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CN-122003246-A - CD70 CAR-T compositions and methods for cell-based therapies

CN122003246ACN 122003246 ACN122003246 ACN 122003246ACN-122003246-A

Abstract

The compositions and methods described herein relate to the use of a CRISPR/Cas9 system in combination with CAR technology for improving the activity of anti-CD 70 CