, and an ITAM3 variant containing one or more (e.g., two) loss-of-function mutations. In certain embodiments, the intracellular signaling domain of CAR comprises a modified CD3ζ polypeptide comprising native ITAM1, native ITAM2, and an ITAM1 variant containing two loss-of-function mutations. In certain embodiments, the intracellular signaling domain of CAR comprises a modified CD3ζ polypeptide comprising native ITAM1 having the amino acid sequence described in SEQ ID NO: 23, native ITAM2 having the amino acid sequence described in SEQ ID NO: 27, and an ITAM3 variant having the amino acid sequence described in SEQ ID NO: 33 (e.g., a construct named "12X").

[0198] In certain embodiments, the intracellular signaling domain of CAR comprises a modified CD3ζ polypeptide comprising native ITAM1, an ITAM2 variant containing one or more (e.g., two) loss-of-function mutations, and native ITAM3. In certain embodiments, the intracellular signaling domain of CAR comprises a modified CD3ζ polypeptide comprising native ITAM1, an ITAM2 variant containing two loss-of-function mutations, and native ITAM3. In certain embodiments, the intracellular signaling domain of CAR comprises a modified CD3ζ polypeptide comprising native ITAM1 having the amino acid sequence described in SEQ ID NO: 23, an ITAM2 variant having the amino acid sequence described in SEQ ID NO: 29, and a native ITAM3 variant having the amino acid sequence described in SEQ ID NO: 31 (e.g., a construct named "1X3").

[0199] In certain embodiments, the intracellular signaling domain of CAR comprises a modified CD3ζ polypeptide containing deletions of one or two ITAMs. In certain embodiments, the modified CD3ζ polypeptide comprises deletions of ITAM1 and ITAM2; for example, the modified CD3ζ polypeptide comprises natural ITAM3 or an ITAM3 variant, but does not contain ITAM1 or ITAM2. In certain embodiments, the modified CD3ζ polypeptide comprises natural ITAM3 having the amino acid sequence described in SEQ ID NO: 31, but does not contain ITAM1 (natural or modified) or ITAM2 (natural or modified) (e.g., D12).

[0200] In certain embodiments, the modified CD3ζ polypeptide includes deletions of ITAM2 and ITAM3, for example, the modified CD3ζ polypeptide includes natural ITAM1 or an ITAM1 variant, but does not include ITAM2 or ITAM3. In certain embodiments, the modified CD3ζ polypeptide includes natural ITAM1 having the amino acid sequence described in SEQ ID NO: 23, but does not include ITAM2 (natural or modified) or ITAM3 (natural or modified) (e.g., D23).

[0201] In certain embodiments, the modified CD3ζ polypeptide includes deletions of ITAM1 and ITAM3, for example, the modified CD3ζ polypeptide includes natural ITAM2 or an ITAM2 variant, but does not include ITAM1 or ITAM3. In certain embodiments, the modified CD3ζ polypeptide includes natural ITAM2 having the amino acid sequence described in SEQ ID NO: 27, but does not include ITAM1 (natural or modified) or ITAM3 (natural or modified) (e.g., D13).

[0202] In certain embodiments, the modified CD3ζ polypeptide includes a deletion of ITAM1, for example, the modified CD3ζ polypeptide includes natural ITAM2 or an ITAM2 variant, and natural ITAM3 or an ITAM3 variant, but does not include ITAM1 (natural or modified).

[0203] In certain embodiments, the modified CD3ζ polypeptide includes a deletion of ITAM2, for example, the modified CD3ζ polypeptide includes natural ITAM1 or an ITAM1 variant, and natural ITAM3 or an ITAM3 variant, but does not include ITAM2 (natural or modified).

[0204] In certain embodiments, the modified CD3ζ polypeptide includes a deletion of ITAM3, for example, the modified CD3ζ polypeptide includes natural ITAM1 or an ITAM1 variant, and natural ITAM2 or an ITAM2 variant, but does not include ITAM3 (natural or modified).

[0205] Basic residue-rich extension (BRS) region In certain non-limiting embodiments, the intracellular signaling domain of CAR comprises a modified CD3ζ polypeptide containing one, two, or three BRS regions (i.e., BRS1, BRS2, ​​and BRS3). The BRS regions may be native BRS or modified BRS (e.g., BRS variants). In certain embodiments, the modified CD3ζ polypeptide comprises native BRS1 containing the amino acid sequence described in SEQ ID NO: 35, provided below. KRRGR[SEQ ID NO: 35]

[0206] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 35 is described in SEQ ID NO: 36, provided below. aagagacgtggccgg[Sequence ID 36]

[0207] In certain embodiments, the modified CD3ζ polypeptide comprises a BRS1 variant containing one or more loss-of-function mutations.

[0208] In certain embodiments, the modified CD3ζ polypeptide comprises natural BRS2 having the amino acid sequence described in SEQ ID NO: 37. KPRRK[SEQ ID NO: 37]

[0209] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 37 is described in SEQ ID NO: 38, provided below. aagccgagaaggaag[Sequence ID 38]

[0210] In certain embodiments, the modified CD3ζ polypeptide comprises a BRS2 variant containing one or more loss-of-function mutations.

[0211] In certain embodiments, the modified CD3ζ polypeptide comprises natural BRS3 having the amino acid sequence described in SEQ ID NO: 39. KGERRRGK[Sequence ID 39]

[0212] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 39 is described in SEQ ID NO: 40, provided below. aaaggcgagcgccggaggggcaag[Sequence ID 40]

[0213] In certain embodiments, the modified CD3ζ polypeptide comprises a BRS3 variant containing one or more loss-of-function mutations.

[0214] In certain embodiments, the intracellular signaling domain of CAR includes a modified CD3ζ polypeptide comprising all three BRS regions, namely the BRS1 region, the BRS2 region, and the BRS3 region. In certain embodiments, the intracellular signaling domain of CAR includes a modified CD3ζ polypeptide comprising natural BRS1, natural BRS2, ​​and natural BRS3. In certain embodiments, the intracellular signaling domain of CAR includes a modified CD3ζ polypeptide comprising natural BRS1 having the amino acid sequence described in SEQ ID NO: 35, natural BRS2 having the amino acid sequence described in SEQ ID NO: 37, and natural BRS3 having the amino acid sequence described in SEQ ID NO: 39, for example, the modified CD3ζ polypeptide contained in construct 1XX.

[0215] In certain embodiments, the intracellular signaling domain of CAR includes a modified CD3ζ polypeptide containing one or two, but not all three, BRS regions. In certain embodiments, the modified CD3ζ polypeptide includes the BRS1 and BRS2 regions but does not include the BRS3 region. In certain embodiments, the modified CD3ζ polypeptide includes the BRS1 and BRS3 regions but does not include the BRS2 region. In certain embodiments, the modified CD3ζ polypeptide includes the BRS2 and BRS3 regions but does not include the BRS1 region.

[0216] In certain embodiments, the modified CD3ζ polypeptide includes the BRS1 region but does not include the BRS2 or BRS3 region. In certain embodiments, the modified CD3ζ polypeptide includes the natural BRS1 having the amino acid sequence described in SEQ ID NO: 35 but does not include the BRS2 or BRS3 region; for example, the modified CD3ζ polypeptide is included in construct D23. In certain embodiments, the modified CD3ζ polypeptide includes the BRS2 region but does not include the BRS1 or BRS3 region. In certain embodiments, the modified CD3ζ polypeptide includes the BRS3 region but does not include the BRS1 or BRS2 region.

[0217] In certain embodiments, the modified CD3ζ polypeptide does not contain the BRS region (natural or modified BRS1, BRS2, ​​or BRS3), for example, all three BRS regions are deleted, for example, the modified CD3ζ polypeptide is included in construct D12.

[0218] In certain non-limiting embodiments, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain including a modified CD3ζ polypeptide, wherein the modified CD3ζ polypeptide lacks all or part of an immunoreceptor-activating tyrosine motif (ITAM), and the ITAMs are ITAM1, ITAM2, and ITAM3. In certain embodiments, the modified CD3ζ polypeptide lacks ITAM2 or part thereof. In certain embodiments, the modified CD3ζ polypeptide further lacks ITAM3 or part thereof. In certain embodiments, the modified CD3ζ polypeptide further lacks ITAM1 or part thereof. In certain embodiments, the modified CD3ζ polypeptide lacks ITAM1 or part thereof. In certain embodiments, the modified CD3ζ polypeptide further lacks ITAM3 or part thereof. In certain embodiments, the modified CD3ζ polypeptide lacks ITAM3 or part thereof. In certain embodiments, the modified CD3ζ polypeptide lacks all or part of the basic residue-rich extension (BRS) region, where the BRS region is BRS1, BRS2, ​​and BRS3. In certain embodiments, the modified CD3ζ polypeptide lacks BRS2 or part thereof. In certain embodiments, the modified CD3ζ polypeptide further lacks BRS3 or part thereof. In certain embodiments, the modified CD3ζ polypeptide further lacks BRS1 or part thereof. In certain embodiments, the modified CD3ζ polypeptide lacks BRS1 or part thereof. In certain embodiments, the modified CD3ζ polypeptide further lacks BRS3 or part thereof. In certain embodiments, the modified CD3ζ polypeptide lacks BRS3 or part thereof. In certain embodiments, the modified CD3ζ polypeptide lacks BRS1 or part thereof, BRS2 or part thereof, and BRS3 or part thereof. In certain embodiments, the modified CD3ζ polypeptide lacks ITAM2, ITAM3, BRS2, ​​and BRS3. In certain embodiments, the CAR comprises the amino acid sequence described in SEQ ID NO: 45 or SEQ ID NO: 47.In certain embodiments, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a modified CD3ζ polypeptide, wherein the modified CD3ζ polypeptide lacks all or part of a basic residue-rich extension (BRS) region, and the BRS region is BRS1, BRS2, ​​and BRS3. In certain embodiments, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a modified CD3ζ polypeptide, wherein the modified CD3ζ polypeptide comprises a BRS variant selected from BRS1 variant, BRS2 variant, and BRS3 variant, and the BRS variant comprises one or more loss-of-function mutations.

[0219] Co-stimulatory signaling region In certain non-limiting embodiments, the intracellular signaling domain of CAR further comprises at least a co-stimulatory signaling region. In certain embodiments, the co-stimulatory signaling region comprises at least one co-stimulatory molecule capable of providing optimal lymphocyte activation.

[0220] As used herein, “costimulatory molecule” refers to a cell surface molecule other than the antigen receptor or its ligand required for an efficient lymphocyte response to an antigen. At least one costimulatory signaling region may include the CD28 polypeptide, 4-1BB polypeptide, OX40 polypeptide, ICOS polypeptide, DAP-10 polypeptide, CD27 peptide, CD40 / My88 peptide, NKGD2 peptide, or a combination thereof. Upon binding to its receptor, a costimulatory molecule can bind to a costimulatory ligand, which is a protein expressed on the cell surface that generates a costimulatory response, i.e., an intracellular response that provides the stimulus when an antigen binds to its CAR molecule. Examples of costimulatory ligands, but not limited to, include CD80, CD86, CD70, OX40L, and 4-1BBL. For example, the 4-1BB ligand (i.e., 4-1BBL) can, along with the CAR signal, +It can bind to 4-1BB (also known as "CD137") to provide intracellular signals that induce effector cell function of T cells. CARs comprising an intracellular signaling domain including a costimulatory signaling region containing 4-1BB, ICOS, or DAP-10 are disclosed in U.S. Patent No. 7,446,190, which is incorporated herein by reference in its entirety.

[0221] In certain embodiments, the intracellular signaling domain of CAR includes a co-stimulatory signaling region comprising a CD28 polypeptide. The CD28 polypeptide comprises, or may comprise, an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homologous or identical to the sequence or fragment thereof having NCBI reference number: P10747 or NP_006130 (SEQ ID NO: 90), and / or may optionally comprise up to one, two, or three conservative amino acid substitutions. In certain non-limiting embodiments, the CD28 polypeptide comprises, or comprises, an amino acid sequence that is a contiguous portion of SEQ ID NO: 90 having a length of at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids. Alternatively, or further, in various non-limiting embodiments, the CD28 polypeptide comprises or has the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 114-220, 150-200, or 200-220 of SEQ ID NO: 90. In certain embodiments, the intracellular signaling domain of CAR comprises a co-stimulatory signaling region comprising a CD28 polypeptide comprising or having the amino acid sequence of amino acids 180-220 of SEQ ID NO: 90.

[0222] In certain embodiments, the CD28 polypeptide contains or has an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homologous or identical to the sequence or fragment thereof having NCBI reference number: NP_031668.3 (Sequence ID 97), and / or may optionally contain up to one, two, or three conservative amino acid substitutions. In certain non-limiting embodiments, the CD28 polypeptide contains or has an amino acid sequence that is a contiguous portion of Sequence ID 97, having at least 20, or at least 30, or at least 40, or at least 50, and up to 218 amino acid lengths. Alternatively, or further, in various non-limiting embodiments, the CD28 polypeptide comprises or has the amino acid sequence of amino acids 1-218, 1-50, 50-100, 100-150, 114-220, 150-200, 178-218, or 200-220 of SEQ ID NO: 97. In certain embodiments, the co-stimulatory signaling region of the CAR of the present disclosure comprises a CD28 polypeptide comprising or having amino acids 178-218 of SEQ ID NO: 97. Sequence ID 97 is provided below: [ka]

[0223] According to the subject matter of this disclosure, “CD28 nucleic acid molecule” refers to a polynucleotide encoding the CD28 polypeptide. In certain embodiments, the CD28 nucleic acid molecule encoding the CD28 polypeptide contained in the co-stimulatory signaling region of the CAR of this disclosure (e.g., amino acids 178-218 of SEQ ID NO: 97) comprises or has the nucleotide sequence described in SEQ ID NO: 98 provided below. [ka]

[0224] In certain embodiments, the intracellular signaling domain of CAR comprises the mouse intracellular signaling domain of CD28. The mouse intracellular signaling domain of CD28 comprises, or may comprise, an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homologous or identical to SEQ ID NO: 99 or a fragment thereof, and / or may optionally comprise up to one, two, or three conserved amino acid substitutions. SEQ ID NO: 99 is provided below:

[0225] [ka]

[0226] An example nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 99 is described in SEQ ID NO: 100, provided below. [ka]

[0227] In certain embodiments, the intracellular signaling domain of CAR includes the human intracellular signaling domain of CD28. The human intracellular signaling domain of CD28 contains, or may contain, an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homologous or identical to SEQ ID NO: 101 or a fragment thereof, and / or may optionally contain up to one, two, or three conserved amino acid substitutions. SEQ ID NO: 101 is provided below:

[0228] [ka]

[0229] An exemplary nucleic acid sequence encoding the amino acid sequence of Sequence ID No. 70 is described in Sequence ID No. 102, provided below. [ka]

[0230] In certain embodiments, the intracellular signaling domain of CAR includes the deimmunized human intracellular signaling domain of CD28. The deimmunized human intracellular signaling domain of CD28 contains, or may contain, an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homologous or identical to SEQ ID NO: 108 or a fragment thereof, and / or may optionally contain up to one, two, or three conserved amino acid substitutions. SEQ ID NO: 108 is provided below: [ka]

[0231] In certain embodiments, the intracellular signaling domain of CAR includes a co-stimulatory signaling region comprising two co-stimulatory molecules, for example, a CD28 and 4-1BB co-stimulatory signaling region or a CD28 and OX40 co-stimulatory signaling region.

[0232] 4-1BB may act as a tumor necrosis factor (TNF) ligand and may have stimulating activity. The 4-1BB polypeptide contains, or may contain, an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homologous or identical to the sequence or fragment thereof having NCBI reference number: P41273 or NP_001552 (SEQ ID NO: 103), and / or may contain up to one, two, or three conservative amino acid substitutions as necessary. Sequence ID 103 is provided below: [ka]

[0233] According to the subject matter of this disclosure, “4-1BB nucleic acid molecule” refers to a polynucleotide that encodes a 4-1BB polypeptide.

[0234] In certain embodiments, the intracellular signaling domain of CAR includes the intracellular signaling domain 4-1BB. The intracellular signaling domain 4-1BB contains, or may contain, an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homologous or identical to SEQ ID NO: 104 or a fragment thereof, and / or may contain, optionally, up to one, two, or three conserved amino acid substitutions. SEQ ID NO: 104 is provided below:

[0235] [ka]

[0236] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 104 is described in SEQ ID NO: 105, provided below. [ka]

[0237] The OX40 polypeptide contains, or may contain, an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homologous or identical to the sequence or fragment thereof having NCBI reference number: P43489 or NP_003318 (SEQ ID NO: 106), and / or may contain, as necessary, up to one, two, or three conservative amino acid substitutions. Sequence ID 106 is provided below: [ka]

[0238] According to the subject matter of this disclosure, “OX40 nucleic acid molecule” refers to a polynucleotide that encodes an OX40 polypeptide.

[0239] The ICOS polypeptide contains, or may contain, an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homologous or identical to the sequence or fragment thereof having NCBI reference number: NP_036224 (Sequence ID 65), and / or may contain, as necessary, up to one, two, or three conservative amino acid substitutions. Sequence ID 65 is provided below: [ka]

[0240] According to the subject matter of this disclosure, “ICOS nucleic acid molecule” refers to a polynucleotide that encodes the ICOS polypeptide.

[0241] In certain embodiments, the CAR of this disclosure further comprises an inducible promoter for expressing a nucleic acid sequence in human cells. The promoter for use in the expression of the CAR gene may be a constitutive promoter, such as a ubiquitin C (UbiC) promoter.

[0242] In certain embodiments, mutation sites and / or junctions between domains / motifs / regions of CARs derived from different proteins are deimmunized. The immunogenicity of junctions between different CAR portions can be predicted using a NetMHC4.0 server. For each peptide containing at least one amino acid derived from the following portions, the binding affinity to HLA A, B, and C for all alleles can be predicted. An immunogenicity score for each peptide can be assigned to each peptide. The immunogenicity score is given by the formula: Immunogenicity Score = [(50 - Binding Affinity)] * HLA frequency] n This can be calculated using the following method, where n is the number of predictions for each peptide.

[0243] 1928z WT constructs In certain embodiments, the CAR of this disclosure comprises an extracellular antigen-binding domain that binds to a CD19 polypeptide (e.g., human CD19 polypeptide), a transmembrane domain and hinge / spacer region derived from a CD28 polypeptide, an intracellular signaling domain comprising a modified CD3ζ polypeptide (e.g., modified human CD3ζ polypeptide) including native ITAM1, native ITAM2, natural ITAM3, natural BRS1, natural BRS2, ​​and natural BRS3, and a co-stimulatory signaling region comprising a CD28 polypeptide (e.g., human CD28 polypeptide). In certain embodiments, the CAR is named "1928z WT". In certain embodiments, the CAR (e.g., 1928z WT) comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence described in Sequence ID No. 41 provided below. Sequence ID 41 contains a CD8 reader sequence in amino acids 1-18 and can bind to CD19 (e.g., human CD19).

[0244] [ka]

[0245] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 41 is described in SEQ ID NO: 42, provided below.

[0246] [ka]

[0247] 1XX Structure In certain embodiments, the CAR of this disclosure comprises an extracellular antigen-binding domain that binds to a CD19 polypeptide (e.g., human CD19 polypeptide), a transmembrane domain and hinge / spacer region derived from a CD28 polypeptide, an intracellular signaling domain comprising a modified CD3ζ polypeptide (e.g., modified human CD3ζ polypeptide) including native ITAM1, native BRS1, native BRS2, ​​native BRS3, an ITAM2 variant having two loss-of-function mutations, and an ITAM3 variant having two loss-of-function mutations, and a co-stimulatory signaling region comprising a CD28 polypeptide (e.g., human CD28 polypeptide). In certain embodiments, the CAR is named "1XX". In certain embodiments, the CAR (e.g., 1XX) comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence described in Sequence ID No. 43 provided below. Sequence ID 43 contains a CD8 reader sequence in amino acids 1-18 and can bind to CD19 (e.g., human CD19).

[0248] [ka]

[0249] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 43 is described below in SEQ ID NO: 44.

[0250] [ka]

[0251] D12 Structure In certain embodiments, the CAR of this disclosure comprises an extracellular antigen-binding domain that binds to a CD19 polypeptide (e.g., human CD19 polypeptide), a transmembrane domain and hinge / spacer region derived from a CD28 polypeptide, an intracellular signaling domain comprising a modified CD3ζ polypeptide (e.g., modified human CD3ζ polypeptide), and a co-stimulatory signaling region comprising a CD28 polypeptide (e.g., human CD28 polypeptide), wherein the modified CD3ζ polypeptide comprises natural ITAM3 and not ITAM1 (natural or modified), ITAM2 (natural or modified), BRS1 (natural or modified), BRS2 (natural or modified), or BRS3 (natural or modified). In certain embodiments, the CAR is named "D12". In certain embodiments, the CAR (e.g., D12) comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous to the amino acid sequence described in Sequence ID No. 45 provided below. Sequence ID 45 contains a CD8 reader sequence in amino acids 1-18 and can bind to CD19 (e.g., human CD19).

[0252] [ka]

[0253] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 45 is described in SEQ ID NO: 46, provided below.

[0254] [ka]

[0255] D23 Structure In certain embodiments, the CAR of the Disclosure comprises an extracellular antigen-binding domain that binds to a CD19 polypeptide (e.g., human CD19 polypeptide), a transmembrane domain and hinge / spacer region derived from a CD28 polypeptide, an intracellular signaling domain comprising a modified CD3ζ polypeptide (e.g., modified human CD3ζ polypeptide) including deletions of ITAM1, BRS1, and ITAM2, ITAM3, BRS2, ​​and BRS3, and a co-stimulatory signaling region comprising a CD28 polypeptide (e.g., human CD28 polypeptide), wherein the modified CD3ζ polypeptide comprises natural ITAM1 and natural BRS1, but not ITAM2 (natural or modified), ITAM3 (natural or modified), BRS2 (natural or modified), or BRS3 (natural or modified). In certain embodiments, the CAR is named "D23". In certain embodiments, the CAR (e.g., D23) includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence described in Sequence ID No. 47 provided below. Sequence ID No. 47 includes a CD8 reader sequence in amino acids 1-18 and can bind to CD19 (e.g., human CD19).

[0256] [ka]

[0257] An exemplary nucleic acid sequence encoding the amino acid sequence of Sequence ID No. 47 is described in Sequence ID No. 48, provided below.

[0258] [ka]

[0259] XX3 Structure In certain embodiments, the CAR of this disclosure comprises an extracellular antigen-binding domain that binds to a CD19 polypeptide (e.g., human CD19 polypeptide), a transmembrane domain and hinge / spacer region derived from a CD28 polypeptide, an intracellular signaling domain comprising a modified CD3ζ polypeptide (e.g., modified human CD3ζ polypeptide) including native ITAM3, native BRS1, native BRS2, ​​native BRS3, an ITAM1 variant having two loss-of-function mutations, and an ITAM2 variant having two loss-of-function mutations, and a co-stimulatory signaling region comprising a CD28 polypeptide (e.g., human CD28 polypeptide). In certain embodiments, the CAR is named "XX3". In certain embodiments, the CAR (e.g., XX3) comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous to the amino acid sequence described in Sequence ID No. 49 provided below. Sequence ID 49 contains a CD8 reader sequence in amino acids 1-18 and can bind to CD19 (e.g., human CD19).

[0260] [ka]

[0261] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 49 is described in SEQ ID NO: 50, provided below.

[0262] [ka]

[0263] X23 Structure In certain embodiments, the CAR of this disclosure comprises an extracellular antigen-binding domain that binds to a CD19 polypeptide (e.g., human CD19 polypeptide), a transmembrane domain and hinge / spacer region derived from a CD28 polypeptide, an intracellular signaling domain comprising a modified CD3ζ polypeptide (e.g., modified human CD3ζ polypeptide) containing native ITAM2, native ITAM3, native BRS1, native BRS2, ​​native BRS3, and an ITAM1 variant having two loss-of-function mutations, and a co-stimulatory signaling region comprising a CD28 polypeptide (e.g., human CD28 polypeptide). In certain embodiments, the CAR is named "X23". In certain embodiments, the CAR (e.g., X23) comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous to the amino acid sequence described in Sequence ID No. 51 provided below. Sequence ID 51 contains a CD8 reader sequence in amino acids 1-18 and can bind to CD19 (e.g., human CD19).

[0264] [ka]

[0265] An exemplary nucleic acid sequence encoding the amino acid sequence of Sequence ID No. 51 is described below in Sequence ID No. 52.

[0266] [ka]

[0267] X2X Structures In certain embodiments, the CAR of this disclosure comprises an extracellular antigen-binding domain that binds to a CD19 polypeptide (e.g., human CD19 polypeptide), a transmembrane domain and hinge / spacer region derived from a CD28 polypeptide, an intracellular signaling domain comprising a modified CD3ζ polypeptide (e.g., modified human CD3ζ polypeptide) including native ITAM2, native BRS1, native BRS2, ​​native BRS3, an ITAM1 variant having two loss-of-function mutations, and an ITAM3 variant having two loss-of-function mutations, and a co-stimulatory signaling region comprising a CD28 polypeptide (e.g., human CD28 polypeptide). In certain embodiments, the CAR is named "X2X". In certain embodiments, the CAR (e.g., X2X) comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence described in Sequence ID No. 53 provided below. Sequence ID 53 contains a CD8 reader sequence in amino acids 1-18 and can bind to CD19 (e.g., human CD19).

[0268] [ka]

[0269] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 53 is described below in SEQ ID NO: 54.

[0270] [ka]

[0271] 12X Structures In certain embodiments, the CAR of the Disclosure comprises an extracellular antigen-binding domain that binds to a CD19 polypeptide (e.g., human CD19 polypeptide), a transmembrane domain and hinge / spacer region derived from a CD28 polypeptide, an intracellular signaling domain comprising a modified CD3ζ polypeptide (e.g., modified human CD3ζ polypeptide) containing native ITAM1, native ITAM2, native BRS1, native BRS2, ​​native BRS3, and an ITAM3 variant having two loss-of-function mutations, and a co-stimulatory signaling region comprising a CD28 polypeptide (e.g., human CD28 polypeptide). In certain embodiments, the CAR is named "12X". In certain embodiments, the CAR (e.g., 12X) comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence described in Sequence ID No. 55 provided below. Sequence ID 55 contains a CD8 reader sequence in amino acids 1-18 and can bind to CD19 (e.g., human CD19).

[0272] [ka]

[0273] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 55 is described below in SEQ ID NO: 56.

[0274] [ka]

[0275] D3 Structure In certain embodiments, the CAR of this disclosure comprises an extracellular antigen-binding domain that binds to a CD19 polypeptide (e.g., human CD19 polypeptide), a transmembrane domain and hinge / spacer region derived from a CD28 polypeptide, an intracellular signaling domain comprising a modified CD3ζ polypeptide (e.g., modified human CD3ζ polypeptide) including deletions of ITAM1, ITAM2, BRS1, BRS2, ​​and parts of ITAM3 and BRS3, and a co-stimulatory signaling region comprising a CD28 polypeptide (e.g., human CD28 polypeptide), wherein the modified CD3ζ polypeptide comprises natural ITAM1, natural ITAM2, natural BRS1 and natural BRS2, ​​but not ITAM3 (natural or modified) or natural BRS3. In certain embodiments, the CAR is named "D3". In certain embodiments, the CAR (e.g., D3) includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence described in Sequence ID No. 57 provided below. Sequence ID No. 57 includes a CD8 reader sequence in amino acids 1-18 and can bind to CD19 (e.g., human CD19).

[0276] [ka]

[0277] An exemplary nucleic acid sequence encoding the amino acid sequence of Sequence ID No. 57 is described below in Sequence ID No. 58.

[0278] [ka]

[0279] 19-166-28z Structure In certain embodiments, the CAR of this disclosure comprises an extracellular antigen-binding domain that binds to a CD19 polypeptide (e.g., human CD19 polypeptide), a transmembrane domain and hinge / spacer region derived from a CD166 polypeptide, an intracellular signaling domain comprising a modified CD3ζ polypeptide (e.g., modified human CD3ζ polypeptide) including native ITAM1, native ITAM2, natural ITAM3, natural BRS1, natural BRS2, ​​and natural BRS3, and a co-stimulatory signaling region comprising a CD28 polypeptide (e.g., human CD28 polypeptide). In certain embodiments, the CAR is named "19-166-28z". In certain embodiments, the CAR (e.g., 19-166-28z) comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence described in Sequence ID No. 59 provided below. Sequence ID 59 contains a CD8 reader sequence in amino acids 1-18 and can bind to CD19 (e.g., human CD19).

[0280] [ka]

[0281] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 59 is described in SEQ ID NO: 60, provided below.

[0282] [ka]

[0283] 19-166-28z 1XX Construction In certain embodiments, the CAR of this disclosure comprises an extracellular antigen-binding domain that binds to a CD19 polypeptide (e.g., human CD19 polypeptide), a transmembrane domain and hinge / spacer region derived from a CD166 polypeptide, an intracellular signaling domain comprising a modified CD3ζ polypeptide (e.g., modified human CD3ζ polypeptide) including native ITAM1, native BRS1, native BRS2, ​​native BRS3, an ITAM2 variant with two loss-of-function mutations, and an ITAM3 variant with two loss-of-function mutations, and a co-stimulatory signaling region comprising a CD28 polypeptide (e.g., human CD28 polypeptide). In certain embodiments, the CAR is named "19-166-28z 1XX". In certain embodiments, the CAR (e.g., 19-166-28z 1XX) comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence described in Sequence ID No. 61 provided below. Sequence ID No. 61 comprises a CD8 reader sequence in amino acids 1-18 and can bind to CD19 (e.g., human CD19).

[0284] [ka]

[0285] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 61 is described in SEQ ID NO: 62, provided below.

[0286] [ka]

[0287] 19-166-28z D23 Structure In certain embodiments, the CAR of this disclosure comprises an extracellular antigen-binding domain that binds to a CD19 polypeptide (e.g., human CD19 polypeptide), a transmembrane domain and hinge / spacer region derived from a CD166 polypeptide, an intracellular signaling domain comprising a modified CD3ζ polypeptide (e.g., modified human CD3ζ polypeptide) including deletions of ITAM1, BRS1, and ITAM2, ITAM3, BRS2, ​​and BRS3, and a co-stimulatory signaling region comprising a CD28 polypeptide (e.g., human CD28 polypeptide), wherein the modified CD3ζ polypeptide comprises natural ITAM1 and natural BRS1, but not ITAM2 (natural or modified), ITAM3 (natural or modified), BRS2 (natural or modified), or BRS3 (natural or modified). In certain embodiments, the CAR is named "19-166-28z D23". In certain embodiments, the CAR (e.g., 19-166-28z D23) comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence described in Sequence ID No. 63 provided below. Sequence ID No. 63 comprises a CD8 reader sequence in amino acids 1-18 and can bind to CD19 (e.g., human CD19).

[0288] [ka]

[0289] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 63 is described below in SEQ ID NO: 64.

[0290] [ka]

[0291] 3. Immune-responsive cells The subject matter of this disclosure provides immune-responsive cells comprising one or more CARs disclosed herein. In certain embodiments, the CARs can activate immune-responsive cells. In certain embodiments, the CARs are expressed from an endogenous locus (e.g., TRAC).

[0292] In certain embodiments, immune-responsive cells containing one or more CARs of the Disclosure exhibit improved therapeutic efficacy compared to control cells. In certain embodiments, immune-responsive cells containing one or more CARs of the Disclosure exhibit similar cytolytic effects compared to control cells. In certain embodiments, immune-responsive cells containing one or more CARs of the Disclosure exhibit increased cell accumulation when administered to a subject compared to control cells. In certain embodiments, immune-responsive cells containing one or more CARs of the Disclosure exhibit reduced cell waste when administered to a subject compared to control cells. Immune-responsive cell waste markers include, but are not limited to, TIM3, LAG3, and PD1. In certain embodiments, immune-responsive cells containing CARs of the Disclosure maintain a higher contingent of memory immune cells when administered to a subject compared to control cells. Memory immune cell markers include, but are not limited to, CD62L and CD45RA. In certain embodiments, immune-responsive cells containing one or more CARs of the Disclosure secrete cytokines at similar levels compared to control cells. In certain embodiments, cytokines secreted by immune-responsive cells include, but are not limited to, TNFα, IFNγ, and IL2. In certain embodiments, control cells contain CARs containing an intracellular signaling domain comprising a modified CD3ζ polypeptide, wherein the modified CD3ζ polypeptide comprises all natural ITAM1-3 and all natural BRS1-3.

[0293] 3.1 Immune-responsive cells containing two or more CARs In certain embodiments, the immune-responsive cell contains two or more CARs. In certain embodiments, at least one of the two or more CARs is a CAR disclosed herein. In certain embodiments, the immune-responsive cell contains two CARs. In certain embodiments, the immune-responsive cell contains three CARs.

[0294] In certain embodiments, the immune-responsive cell includes a) a first CAR comprising a first extracellular antigen-binding domain for binding to a first antigen, a first transmembrane domain, and a first intracellular signaling domain comprising a modified CD3ζ polypeptide (e.g., one modified CD3ζ polypeptide disclosed herein); and b) a second CAR comprising a second extracellular antigen-binding domain for binding to a second antigen, a second transmembrane domain, and a second intracellular signaling domain. In certain embodiments, the first CAR further comprises a first hinge / spacer region. In certain embodiments, the second CAR further comprises a second hinge / spacer region.

[0295] In certain embodiments, the second intracellular signaling domain includes a modified CD3ζ polypeptide (e.g., one modified CD3ζ polypeptide disclosed herein). In certain embodiments, the second intracellular signaling domain includes a native CD3ζ polypeptide. In certain embodiments, the modified CD3ζ polypeptide included in the second intracellular signaling domain is the same as the modified CD3ζ polypeptide included in the first intracellular signaling domain. In certain embodiments, the modified CD3ζ polypeptide included in the second intracellular signaling domain is different from the modified CD3ζ polypeptide included in the first intracellular signaling domain. In certain embodiments, the modified CD3ζ polypeptide is selected from the group consisting of a CD3ζ polypeptide containing one natural ITAM, a CD3ζ polypeptide containing two natural ITAMs, a CD3ζ polypeptide containing three natural ITAMs, a CD3ζ polypeptide containing one ITAM variant disclosed herein, a CD3ζ polypeptide containing two ITAM variants disclosed herein, a CD3ζ polypeptide containing one natural BRS region, a CD3ζ polypeptide containing two natural BRS regions, a CD3ζ polypeptide containing three natural BRS regions, a CD3ζ polypeptide lacking all or part of ITAM1, ITAM2, ITAM3 and / or any part thereof, and any combination thereof.

[0296] In certain embodiments, two or more CARs contained in an immune-responsive cell are different (e.g., the first CAR is different from the second CAR). In certain embodiments, two or more CARs contained in an immune-responsive cell are the same (e.g., the first CAR is the same as the second CAR).

[0297] In certain embodiments, two or more CARs bind to different antigens (for example, the first antigen is different from the second antigen).

[0298] In certain embodiments, two or more CARs may contain different intracellular signaling domains (e.g., the first intracellular signaling domain may be different from the second intracellular signaling domain). In certain embodiments, two or more CARs may contain the same intracellular signaling domain (e.g., the first intracellular signaling domain may be the same as the second intracellular signaling domain).

[0299] In certain embodiments, two or more intracellular signaling domains of a CAR include different co-stimulatory signaling regions. In certain embodiments, the co-stimulatory signaling region is selected from the group consisting of CD28 polypeptide, 4-1BB polypeptide, OX40 polypeptide, ICOS polypeptide, DAP-10 polypeptide, CD27 peptide, CD40 / My88 peptide, NKGD2 peptide, and combinations thereof. In certain embodiments, two or more intracellular signaling domains of a CAR include the same co-stimulatory signaling region.

[0300] In certain embodiments, the immune-responsive cells include two, three or more CARs of the Disclosure. In certain embodiments, the intracellular signaling domains of the CARs are independently selected from the group consisting of the intracellular signaling domains 1928ζ, 19ζ, 1XX, X2X, XX3, X23, 12X, D3, D12, and D23.

[0301] The selection of CARs within a cell may depend on the density of the antigen targeted by the CAR, the sum of all ITAMs, the distance between each ITAM, the transmembrane domain of the CAR, and / or the co-stimulatory signaling domain of the CAR, as this can determine the intensity of the activation signal produced by each CAR.

[0302] In a particular embodiment, the immune-responsive cell comprises two CARs, where the first CAR comprises a first intracellular signaling domain and the second CAR comprises a second intracellular signaling domain. In a particular embodiment, the first and second intracellular signaling domains are each selected from the group consisting of intracellular signaling domains 1928ζ, 19ζ, 1XX, X2X, XX3, 12X, X23, D3, D12, and D23.

[0303] In certain embodiments, the first intracellular signaling domain is the same as the second intracellular signaling domain. In certain embodiments, the first and second intracellular signaling domains are each the 1XX intracellular signaling domain. In certain embodiments, the first and second intracellular signaling domains are the 1XX intracellular signaling domain and the D23 intracellular signaling domain. In certain embodiments, the first and second intracellular signaling domains are the 1XX intracellular signaling domain and the XX3 intracellular signaling domain. In certain embodiments, the first and second intracellular signaling domains are the D23 intracellular signaling domain and the XX3 intracellular signaling domain. In certain embodiments, the first and second intracellular signaling domains are the 1XX intracellular signaling domain and the X2X intracellular signaling domain. In certain embodiments, the first and second intracellular signaling domains are the 1XX intracellular signaling domain and the D12 intracellular signaling domain. In certain embodiments, the first intracellular signaling domain and the second intracellular signaling domain are 1XX intracellular signaling domains and 12X intracellular signaling domains. In certain embodiments, the first intracellular signaling domain and the second intracellular signaling domain are 1XX intracellular signaling domains and D3 intracellular signaling domains. In certain embodiments, the first intracellular signaling domain and the second intracellular signaling domain are X2X intracellular signaling domains and X2X intracellular signaling domains. In certain embodiments, the first intracellular signaling domain and the second intracellular signaling domain are 1928z intracellular signaling domains and 1XX intracellular signaling domains.

[0304] In certain embodiments, the first intracellular signaling domain includes or has ITAM2 variants and ITAM3 variants, and the second intracellular signaling domain includes or has deletions of ITAM2 or a portion thereof and ITAM3 or a portion thereof. In certain embodiments, the first intracellular signaling domain includes or has ITAM2 variants and ITAM3 variants, and the second intracellular signaling domain includes or has ITAM1 variants and ITAM2 variants.

[0305] In a particular embodiment, the total amount of natural ITAM contained in the two CARs is approximately 5 or less, approximately 4 or less, approximately 3 or less, or approximately 2 or less.

[0306] In a particular embodiment, the immune-responsive cell comprises three CARs, where the first CAR comprises a first intracellular signaling domain, the second CAR comprises a second intracellular signaling domain, and the third CAR comprises a third intracellular signaling domain. In a particular embodiment, the first, second, and third intracellular signaling domains are each independently selected from the group consisting of intracellular signaling domains 1928ζ, 19ζ, 1XX, X2X, XX3, X23, 12X, D3, D12, and D23.

[0307] In a particular embodiment, the first intracellular signaling domain, the second intracellular signaling domain, and the third intracellular signaling domain are the 1XX intracellular signaling domain, the D23 intracellular signaling domain, and the XX3 intracellular signaling domain. In a particular embodiment, the first intracellular signaling domain, the second intracellular signaling domain, and the third intracellular signaling domain are the D23 intracellular signaling domain, the D23 intracellular signaling domain, and the XX3 intracellular signaling domain. In a particular embodiment, the first intracellular signaling domain, the second intracellular signaling domain, and the third intracellular signaling domain are the D23 intracellular signaling domain, the XX3 intracellular signaling domain, and the XX3 intracellular signaling domain. In a particular embodiment, the first intracellular signaling domain, the second intracellular signaling domain, and the third intracellular signaling domain are the XX3 intracellular signaling domain, the XX3 intracellular signaling domain, and the XX3 intracellular signaling domain. In a particular embodiment, the first intracellular signaling domain, the second intracellular signaling domain, and the third intracellular signaling domain are 1XX intracellular signaling domains, 1XX intracellular signaling domains, and 1XX intracellular signaling domains. In a particular embodiment, the first intracellular signaling domain, the second intracellular signaling domain, and the third intracellular signaling domain are 1XX intracellular signaling domains, X2X intracellular signaling domains, and X2X intracellular signaling domains. In a particular embodiment, the first intracellular signaling domain, the second intracellular signaling domain, and the third intracellular signaling domain are 1XX intracellular signaling domains, 1XX intracellular signaling domains, and X2X intracellular signaling domains.In a particular embodiment, the first intracellular signaling domain, the second intracellular signaling domain, and the third intracellular signaling domain are 1XX intracellular signaling domains, 1XX intracellular signaling domains, and D12 intracellular signaling domains. In a particular embodiment, the first intracellular signaling domain, the second intracellular signaling domain, and the third intracellular signaling domain are 1XX intracellular signaling domains, 1XX intracellular signaling domains, and D23 intracellular signaling domains. In a particular embodiment, the first intracellular signaling domain, the second intracellular signaling domain, and the third intracellular signaling domain are 1XX intracellular signaling domains, 1XX intracellular signaling domains, and 12X intracellular signaling domains. In a particular embodiment, the first intracellular signaling domain, the second intracellular signaling domain, and the third intracellular signaling domain are 1XX intracellular signaling domains, 1XX intracellular signaling domains, and D3 intracellular signaling domains. In a particular embodiment, the first intracellular signaling domain, the second intracellular signaling domain, and the third intracellular signaling domain are the intracellular signaling domains of X2X, X2X, and X2X.

[0308] In certain embodiments, the first intracellular signaling domain includes or contains the ITAM2 variant and the ITAM3 variant, the second intracellular signaling domain includes or contains deletions of ITAM2 or a portion thereof and deletions of ITAM3 or a portion thereof, and the third intracellular signaling domain includes or contains the ITAM1 variant and the ITAM2 variant. In certain embodiments, the first intracellular signaling domain includes or has a deletion of ITAM2 or a portion thereof and a deletion of ITAM3 or a portion thereof, the second intracellular signaling domain includes or has an ITAM1 variant and an ITAM2 variant, and the third intracellular signaling domain includes or has an ITAM1 variant and an ITAM2 variant.

[0309] In a particular embodiment, the total amount of natural ITAM contained in the three CARs is approximately 5 or less, approximately 4 or less, and approximately 3 or less.

[0310] In a particular embodiment, the targets of the CAR are different from each other.

[0311] 3.1 Types of immune-responsive cells The immune-responsive cells of the subject matter of this disclosure may be lymphoid cells. The lymphoid system, including B, T, and natural killer (NK) cells, enables antibody production, regulation of the cellular immune system, detection of exogenous substances in the blood, detection of cells foreign to the host, etc. Non-limiting examples of lymphoid immune-responsive cells include T cells, natural killer (NK) cells, embryonic stem cells, and pluripotent stem cells (e.g., those capable of differentiating lymphoid cells). T cells may be lymphocytes that mature in the thymus and primarily play a role in cell-mediated immunity. T cells are involved in the adaptive immune system. The T cells of the subject matter of this disclosure include helper T cells, cytotoxic T cells, memory T cells (central memory T cells, stem cell-like memory T cells (or stem-like memory T cells) and two types of effector memory T cells: e.g., T EM Cells and T EMRA The T cells may be any type of T cell, including, but not limited to, regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosal-associated invariant T cells, and γδ T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells. A patient's own T cells can be genetically modified to target specific antigens through the introduction of CARs. In certain embodiments, the immune-responsive cells are T cells. T cells are CD4 + T cells or CD8 + It may be a T cell. In a particular embodiment, the T cell is a CD4 + These are T cells. In certain embodiments, T cells are CD8 + These are T cells.

[0312] Natural killer (NK) cells are part of cell-mediated immunity and may be lymphocytes that act during innate immune responses. NK cells do not require prior activation to carry out their cytotoxic effects against target cells.

[0313] The types of human lymphocytes that are the subject of this disclosure include, but are not limited to, peripheral donor lymphocytes, e.g., Sadelain, M., et al. 2003 Nat RevCancer3:35-45 (disclosing peripheral donor lymphocytes genetically modified to express CAR), Morgan, RA, et al. 2006 Science314:126-129 (expressing full-length tumor antigen-recognizing T cell receptor complexes containing α and β heterodimers). (Disclosing genetically modified peripheral donor lymphocytes), Panelli, MC, et al. 2000 J Immunol 164:495-504; Panelli, MC, et al. 2000 JImmunol164:4382-4392 (Discloses lymphocyte cultures derived from tumor-infiltrating lymphocytes (TILs) in tumor biopsies), and Dupont, J., et al. 2005 Cancer Res 65:5417-5427; Papanicolaou, GA, et al. 2003 Blood102:2498-2505 (Selection using artificial antigen-presenting cells (AAPCs) or pulsed dendritic cells) This includes disclosing antigen-specific peripheral blood leukocytes selectively enlarged in vitro. Immune-responsive cells (e.g., T cells) may be self, non-self (e.g., allogeneic), or can be induced in vitro from engineered progenitor cells or stem cells.

[0314] The immune-responsive cells of this disclosure are capable of modulating the tumor microenvironment. Tumors have a microenvironment that is adversarial to the host immune response, including a set of mechanisms by malignant cells to protect themselves from immune recognition and elimination. This “adversarial tumor microenvironment” is influenced by invasion-modulating CD4 +This includes the expression of various immunosuppressive factors, including T cells (Tregs), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), immunosuppressive cytokines such as TGF-β, and ligands that target immunosuppressive receptors expressed by activated T cells (CTLA-4 and PD-1). While these mechanisms of immunosuppression play a role in maintaining tolerance and suppressing inadequate immune responses, within the tumor microenvironment, these mechanisms interfere with effective anti-tumor immune responses. In summary, these immunosuppressive factors can induce significant anergy or apoptosis of CAR-modified T cells that are adopted upon encounter with targeted tumor cells.

[0315] The unpurified source of CTLs may be any known in the art, e.g., bone marrow, fetal, neonatal or adult or other hematopoietic cell sources, e.g., fetal liver, peripheral blood or umbilical cord blood. Various techniques can be used to isolate the cells. For example, negative selection methods can remove non-CTLs first. mAbs are particularly useful for identifying markers associated with specific cell lineages and / or differentiation stages for both positive and negative selection.

[0316] Relatively coarse separation allows for the initial removal of a large proportion of terminally differentiated cells. For example, magnetic bead separation can be used first to remove a large number of irrelevant cells. In certain embodiments, at least about 80%, and usually at least 70%, of all hematopoietic cells are removed before cell isolation.

[0317] The separation procedure may include, but is not limited to, density gradient centrifugation; recoagulation (resetting); and cell density. Coupling with modified particles; magnetic separation by antibody-coated magnetic beads; affinity chromatography; cytotoxic agents, including, but not limited to, complement and cytotoxins, linked to or used with mAbs; as well as panning, elatrillation, or any other convenient techniques using antibodies attached to solid matrices, such as plates or chips.

[0318] The separation and analysis techniques include, but are not limited to, flow cytometry, which can have varying degrees of sophistication, for example, multiple color channels, low-angle and obtuse-angle light scattering detection channels, and impedance channels.

[0319] Cells can be selected for dead cells by using dead cell-associated dyes such as propidium iodide (PI). In certain embodiments, cells are collected in a medium containing 2% fetal bovine serum (FCS) or 0.2% bovine serum albumin (BSA), or any other suitable, for example, sterile, isotonic medium.

[0320] 4. Vector Genetic modification of immune-responsive cells (e.g., T cells or NK cells) can be achieved by transducing a substantially homogeneous cell composition with recombinant DNA constructs. In certain embodiments, retroviral vectors (either gamma-retroviruses or lentiviruses) are used to introduce DNA constructs into cells. For example, polynucleotides encoding CARs can be cloned into a retroviral vector, and expression can be induced from its endogenous promoter, from the terminal repeat sequence of the retrovirus, or from a promoter specific to the target cell type of interest. Non-viral vectors can be used in a similar manner.

[0321] When initially genetically modifying immune-responsive cells to include CARs, retroviral vectors are commonly used for transduction, but any other suitable viral vector or nonviral delivery system can be used. CARs can be constructed in a single polycistronic expression cassette, in multiple expression cassettes of a single vector, or in multiple vectors together with auxiliary molecules (e.g., cytokines). Examples of elements for constructing polycistronic expression cassettes include various viral and nonviral intra-sequence ribosome entry sites (IRESs, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-κB IRES, RUNX1) Examples include, but are not limited to, IRESs (IRESs, p53 IRESs, hepatitis A IRESs, hepatitis C IRESs, pestivirus IRESs, aftvirus IRESs, picornavirus IRESs, poliovirus IRESs, and encephalomyocarditis virus IRESs), and cleavable linkers (e.g., 2A peptides, e.g., P2A, T2A, E2A, and F2A peptides). In certain embodiments, any vector or CAR disclosed herein may contain a P2A peptide comprising the amino acid sequence GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 107). A combination of a retroviral vector and an appropriate packaging system is also suitable if the capsid protein is functional for infecting human cells. Various amphitrophic virus-producing cell lines are known, including but not limited to PA12 (Miller, et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller, et al. (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP (Danos, et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464). Non-amphitrophic particles, such as pseudotyped particles with VSVG, RD114, or GALV envelopes, and any other particles known in the art are also suitable.

[0322] Possible methods of transduction include, for example, Bregni, et al. (1992) Blood 80:1418-1422. Direct co-culture of cells with producing cells by the method, or by the method of, for example, Xu, et al. (1994) Exp. Hemat. 22:223-230; and Hughes, et al. (1992) J. Clin. Invest. 89:1817 This includes culturing with viral supernatant alone or with enriched vector stocks, with or without appropriate growth factors and polycations.

[0323] Other transdextrin viral vectors can be used to modify immune-responsive cells. In certain embodiments, the selected vectors exhibit highly efficient infection and stable incorporation and expression (see, for example, Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldinietal., Science 272:263-267, 1996 and Miyoshi et al., Proc. Natl. Acad. Sci.USA 94:10319, 1997). Other viral vectors include, for example, adenovirus, lentivirus and adeno-associated virus vectors, vaccinia virus, bovine papillomavirus or herpesvirus, such as Epstein-Barr virus (e.g., Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitisetal., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990). (See also the vectors in Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; LeGal La Salle et al., Science 259:988-990, 1993 and Johnson, Chest 107:77S-83S, 1995). Retroviral vectors are particularly well-developed and used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Patent No. 5,399,346).

[0324] Non-viral approaches can also be used for genetic modification of immune-responsive cells. For example, by administering nucleic acids in the presence of lipofection (Feigner etal., Proc. Natl. Acad. Sci. USA 84:7413, 1987; Ono etal., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), asialorosomucoid-polylysine conjugate Nucleic acid molecules can be introduced into immune-responsive cells by microinjection (Wuetal., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989) or by microinjection under surgical conditions (Wolffetal., Science 247:1465, 1990). Other non-viral means for gene transfer include in vitro transfection using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes may also be potentially beneficial for DNA delivery to cells. Transplantation of normal genes into the affected tissue of a subject can also be achieved by ex vivo introducing normal nucleic acids into a cultureable cell type (e.g., autologous or xenogeneic primary cells or their progeny), which are then injected into the target tissue or systemically. Recombinant receptors can also be induced or obtained using transposases or targeted nucleases (e.g., zinc finger nucleases, meganucleases, or TALE nucleases, CRISPR). Transient expression can be obtained by RNA electroporation.

[0325] The clustered, regularly arranged short palindromic repeat (CRISPR) system is a genome editing tool discovered in prokaryotic cells. When used for genome editing, the system includes Cas9 (a protein that can modify DNA using crRNA as its guide), CRISPR RNA (a crRNA containing a region that binds to tracrRNA (generally in the form of a hairpin loop) to form an active complex with Cas9, which is used by Cas9 to guide it to the correct part of the host DNA), transactivating crRNA (tracrRNA that binds to crRNA and forms an active complex with Cas9), and a DNA repair template (DNA that induces a cellular repair process that allows for the insertion of a specific DNA sequence), as needed. CRISPR / Cas9 often uses plasmids to transfect target cells. The crRNA is the sequence that Cas9 uses to identify the target DNA in the cell and bind to it directly, so it needs to be designed for each application. The repair template with a CAR expression cassette also needs to be designed for each application, as it must overlap with the sequence on either side of the cleavage and encode the insertion sequence. Multiple crRNAs and tracrRNAs can be packaged together to form a single guide RNA (sgRNA). This sgRNA can then be ligated together with the Cas9 gene to form a plasmid for transfection into cells.

[0326] Zinc finger nucleases (ZFNs) are artificial restriction enzymes produced by combining a zinc finger DNA-binding domain with a DNA-cleaving domain. The zinc finger domain can be manipulated to target specific DNA sequences, enabling the zinc finger nuclease to target desired sequences within the genome. The DNA-binding domain of individual ZFNs typically contains multiple individual zinc finger repeat sequences, each capable of recognizing multiple base pairs. The most common method for generating novel zinc finger domains is by combining known zinc finger "modules" with less specificity. The most common cleavage domain in ZFNs is the nonspecific cleavage domain from the type II restriction endonuclease FokI. ZFNs can be used to insert CAR expression cassettes into the genome using homologous DNA templates with endogenous homologous recombination (HR) mechanisms and CAR expression cassettes. When a target sequence is cleaved by a ZFN, the HR mechanism searches for homology between the damaged chromosome and a homologous DNA template, then copies the template sequence between the two cleaved ends of the chromosome, thereby incorporating the homologous DNA template into the genome.

[0327] Activator-like effector nucleases (TALENs) are restriction enzymes that can be manipulated to cleave specific sequences of DNA. The TALEN system operates on almost the same principle as ZFNs. They are generated by combining an activator-like effector DNA-binding domain with a DNA-cleaving domain. Activator-like effectors (TALEs) consist of a repeating motif of 33-34 amino acids with two variable positions that have strong recognition for specific nucleotides. By assembling arrays of these TALEs, the TALE DNA-binding domain can be manipulated to bind to a desired DNA sequence and thereby induce a nuclease to cleave at a specific site in the genome. cDNA expression for use in polynucleotide therapy can be induced from any suitable promoter (e.g., human cytomegalovirus (CMV), Simian virus 40 (SV40), or metallothionein promoter) and can be regulated by any suitable mammalian regulatory element or intron (e.g., elongation factor 1a enhancer / promoter / intron structure). For example, enhancers known to preferentially induce gene expression in specific cell types may be used to induce nucleic acid expression, if desired. The enhancers used may include, but are not limited to, those characterized as tissue or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation may be mediated by a congeneral regulatory sequence, or optionally by a regulatory sequence derived from a heterologous source containing any of the promoters or regulatory elements described above.

[0328] The resulting cells can be grown under conditions similar to those for unmodified cells, thereby allowing the modified cells to be enlarged and used for various purposes.

[0329] 5. Genome editing methods The CARs of the Disclosure can be positioned at one or more endogenous loci of the immune-responsive cells of the Disclosure using any targeted genome editing method. In certain embodiments, a CRISPR system is used to deliver the CARs of the Disclosure to one or more endogenous loci of the immune-responsive cells of the Disclosure. In certain embodiments, a zinc finger nuclease is used to deliver the CARs of the Disclosure to one or more endogenous loci of the immune-responsive cells of the Disclosure. In certain embodiments, a TALEN system is used to deliver the CARs of the Disclosure to one or more endogenous loci of the immune-responsive cells of the Disclosure.

[0330] The method for delivering genome editing agents / systems may vary depending on the need. In certain embodiments, components of the selected genome editing method are delivered as DNA constructs in one or more plasmids. In certain embodiments, components are delivered through a viral vector. Common delivery methods include, but are not limited to, electroporation, microinjection, gene guns, impalefection, hydrostatic pressure, serial injection, sonication, magnetofection, adeno-associated viruses, envelope protein pseudotyping of viral vectors, cis and trans-acting elements of replicating vectors, herpes simplex viruses, and chemical vehicles (e.g., oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles, and cell-permeable peptides).

[0331] The placement of the CARs in this disclosure can be performed at any endogenous locus. In certain embodiments, the endogenous locus is the TRAC locus, the TRBC locus, or the TRGC locus; in certain embodiments, the endogenous locus is the TRAC locus. In certain embodiments, the placement of the CAR disrupts or inactivates the endogenous expression of the TCR.

[0332] 6. Polypeptides and Analogues CD19, CD8, CD28, CD3ζ, CD40, 4-1BB, OX40, CD84, CD166, CD8a, CD8b, ICOS, ICAM-1, CD27, MY88, NKGD2, and CTLA-4 polypeptides or fragments thereof, modified to enhance their antineoplastic activity when expressed in immune-responsive cells, are also included in the subject matter of this disclosure. The subject matter of this disclosure provides methods for optimizing amino acid sequences or nucleic acid sequences by introducing changes in the sequence. Such changes may include certain mutations, deletions, insertions, or post-translational modifications. The subject matter of this disclosure further includes analogs of any naturally occurring polypeptide disclosed herein (including, but not limited to, CD19, CD8, CD28, CD3ζ, CD40, 4-1BB, OX40, CD27, CD40 / My88, NKGD2, CD84, CD166, CD8a, CD8b, ICOS, ICAM-1, and CTLA-4). The analogs may differ from the naturally occurring polypeptides disclosed herein by differences in amino acid sequence, by post-translational modification, or both. The analogs may exhibit at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher homology to all or part of the naturally occurring amino acid sequences of the subject matter of this disclosure. The length of the sequence comparison is at least 5, 10, 15, or 20 amino acid residues, for example, at least 25, 50, or 75 amino acid residues, or longer than 100 amino acid residues. In this case as well, in an exemplary approach to determining the degree of identity, the BLAST program is used to show closely related sequences. -3 from e -100It may be used as a probability score between the following. Modifications include in vivo and in vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, phosphorylation, or glycosylation, and such modifications may occur during polypeptide synthesis or processing, or after treatment with isolated modified enzymes. Analogues may also differ from naturally occurring polypeptides due to changes in the primary sequence. These include both native and derived gene variants (e.g., resulting from random mutagenesis due to radiation or exposure to ethanemethyl sulfate, or Sambrook, Fritschand Maniatis, Molecular Cloning: A Laboratory Manual) (2nd ed.), CSHPress, 1989, or Ausubel et al., the site-specific mutation described above. This includes (dysinogenic) compounds. It also includes cyclized peptides, molecules, and analogues containing residues other than L amino acids, such as D amino acids or naturally occurring or synthetic amino acids, such as β or γ amino acids.

[0333] In addition to full-length polypeptides, the subject matter of this disclosure also provides fragments of either the polypeptides or peptide domains disclosed herein. As used herein, the term “fragment” means at least 5, 10, 13, or 15 amino acids. In certain embodiments, a fragment comprises at least 20 consecutive amino acids, at least 30 consecutive amino acids, or at least 50 consecutive amino acids. In certain embodiments, a fragment comprises at least 60–80, 100, 200, 300, or more consecutive amino acids. Fragments can be generated by methods known to those skilled in the art, or may result from normal protein processing (e.g., removal of amino acids not necessary for biological activity from a nascent polypeptide, or removal of amino acids by alternative mRNA splicing or alternative protein processing events).

[0334] Non-protein analogs have a chemical structure designed to mimic the functional activity of the proteins disclosed herein. Such analogs may exceed the physiological activity of the original polypeptide. Methods for designing analogs are well known in the art, and the synthesis of analogs can be carried out according to such methods by modifying the chemical structure of the original polypeptide so that the resulting analog increases the antineoplastic activity when expressed in immune-responsive cells. These chemical modifications include, but are not limited to, substituting alternative R groups and altering the degree of saturation of specific carbon atoms of the reference polypeptide. In certain embodiments, protein analogs are relatively resistant to degradation in vivo and, when administered, result in a more sustained therapeutic effect. Assays for measuring functional activity include, but are not limited to, the assays described in the following examples.

[0335] 7. Administration Compositions comprising the immune-responsive cells of this disclosure can be delivered systemically or directly to a subject to induce and / or enhance an immune response to an antigen, and / or to treat and / or prevent neoplasms, pathogen infections, or infectious diseases. In certain embodiments, the immune-responsive cells or compositions comprising the same of this disclosure are injected directly into an organ of interest (e.g., an organ affected by a neoplasm). Alternatively, the immune-responsive cells or compositions comprising the same of this disclosure are delivered indirectly to an organ of interest, for example, by administration into the circulatory system (e.g., the tumor vascular system). To increase the in vitro or in vivo generation of T cells, NK cells, or CTL cells, growth and differentiation agents can be provided before, during, or after administration of the cells or compositions.

[0336] The immune-responsive cells of this disclosure can be administered, typically intravascularly, within any physiologically acceptable vehicle, but they can also be introduced into bone or other convenient sites (e.g., the thymus) where the cells can find a suitable site for regeneration and differentiation. Typically, at least about 1 × 10⁻⁶ 5Individual cells are administered, and ultimately approximately 1 × 10⁶ cells are produced. 10 The number of cells or more can be reached. The immune-responsive cells of this disclosure may include purified cell populations. Those skilled in the art can easily determine the percentage of immune-responsive cells of this disclosure in a population using various well-known methods such as fluorescence-activated cell sorting (FACS). Preferred purity ranges in a population containing immune-responsive cells of this disclosure are about 50% to about 55%, about 5% to about 60%, and about 65% to about 70%. In certain embodiments, the purity is about 70% to about 75%, about 75% to about 80%, or about 80% to about 85%. In certain embodiments, the purity is about 85% to about 90%, about 90% to about 95%, and about 95% to about 100%. The dosage can be easily adjusted by those skilled in the art (for example, a decrease in purity may require an increase in dosage). The cells can be introduced by injection, catheter, etc.

[0337] The compositions of this disclosure may be pharmaceutical compositions comprising immune-responsive cells or their progenitor cells and a pharmaceutically acceptable carrier. Administration may be autologous or heterologous. For example, immune-responsive cells or progenitor cells may be obtained from one subject and administered to the same subject or to different, suitable subjects. Immune-responsive cells or their progeny derived from peripheral blood (e.g., in vivo, ex vivo, or in vitro) may be administered by local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. When a therapeutic composition of the subject of this disclosure (e.g., a pharmaceutical composition comprising immune-responsive cells of this disclosure) is administered, it may be formulated in unit dose injectable form (liquid, suspension, emulsion).

[0338] 8. Formulation The compositions comprising immune-responsive cells of this disclosure can be conveniently provided as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, that can be buffered to a selected pH. Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Furthermore, liquid compositions are slightly more convenient to administer, particularly by injection. On the other hand, viscous compositions can be formulated within a suitable viscosity range to provide a longer contact period with specific tissues. The liquid or viscous composition may contain a carrier, which may be a solvent or dispersion medium containing, for example, water, saline solution, phosphate-buffered saline solution, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof.

[0339] Sterile, injectable solutions can be prepared by incorporating genetically modified immune-responsive cells into the required amount of a suitable solvent along with various amounts of other components as desired. Such compositions may be mixed with suitable carriers, diluents, or excipients, such as sterile water, physiological saline, glucose, or dextrose. The compositions may be lyophilized. Depending on the desired administration route and preparation, the compositions may contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffers, gelling or thickening additives, preservatives, flavoring agents, and colorants. To prepare suitable preparations without excessive experimentation, one can refer to standard texts, such as "REMINGTON'SPHARMACEUTICALSCIENCE", 17th edition, 1985, which is incorporated herein by reference.

[0340] Various additives can be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of microbial activity can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. Long-term absorption of the injectable pharmaceutical form can be achieved by the use of absorption-delaying agents, such as aluminum monostearate and gelatin. However, according to the subject matter of this disclosure, any vehicle, diluent, or additive used must be compatible with genetically modified immune-responsive cells or their progenitor cells.

[0341] The compositions may be isotonic, meaning they can have the same osmotic pressure as blood and tears. The desired isotonicity of the composition can be achieved using sodium chloride or other pharmaceutically acceptable agents, such as dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes. Sodium chloride may be particularly useful for buffers containing sodium ions.

[0342] The viscosity of the composition can be maintained to a selected level using a pharmaceutically acceptable thickener, if desired. For example, methylcellulose is readily and economically available and easy to work with. Other suitable thickeners include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, and carbomer. The concentration of the thickener may depend on the selected agent. The important point is to use an amount that achieves the desired viscosity. Obviously, the selection of a suitable carrier and other additives depends on the exact route of administration and the specific dosage form, for example, the properties of the liquid dosage form (e.g., whether the composition is formulated as a solution, suspension, gel, or another liquid form, such as a time-release form or a liquid-filled form).

[0343] The amount of cells administered varies with respect to the subject being treated. In one embodiment, about 10 4 ~about 10 10 pieces, about 10 5 ~about 109 10, or about 10 6 ~about 10 8 A number of immune-responsive cells of this disclosure are administered to a human subject. More effective cells may be administered in even smaller quantities. In certain embodiments, at least about 1 × 10⁶ cells are administered. 8 pieces, approximately 2×10 8 pieces, about 3×10 8 pieces, about 4×10 8 pieces, or approximately 5 x 10 8 A number of immune-responsive cells of this disclosure are administered to a human subject. In a particular embodiment, approximately 1 × 10⁶ cells are administered to a human subject. 7 ~5×10 8 Individual immunoresponsive cells of this disclosure are administered to human subjects. The precise determination of the amount considered to be an effective dose may be based on individual factors for each subject, including the size, age, sex, weight, and condition of the particular subject. The dosage can be readily determined by those skilled in the art from this disclosure and knowledge in the art.

[0344] Those skilled in the art can easily determine the amounts of cells and, as needed, additives, media, and / or carriers administered in the composition and in the method. Typically, any additives (in addition to active cells and / or activators) are present in phosphate-buffered saline in amounts of 0.001 to 50% (by weight) solution, and the active ingredients are present in amounts on the order of micrograms to milligrams, for example, about 0.0001 to about 5% by weight, about 0.0001 to about 1% by weight, about 0.0001 to about 0.05% by weight, or about 0.001 to about 20% by weight, about 0.01 to about 10% by weight, or about 0.05 to about 5% by weight. With respect to any composition administered to animals or humans, the following can be determined: toxicity by determining the lethal dose (LD) and LD50 in a suitable animal model, e.g., rodents such as mice; the dosage of the composition that elicits a suitable response, the concentration of its components, and the timing of administration of the composition. Such a determination does not require excessive experimentation based on the knowledge of those skilled in the art, this disclosure, and the documents referenced herein. The duration of continuous administration can also be determined without excessive experimentation.

[0345] 9. Treatment Method The subject matter of this disclosure provides methods for inducing and / or increasing an immune response in subjects in need. The immune-responsive cells and compositions comprising the same of this disclosure can be used to treat and / or prevent neoplasms in subjects. The immune-responsive cells and compositions comprising the same of this disclosure can be used to prolong the survival of subjects suffering from neoplasms. The immune-responsive cells and compositions comprising the same of this disclosure can also be used to treat and / or prevent pathogen infections or other infectious diseases in subjects, for example, immunodeficient human subjects. In certain embodiments, immune-responsive cells comprising CARs disclosed herein can be used to treat a subject having a relapse of disease, and the subject has been treated to result in residual tumor cells. In certain embodiments, the residual tumor cells have a low density of target molecules on the surface of the tumor cells. In certain embodiments, the target molecules having a low density on the cell surface have a density of less than about 10,000 molecules / cell, less than about 8,000 molecules / cell, less than about 6,000 molecules / cell, less than about 4,000 molecules / cell, less than about 2,000 molecules / cell, less than about 1,000 molecules / cell, less than about 500 molecules / cell, less than about 200 molecules / cell, or less than about 100 molecules / cell. In certain embodiments, the target molecules having a low density on the cell surface have a density between about 4,000 and about 2,000 molecules / cell, or between about 2,000 and about 1,000 molecules / cell. In certain embodiments, immune-responsive cells containing the CARs disclosed herein can be used to treat subjects having disease relapses, and the subjects received immune-responsive cells (e.g., T cells) containing a CAR (e.g., 4-1BBz CAR) containing an intracellular signaling domain containing a co-stimulatory signaling domain containing a 4-1BB polypeptide. In certain embodiments, tumor cells have a low density of tumor-specific antigens on the surface of the tumor cells. In a particular embodiment, the disease is CD19 +ALL. In certain embodiments, tumor cells have a low density of CD19 on the tumor cells. Such methods involve administering an effective amount of the immune-responsive cells of the present disclosure, or a composition containing them (e.g., a pharmaceutical composition), to achieve a desired effect, mitigation of an existing condition, or prevention of recurrence. In the case of treatment, the amount administered is an effective amount to produce the desired effect. The effective amount may be provided in one or a series of doses. The effective amount may be provided by a bolus or continuous perfusion.

[0346] An effective dose (or therapeutic effective dose) is the amount sufficient to achieve a beneficial or desired clinical outcome through treatment. An effective dose may be administered to a subject in one or more doses. With respect to treatment, an effective dose is sufficient to alleviate, improve, stabilize, reverse or slow the progression of a disease, or otherwise reduce the pathological outcomes of the disease. The effective dose is generally determined on a case-by-case basis by a physician and is within the scope of the skill of those skilled in the art. Several factors are generally considered when determining an appropriate dosage to achieve an effective dose. These factors include the subject's age, sex, and weight, the condition being treated, the severity of the condition, and the morphology and effective concentration of the immune-responsive cells being administered.

[0347] For adoptive immunotherapy using antigen-specific T cells, approximately 10 6 ~10 10 (For example, about 10 9 Cell doses within the range of ) are generally injected. Upon administration of the cells of this disclosure to the host and subsequent differentiation, T cells that are specifically directed toward specific antigens are induced. Immune-responsive cells can be administered by any method known in the art, including, but not limited to, intravenous, subcutaneous, intranodal, intratumoral, intrathecal, intrapleural, intraperitoneal, and directly to the thymus.

[0348] The subject matter of this disclosure provides a method for treating and / or preventing neoplasms in a subject. The method may include administering an effective amount of the immune-responsive cells of this disclosure or a composition comprising them to a subject having a neoplasm.

[0349] Non-exclusive examples of neoplasms include blood cancers (e.g., leukemia, lymphoma, and myeloma), ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, glioblastoma, throat cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma, and various carcinomas (including prostate and small cell lung cancer). Suitable carcinomas include astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymal cell carcinoma, medulloblastoma, undifferentiated neuroectodermal tumor (PNET), chondrosarcoma, osteogenic sarcoma, pancreatic ductal adenocarcinoma, small and large cell lung adenocarcinoma, chordoma, angiosarcoma, endosarcoma, squamous cell carcinoma, bronchoalveolar carcinoma, epithelial adenocarcinoma and its liver metastases, lymphangiosarcoma, lymphangiosarcoma, hepatocellular carcinoma, cholangiocarcinoma, synoviomas, mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon cancer, basal cell carcinoma, sweat gland carcinoma, papillary carcinoma, sebaceous carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, fetal cancer, Wilms' tumor, and testicular cancer. This further includes any known tumors in the field of oncology, without limitation, including medulloblastoma, craniopharyngioma, ependymocyteoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, Waldenström hypergammaglobulinemia, and heavy chain disease, breast tumors such as tubular and lobular adenocarcinoma, squamous and adenocarcinoma of the cervix, epithelial carcinoma of the uterus and ovaries, prostate adenocarcinoma, transitional squamous cell carcinoma of the bladder, B and T cell lymphoma (nodular and diffuse), plasmacytoma, acute and chronic leukemia, malignant melanoma, soft tissue sarcoma and leiomyosarcoma. In certain embodiments, the neoplasm is selected from the group consisting of blood cancers (e.g., leukemia, lymphoma, and myeloma), ovarian cancer, prostate cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, gastric cancer, glioblastoma, and throat cancer. In certain embodiments, the immune-responsive cells of the Disclosure and compositions comprising them can be used to treat and / or prevent blood cancers (e.g., leukemia, lymphoma, and myeloma) or ovarian cancers that are not suitable for conventional therapeutic interventions.

[0350] The subject may have an advanced form of the disease, in which case the objective of treatment may include mitigating or reversing disease progression and / or improving side effects. The subject may have a history of a condition already treated, in which case the objective of treatment generally includes reducing or delaying the risk of recurrence.

[0351] Suitable human subjects for therapy generally include two treatment groups that can be distinguished by clinical criteria. Subjects with “progressive disease” or “high tumor burden” are those with clinically measurable tumors. Clinically measurable tumors are those that can be detected based on tumor mass (e.g., palpation, CAT scan, sonograph, mammography or radiography; positive biochemical or histopathological markers alone are insufficient to identify this population). Pharmaceutical compositions are administered to these subjects to elicit an antitumor response with the aim of alleviating the subject’s condition. Ideally, a reduction in tumor mass occurs as a result, but any clinical improvement constitutes a benefit. Clinical improvement includes a reduction in the risk or rate of tumor progression or a reduction in pathological outcomes.

[0352] A second group of suitable subjects is known in the art as the “adjuvant group.” These are individuals with a history of neoplasm but who are responsive to a different mode of therapy. The previous therapy could include, but not limited to, surgical resection, radiotherapy, and traditional chemotherapy. As a result, these individuals do not have a clinically measurable tumor. However, they are suspected to be at risk of disease progression near the original tumor site or due to metastasis. This group can be further subdivided into high-risk and low-risk individuals. Subdivision is based on features observed before and after initial treatment. These features are known in the clinical field and are appropriately defined for each different neoplasm. A common feature in the high-risk subgroup is that the tumor has invaded adjacent tissue or shows lymph node involvement.

[0353] Another group has a genetic predisposition to neoplasms but does not yet have demonstrated clinical signs of neoplasms. For example, a woman who tests positive for a gene mutation associated with breast cancer but is still of childbearing age may wish to receive one or more of the immune-responsive cells described herein as a preventive measure to prevent the development of neoplasms until it is appropriate to perform preventive surgery.

[0354] Furthermore, the subject matter of this disclosure provides a method for treating and / or preventing a pathogenic infection (e.g., a viral, bacterial, fungal, parasitic, or protozoan infection) in a subject, such as an immunodeficient subject. The method may include administering an effective amount of the immune-responsive cells of this disclosure or a composition comprising them to a subject having a pathogenic infection. Exemplary viral infections that are susceptible to treatment include, but are not limited to, cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), and influenza virus infections.

[0355] Further modifications can be introduced into the immune-responsive cells (e.g., T cells) of this disclosure to avoid or minimize the risk of immunological complications (known as “malignant T-cell transformation”), such as graft-versus-host disease (GvHD), or when healthy tissue expresses the same target antigens as tumor cells, leading to outcomes similar to GvHD. A potential solution to this problem is to incorporate a suicide gene into the immune-responsive cells of this disclosure. Suitable suicide genes include, but are not limited to, herpes simplex virus thymidine kinase (hsv-tk), an inducible caspase 9 suicide gene (iCasp-9), and a truncated human epidermal growth factor receptor (EGFRt) polypeptide. In certain embodiments, the suicide gene is the EGFRt polypeptide. The EGFRt polypeptide enables T-cell elimination by administering an anti-EGFR monoclonal antibody (e.g., cetuximab). EGFRt can be covalently bound upstream of the CAR of this disclosure. The suicide gene may be included in a vector containing the nucleic acid encoding the CAR of this disclosure. Thus, administration of a prodrug designed to activate a suicide gene (e.g., a prodrug capable of activating iCasp-9 (e.g., AP1903)) during malignant T cell transformation (e.g., GVHD) induces apoptosis in suicide gene-activated CAR-expressing T cells. The integration of the suicide gene into the CAR of this disclosure provides an additional level of safety by its ability to eliminate most CAR T cells in a very short time. The immune-responsive cells (e.g., T cells) of this disclosure with the suicide gene incorporated are CAR It may be possible to preemptively eliminate the T cells at a given point in time after injection, or eradicate them at the earliest signs of toxicity.

[0356] 10. Kit The subject matter of this disclosure provides kits for inducing and / or enhancing an immune response, and / or treating and / or preventing neoplasm or pathogen infection in a subject. In certain embodiments, the kit comprises an effective amount of the immune-responsive cells of this disclosure or a pharmaceutical composition comprising them. In certain embodiments, the kit comprises a sterile container, such container may be in the form of a box, ampoule, bottle, vial, tube, bag, pouch, blister pack, or other suitable container known in the art. Such container may be made of plastic, glass, laminated paper, metal foil, or other material suitable for holding pharmaceuticals. In certain non-limiting embodiments, the kit comprises an isolated nucleic acid molecule encoding the CAR of this disclosure against an antigen of interest, in an expressible form, which may optionally be contained in one or more vectors.

[0357] If desired, immune-responsive cells and / or nucleic acid molecules are provided with instructions for administering the cells or nucleic acid molecules to subjects who have or are at risk of developing neoplasms, pathogens, or immune disorders. The instructions generally include information regarding the use of the composition for the treatment and / or prevention of neoplasms or pathogen infections. In certain embodiments, the instructions include at least one of the following: a description of the therapeutic agent; a dosage plan and administration for the treatment or prevention of neoplasms, pathogen infections, or immune disorders or their symptoms; precautions; warnings; indications; contraindications (counter-indications); overdose information; adverse reactions; animal pharmacology; clinical trials; and / or references. The instructions may be printed directly on the container (if any), as a label affixed to the container, or as a separate sheet, pamphlet, card, or folder supplied inside or with the container. [Examples]

[0358] The implementation of this disclosure will, unless otherwise indicated, utilize ordinary techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology that are well within the skill of a person skilled in the art. Such techniques are well described in literature such as "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); and "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of polynucleotides and polypeptides disclosed herein and can therefore be considered in the preparation and implementation of the subject matter of this disclosure. Techniques that are particularly useful for specific embodiments will be discussed in the following sections.

[0359] The following examples are provided to give a complete disclosure and explanation of the methods for preparing and using the cells and compositions of this disclosure to those skilled in the art, and are not intended to limit the scope that the inventors consider to be part of the present invention.

[0360] (Example 1) Introduction Multiple ITAMs in the CD3ζ and TCR complex have been proposed to amplify TCR signaling (PMID:20516133). Meanwhile, CD28 provides quantitative support for TCR signaling (PMID:14647476). Therefore, the number and type of signaling domains are important in TCR signaling. In human T cells, CD28 and TCR / CD3ζ amplify approximately 6 × 10⁶ 4 and approximately 2 × 10 4 It is expressed in molecules / cells (PMID:14647476). Therefore, three molecules of CD28 can provide signaling support for one molecule of TCR / CD3ζ with three ITAMs. However, the second-generation CAR 1928z has a design of fused CD28 and CD3ζ cytoplasmic domains, fixing their stoichiometric ratio to 1. Due to its superior killing and proliferative capabilities, the 1928z CAR enables rapid elimination of CD19-bearing cells in vitro, in mouse models, and in patients. However, improvements to the current 1928z CAR are needed to overcome several problems in clinical trials. One problem is cytokine release syndrome (CRS), characterized by massive synchronized T cell activation and massive cytokine release. The mechanism of CRS is still unclear, but it may be related to excessive signaling of 1928z CAR T cells. Another problem is the depletion and persistence of CAR T cells. 1928z CAR T cells have been found to be present in small numbers and / or in a dysfunctional state expressing depletion markers, such as PD-1, weeks or months after injection, which may explain some cases of relapse. It has been hypothesized that depletion is linked to excessive activation, which may be due to the inherent properties of the 1928z CAR design (PMID:26331345).

[0361] Comparing 1928z CAR signaling to TCR signaling reveals several key differences due to the fusion of the two cytoplasmic domains. First, in CAR, the CD28 signaling domain is cis-localized with the CD3ζ signaling domain, whereas CD28 is recruited to the synapse and co-localized with CD3ζ on the trans side. Second, CD28 activation is simultaneous with CD3ζ activation in CAR, while CD28 co-stimulation occurs several seconds after TCR ligation. Third, three CD28 molecules assist one TCR, but the ratio is 1:1 in CAR. The first two differences are not easily altered under current CAR designs, but the third difference can be addressed by maintaining a balance between the co-stimulatory and activating signals, which could help solve the current problem of hyperstimulation. The CD28 / CD3ζ ratio cannot be directly altered by the fusion design, but the number of ITAMs in CD3ζ can be mutated to mimic the ratio in TCR signaling. The three ITAMs in CD3ζ differ in their primary amino acid sequences and their positions relative to the plasma membrane (i.e., ITAM1, ITAM2, ITAM3 from proximal to distal to the membrane), and therefore differ in their ability to be phosphorylated by Lck or their binding ability to ZAP70 when phosphorylated (PMID:23555234). Previous studies on CD28-based ErbB2 CARs with only one ITAM at the second position showed reduced apoptosis in vitro (PMID:19843940), but further CAR characterization and in vivo studies were still lacking. Therefore, we designed novel CARS based on the 1928z CAR design, introducing a predetermined number and position of ITAMs, to evaluate their functionality with respect to the above issues, particularly their effects on T cell persistence, differentiation, depletion, and antitumor activity. We analyzed how each ITAM coupled to CD28 signaling in the context of the 1928z CAR affects overall CAR function in vitro and in vivo.CD28-coupled ITAM1 was not only superior to CD28-coupled ITAM2 or ITAM3, but more importantly, it was superior to the original 1928z CAR in terms of in vivo antitumor efficacy, making it an excellent candidate for clinical application. All trials were conducted in human peripheral blood T cells.

[0362] result The CD3ζ ITAM domain in 1928ζ CAR is qualitatively a differential CAR Exercising T cell function The first step was to explore whether each CD3ζ ITAM in the second-generation 1928z-CAR constructs contributed to a qualitatively specific function, or whether the individual ITAMs exhibited overlapping functional redundancy. To evaluate the contribution of each individual ITAM to the function of the 1928z CAR, 1928z CARs with only one single functional ITAM domain were generated (1XX, X2X, and XX3; see Figure 1A). Signaling of the remaining two ITAMs was disrupted by insertion of point mutations converting two tyrosine (Y) to phenylalanine (F), thus invalidating the phosphorylation and sequential recruitment of ZAP70 for full activation of downstream signaling pathways. T cells were effectively transduced using the SFG retroviral vector at comparable transduction rates between the different constructs (Figure 1B).

[0363] To compare the therapeutic activity of the generated 1928z mutant, a suboptimal CAR T cell dose of 5 × 10⁶ was used. 4 individual CAR + T cells were administered to the previously described pre-B acute lymphoblastic leukemia NALM-6 mouse model, and the efficacy of the treatment was compared to that of 1928z wild-type (1928z WT) mice (Figure 2).

[0364] Regarding the associated functional ITAMs, graded differences were found in tumor eradication and mouse survival: unmodified ITAM1(1XX) or ITAM2(X2X) at their original 1928z CAR site showed improved antitumor activity compared to the three functional ITAMs, as well as the original 1928z wild-type (WT). In contrast, ITAM3(XX3) as the sole functional ITAM combined with dysfunctional ITAM1 and 2 performed poorly, with reduced tumor eradication and decreased mouse survival (Figure 3).

[0365] Therefore, individual ITAMs in 1928z CARs exhibited qualitatively differential function when they maintained their original positions in the second-generation 1928z structure. The efficacy of tumor eradication gradually decreased as the location of each functional ITAM became more distal: 1XX consistently showed rapid tumor eradication and achieved long-term complete remission, while treatment with X2X slowed tumor progression and was superior to 1928z WT, but ultimately recurrence occurred. XX3, with its non-mutated (natural) ITAM at the most distal position, did not achieve tumor control and resulted in rapid tumor progression and reduced survival (Figure 3). In conclusion, 1928z CARs (XX3, X2X, X23) with one or two ITAMs at the second and third ITAM positions were less active than 1928z wild-type, or even not therapeutically active (in vivo).

[0366] A single, functional CD3ζ ITAM in the correct location is sufficient for potent antitumor activity. The next question to address was whether a combination of two functional ITAMs at distal locations—ITAM2 and ITAM3 (X23, Figure 1A), which are presumed to have a lower affinity for ZAP70 than ITAM1—could both improve the efficacy of their associated single ITAM 1928ζ mutant (X2X / XX3). In vivo analysis revealed that distal tumor clearance and survival in mice treated with X23 were comparable to the outcomes of mice treated with XX3 (Figures 2 and 3). In contrast, 1928z mutant CARs with only one functional ITAM at either the first (1XX) or second (X2X) location were found to be superior to 1928z CARs with two (X23) or three (WT) functional ITAMs, as reflected in tumor burden and survival outcomes. 1XX consistently proved to be the most potent 1928z mutant, achieving rapid and sustainable tumor eradication even at very low treatment doses. The results thus demonstrate that a single ITAM is sufficient for efficient killing, but also reveal significant differences in the therapeutic efficacy of CAR T cells depending on which ITAM within CD3ζ is functional.

[0367] Next, we analyzed whether the reduction in CAR function in XX3 was due to the individual specificity of ITAM3 in the context of second-generation CARs compared to its native location, or to the more distal location of ITAM3. Therefore, we generated 1928z mutant CARs with ITAM1 or ITAM3 at exactly the same proximal CAR location (D12 and D23) and a deletion of the remaining CD3ζ chain, enabling a direct comparison of the two ITAMs in the context of 1928-CARs (Figure 1). The results demonstrated that ITAM3 at the more proximal location (D12) was sufficient for rapid and efficient long-term tumor clearance (Figures 2 and 3), showing comparable results to D23. Despite sharing the same ITAM (ITAM3), D12 was clearly superior to XX3, achieving effective antitumor efficacy. Thus, the significant difference in therapeutic efficacy between D12 and XX3 demonstrates the influence of ITAM location within second-generation CARs. In conclusion, 1928z CARs with a single ITAM at the first ITAM position (ITAM1 in 1XX and D23, or ITAM3 in D12) were more therapeutically active than 1928z wild-type (in vivo).

[0368] While D23 and 1XX both possess ITAM1 as the sole functional ITAM, 1XX exhibited improved functional properties compared to D23, resulting in higher T cell proliferation and a more favorable T cell phenotype (Figures 4 and 5). This supports the idea that basic residue-rich elongation (BRS) within the CD3ζ chain, which has been previously shown to mediate membrane association and modulate signaling, may be attributable to the functional properties of the 1928z CAR (Figure 1B).

[0369] Therefore, additional mutations are inserted into the structure of 1XX CAR, thereby creating BRS-2 and -3 (=1XX BRS). negative ) disrupted the signaling pathway. 1XX BRS negativeHowever, the reduced proliferation and killing functions observed in vitro and in vivo demonstrated that the BRS region is essential for the function of 1XX. Therefore, the inventors demonstrate the functional importance of the BRS region in 1XX.

[0370] CD3ζ ITAM mutations allow for enhanced T cell proliferation and limit T cell differentiation and depletion in 1928ζ CARs. In vivo studies revealed enhanced CAR T cell accumulation for all CAR groups with one functional ITAM and two mutant ITAM regions: XX3, X2X, and 1XX cell counts achieved significantly higher T cell accumulation at tumor sites after 17 days compared to 1928z WT (Figure 4). T cells expressing mutant 1928z CARs containing a single ITAM (XX3, X2X, 1XX) accumulated at higher levels in vivo than T cells expressing wild-type 28z CARs. CARs containing two or more ITAMs (1928z and X23) showed reduced accumulation compared to T cells expressing single ITAM CARs (XX3, X2X, 1XX, D23, and D12).

[0371] Higher T cell accumulation is CD4 + and CD8 + Central memory in CAR T cells (CD62L+CD45RA-)(T CM ) and effect pedals (CD62L-CD45RA+) (T EFF ) As reflected in the percentage of cells, :1XX is a T cell phenotype associated with less T cell differentiation and improved in vivo proliferative capacity compared to 1928z WT. CM It showed a significantly higher percentage, and terminal differentiation T EFF It showed a significantly lower percentage (Figure 5). Furthermore, 1XX showed a significantly larger number of T in the bone marrow of treated mice 17 days after CAR injection. CMThe study showed T cells expressing the cellular and memory-related marker IL17R. Furthermore, both T cell populations showed a significant increase from day 10 to day 17 after CAR administration (Figure 6). Overall, 1XX induced the largest fraction of memory T cells, while D23 was the second best in long-term in vitro assays. Delayed differentiation and an increase in IL7R-expressing CAR T cells were also observed in vitro with repeated exposure to the antigen (data not shown).

[0372] Furthermore, the 1928z mutants exhibited different degrees of T cell depletion, as determined by the co-expression of inhibitory molecules PD1, TIM3, and LAG3, which are associated with reduced antitumor activity. The 1928z mutant group (D12 and D23), which had deletions of one single functional ITAM and two other ITAMs, showed significantly lower expression of depletion markers (Figure 7).

[0373] XX3 exceeded 1928z WT in terms of the number of CARs at the tumor site (Figure 4), T CM Although they showed a high percentage of cytotoxicity, these cells were unable to prevent tumor progression. XX3-treated mice demonstrated an initial treatment response but relapsed shortly thereafter. In vitro functional analysis demonstrated reduced cytotoxic activity and decreased secretion of Th1 cytokines and granzyme B (GrB) in XX3 (Figures 8 and 9). Figure 8 shows that in a standard in vitro cytotoxicity assay, all CARs similarly lysed tumor cells, except for XX3, which showed reduced cytolytic activity. All constructs showed similar and potent cytotoxic activity after initial T cell transduction, but differences were observed after repeated Ag exposure increased CAR levels (Figure 8). Figure 9 shows that a standard in vitro cytokine assay did not correlate with in vivo functional characteristics, but 1XX and D23 showed desirable profiles (IL2 and antitumor cytokines, e.g., IFNg and TNFα).

[0374] These findings indicate that effector function and proliferative function do not couple in XX3, and that the increase and persistence of CAR T cells were insufficient for effective tumor eradication. In contrast, 1928z WT exhibited high cytotoxic activity, but achieved early differentiation into effector cells, increased upregulation of inhibitory molecules, and resulted in reduced depletion and persistence of 1928z CARs.

[0375] conclusion The combination of CD3ζ activation and CD28 co-stimulation in second-generation 1928z CARs leads to increased functional redundancy and signaling, resulting in premature T cell differentiation and depletion, thus potentially reducing antitumor activity. Therefore, the contribution of a single ITAM to CAR function was analyzed. The location, affinity, and number of ITAMs within the CD3ζ chain differentially influenced the functional properties of 1928z CAR T cells. Overall, 1928z CARs containing a single ITAM directed towards superior T cell accumulation, memory formation, and reduced depletion (Figures 4-7), however, the single ITAM must be placed in a primary position to provide optimal therapeutic (antitumor) activity (Figures 2-3). 1XX contained the most favorable properties for both effector and memory cells, thereby maintaining a balance between activation and differentiation. 1XX modulated the strong activation of the combination of CD3ζ signaling and CD28 co-stimulation, fine-tuning the intensity to appropriate intracellular signaling, thus modulating CAR-mediated signaling and subsequently leading to superior long-term tumor eradication.

[0376] (Example 2: Alternative hinge / spacer region and transmembrane domain of CAR) Alternative hinge / spacer regions and transmembrane domains were tested in CAR constructs. Schematic diagrams of 1XX CARs with different hinge (H) and transmembrane domain (TM) are shown in Figure 10. All tested hinge and transmembrane domains belong to the immunoglobulin superfamily (IgSF) and can form cell-surface homodimers. All constructs were cloned into P2A bicistronic oncholoterovirus vectors (SFG) encoding CAR and LNGFR. Flow cytometry profiles show CAR and LNGFR expression using goat IgG anti-mouse IgG(F(ab')2) fragments and anti-LNGFR, respectively. T cells were obtained from healthy donor PBMCs (peripheral blood mononuclear cells), stably transduced 48 hours after activation, and analyzed for CAR expression at multiple time points. 1928z-LNGFR containing the CD28 / CD28 H / TM region was also analyzed. CAR was used as a comparison.

[0377] Replacing the CD28 / CD28(H / TM) hinge with ICOS / ICOS(H / TM), CTLA-4 / CTLA-4(H / TM), or ICAM-1 / ICAM-1(H / TM) hinges inactivated CAR cell-surface expression. However, fusing the CD28 hinge with ICOS transmembrane (CD28 / ICOS;H / TM) restored CAR expression. However, the CD28 / CTLA-4(H / TM) configuration did not rescue CAR expression. These findings indicate that discovering H / TM combinations that enable efficient CAR expression is not trivial. In conclusion, 1XX CARs possessing any of the CD84 / CD84, CD166 / CD166, CD8a / CD8a, CD8b / CD8b, or CD28 / ICOS(H / TM) regions were similarly expressed on the T cell surface, but three of them—CD166-28z, CD8a-28z, and CD8b-28z—were unable to form dimers with CD28.

[0378] 10 6Figure 11 shows the cumulative CAR T cell count when stimulated weekly, starting from 100 cells / ml of CAR T cells. CAR T cells induced by weekly exposure to CD19. T cell proliferation was initially evaluated in vitro for 21 days. CAR T cells were irradiated with CD19 without the addition of any exogenous cytokines. + Co-cultured with NIH 3T3. 10 6 CARs of individual cells / ml + T cells, radiation and fresh CD19 + It was added to NIH 3T3. A 1928z-1XX-LNGFR CAR containing CD28 / CD28(H / TM) was used as a reference. In vitro growth of 19-CD166-28z was similar to that of 1928z wild-type.

[0379] Data was constructed based on CAR T cell surface detection. T cells transduced with CARs containing ICOS / ICOS(H / TM), CTLA-4 / CTLA-4(H / TM), ICAM-1 / ICAM-1 (or CD28 / CTLA-4(H:TM)) were CD19+ After co-culture with NIH 3T3 cells, accumulation was not achieved. CAR T cells possessing CD84 / CD84 and CD8b / CD8b(H / TM) were able to accumulate after two rounds of stimulation. However, these cells lost their accumulation ability after a third round of stimulation. CAR T cells possessing CD166(H / TM), CD8a(H / TM) domains, and CD28 / ICOS(H / TM) accumulated as effectively as control 1928Z-1XX CAR T cells.

[0380] The cytotoxic activity of CAR T cells was measured at the end of the third stimulation (D21) for 4 hours. 51The results were measured using a Cr release assay and are shown in Figure 12. T cells and EL-4-CD19+ target cells were used with different effector:target ratios (E:T). EL-4-PSMA cells were used as a control. The in vitro cytotoxicity of all well-expressed H / TM CD28 / CD3z variants was similar, as expected. The cytotoxicity of CAR T cells with CD166(H / TM), CD8a(H / TM) domains, and CD28 / ICOS(H / TM) was comparable to that of CAR T cells with CD28 / CD28(H / TM).

[0381] Figure 13 shows the in vivo antitumor efficacy of different H / TM CAR T cells using NOD.Cg PrkdcscidIl2rgtm1Wjl / SzJ (immunodeficient NSG) mice. 5 × 10⁶ cells were injected into the tail vein of the mice. 5 Inject 10 FFLuc-GFP NALM6 cells (pre-B ALL cell line), and 4 days later 2 x 10 5 Individual CAR T cells were injected. Tumor burden was tracked by weekly quantification of bioluminescence signals. All constructs selected for the in vivo study, CD166 / CD166(H / TM), CD8a / CD8a(H / TM), and CD28 / ICOS(H / TM), were able to completely eradicate tumor cells. No significant differences in tumor burden or survival were observed under any conditions compared to the control group, 1928z CAR T cells.

[0382] (Example 3: Deimmunization Strategy) Proximal or mutational locations in the human sequence carry the risk of generating neoantigens. Therefore, novel CD166 junctions and mutations in 1XX were deimmunized. Immunogenicity of junctions between different CAR regions was predicted using the NetMHC 4.0 server. A total of 26 amino acids were selected (13 from the first region and 13 from the second region). For each generated peptide (14, 13, 12, 11, 10, 9, and 8 mers) containing at least one amino acid from the adjacent region, the binding affinity to HLA A, B, and C for all alleles was predicted. An immunogenicity score was assigned to each peptide. The immunogenicity score was expressed using the formula: Immunogenicity score = [(50 - binding affinity)] * HLA frequency [n] was used for calculation. 50 nm was used as the cutoff for strong binding affinity. HLA frequencies in the Caucasian population were used. n = number of predictions for each peptide. HLA frequencies less than 1% of the total population were excluded. For junction deimmunization, shuffling of both amino acids forming the junction or deletion of amino acids on both sides of the junction were tested. Previously described strategies for predicting immunogenicity were used for each novel-generated peptide. Deimmunized CARs were constructed using junctions with the lowest immunogenicity scores. Exemplary deimmunization strategies are shown in Figure 14.

[0383] (Example 4: Rescue of relapsed CD19-low ALL after treatment with 4-1BBz CAR T cells) Introduction Adoptive immunotherapy using chimeric antigen receptors (CARs) has shown remarkable clinical outcomes in the treatment of leukemia and lymphoma and is a promising immunotherapy applicable in principle to a wide range of cancers. Two promising CAR designs have been successfully introduced into clinical practice: one utilizing the cytoplasmic domain of CD28 as a co-stimulatory component, and the other using the cytoplasmic domain of 4-1BB. In both examples, T cell activation is initiated through the cytoplasmic domain fused with the CD3 zeta chain. While both designs have achieved remarkable results, clinical outcomes are limited by the shortcomings of these CAR structures. T cells expressing CD28-based CARs are potent but short-lived, while T cells expressing 4-1BB-based CARs are long-lived but allow antigen escape from tumor cells expressing low levels of the target antigen. Thus, there is a need for novel CAR designs that extend T cell persistence without impairing function.

[0384] result The CD28z CAR T cells of this disclosure can rescue relapses of CD19-low ALL after treatment with 4-1BBz CAR T cells. This may be a major rescue pathway for many relapses that occur after treatment with 4-1BBz CAR T cells, excluding cells that are stably CD19-negative.

[0385] 5 × 10⁶ of the tail vein in NSG mice 5 Individual FFLuc-GFP NALM6 cells (pre-B) Inject the ALL cell line, and 4 days later, 2 x 10 5 Each mouse was injected with 19BBz CAR T cells. Ten days after the initial T cell injection (a non-effective dose that only slowed tumor progression), the mice were again injected with 19BBz CAR T cells or 1928z or (5 × 10¹⁶) cells. 5 (19BBz cells / mouse) were injected. Various survival curves are shown in Figure 15. Only a new injection of 1928z-based CAR T cells (second injection) was able to rescue mice treated with NALM-6 from relapse. No significant increase in survival rate was observed after the injection of new 19BBz CAR T cells.

[0386] In conclusion, CD28z CAR T cells can rescue dysfunctional 4-1BBz CAR T cells. Considering the above examples, modified CD28z CAR T cells disclosed herein, such as 1XX, D23, and D12 CAR T cells (having various hinge / TM domains, such as the CD166 hinge / TM domain), may be even more effective than 1928z CAR T cells in rescuing dysfunctional 4-1BBz CAR T cells.

[0387] (Example 5) This embodiment is an updated and further investigation of a particular aspect of Example 1.

[0388] Chimeric antigen receptors (CARs) are synthetic receptors that reprogram T cells to target them and acquire enhanced antitumor properties. 1 CD28 and CD3ζ signaling motifs 2 CD19-specific CARs, including those mentioned above, are used in refractory leukemia. 3-5 and lymphoma 6 It induces a significant response in patients with and has recently been approved by the U.S. Food and Drug Administration. 7 These CARs have limited persistence on T cells. 3-6,8 , mediates powerful tumor elimination 4,8 Program high-performance effector functions. Extending the duration of these functions without compromising their effectiveness should improve current CAR therapy. Strong T-cell activation accelerates depletion, however. 9,10 This is due to the redundancy of CD28 and CD3ζ signaling. 11,12 and second-generation CAR 2This can be emphasized by the spatial and temporal constraints imposed by the structure. Thus, we hypothesize that calibrating the CD28-based activating ability of CARs differentially reprograms T cell function and differentiation. Herein, we propose that CARs encoding a single immunoreceptor-activating tyrosine motif direct T cells toward different fates by balancing effector and memory programs, thereby resulting in CAR designs with enhanced therapeutic profiles.

[0389] All three CD3 immunoreceptor-activated tyrosine motifs (ITAMs) 11,13 The redundancy of CD28 and CD3 signaling in the design of chimeric antigen receptors (CARs) incorporating these redundancies leads to counterproductive T cell differentiation and depletion. 9,10 We hypothesized that this could promote ITAM activity, thereby inhibiting its phosphorylation and downstream signaling. 14-17 Calibration was performed by mutating tyrosine residues. To investigate the contribution of each of the three CD3 ITAMs to the function, differentiation, and therapeutic efficacy of 1928z-modified T cells, 1928z mutants containing a single ITAM, named 1XX, X2X, and XX3, were generated (Figure 16A). In one additional mutant, named X23, two distal ITAMs (ITAM2 and ITAM3) were retained, both of which were shown to exhibit lower binding affinity to the tyrosine protein kinase ZAP-70 compared to ITAM1. 13,18 All mutant CARs were similarly expressed in human peripheral blood T cells transduced with retrovirus (Figure 16B); they were also expresse...

Claims

1. A composition comprising a polynucleotide encoding a chimeric antigen receptor (CAR) for use in a) reducing tumor burden in a subject, b) treating and / or preventing tumors in a subject, or c) treating a subject with recurrent tumors, The CAR comprises an extracellular antigen-binding domain, a hinge / spacer region, a transmembrane domain, and an intracellular signaling domain including a modified CD3ζ polypeptide. The modified CD3ζ polypeptide comprises an ITAM2 variant containing one or more loss-of-function mutations and an ITAM3 variant containing one or more loss-of-function mutations. (a) The hinge / spacer region comprises a CD28 polypeptide, and the transmembrane domain comprises a CD28 polypeptide; (b) The hinge / spacer region comprises the CD166 polypeptide, and the transmembrane domain comprises the CD166 polypeptide; (c) The hinge / spacer region comprises a CD8α polypeptide, and the transmembrane domain comprises a CD8α polypeptide; or (d) A composition in which the hinge / spacer region comprises a CD28 polypeptide and the transmembrane domain comprises an ICOS polypeptide.

2. The composition for use according to claim 1, wherein both the ITAM2 variant and the ITAM3 variant contain two loss-of-function mutations.

3. The composition for use according to claim 2, wherein both of the loss-of-function mutations are located at tyrosine amino acid residues.

4. The composition for use according to any one of claims 1 to 3, wherein the ITAM2 variant comprises the amino acid sequence described in SEQ ID NO:

29.

5. The composition for use according to any one of claims 1 to 4, wherein the ITAM3 variant comprises the amino acid sequence described in SEQ ID NO:

33.

6. The composition for use according to any one of claims 1 to 5, wherein the modified CD3ζ polypeptide comprises or comprises amino acids 374 to 485 of SEQ ID NO:

43.

7. The composition for use according to any one of claims 1 to 6, wherein the transmembrane domain comprises a CD166 polypeptide, and the CAR further comprises a hinge / spacer region comprising a CD166 polypeptide.

8. (a) The transmembrane domain comprises the amino acid sequence described in Sequence ID No. 92; or (b) The CAR contains amino acids 489 to 553 of SEQ ID NO: 3 A composition for use according to claim 7.

9. The composition for use according to any one of claims 1 to 8, wherein the intracellular signaling domain further comprises a co-stimulatory signaling domain.

10. The composition for use according to claim 9, wherein the co-stimulus signaling domain comprises a CD28 polypeptide.

11. The composition for use according to any one of claims 1 to 10, wherein the extracellular antigen-binding domain binds to an antigen.

12. The antigens mentioned above are CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, erb-B2, erb-B3, erb-B4, FBP, fetal acetylcholine receptor, folate receptor-a, GD2, GD3, HER-2, hTERT, IL-13R-a2, K-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, ERBB2, MAGEA3, p53, MART1, GP100, protein A composition for use according to claim 11, wherein the tumor antigen is selected from Nase 3 (PR1), tyrosinase, Survivin, hTERT, EphA2, NKG2D ligand, NY-ESO-1, fetal carcinoma antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME, CCR4, CD5, CD3, TRBC1, TRBC2, TIM-3, integrin B7, ICAM-1, CD70, Tim3, CLEC12A, and ERBB.

13. The composition for use according to any one of claims 1 to 12, wherein the polynucleotide is contained in the vector.

14. The aforementioned polynucleotides genetically modify cells, (a) The cells are selected from the group consisting of T cells, natural killer (NK) cells, myeloid cells, and pluripotent stem cells that can differentiate into lymphoid cells; (b) The cell is a T cell; (c) The cells are T cells selected from cytotoxic T lymphocytes (CTLs), regulatory T cells, and natural killer T (NKT) cells; (d) The cells are human pluripotent stem cells; or (e) The cells are human embryonic stem cells, A composition for use according to any one of claims 1 to 13.

15. A composition for use according to any one of claims 1 to 13, further comprising a second polynucleotide encoding a second CAR, comprising a second extracellular antigen-binding domain for binding to a second antigen, a second transmembrane domain, and a second intracellular signaling domain.

16. The composition for use according to claim 15, further comprising a third polynucleotide encoding a third CAR, comprising a third extracellular antigen-binding domain for binding to a third antigen, a third transmembrane domain, and a third intracellular signaling domain.

17. (a) The second intracellular signaling domain comprises the ITAM2 variant and the ITAM3 variant; and / or (b) The third intracellular signaling domain comprises an ITAM2 variant and an ITAM3 variant, A composition for use according to claim 15 or 16.

18. The composition for use according to any one of claims 1 to 17, wherein the subject has been treated to induce residual tumor cells, or the subject has been treated with immune-responsive cells comprising antigen-recognizing receptors comprising a 4-1BB costimulatory signal.

19. The composition for use according to any one of claims 1 to 18, wherein the tumor is selected from hematological cancer, B-cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, non-Hodgkin lymphoma, and ovarian cancer.

20. (a) The CAR is bound to CD19, and the tumor is B-cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, or non-Hodgkin lymphoma; (b) The CAR binds to CD19, and the tumor is CD19 ＋ It is ALL; (c) The CAR binds to CD19, and the tumor comprises tumor cells having a low density of tumor-specific antigens on the surface of the tumor cells; or (d) The CAR is bound to BCMA, ADGRE2, MSLN, PSMA or a combination thereof, and the tumor is B-cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, or non-Hodgkin lymphoma. A composition for use according to claim 19.

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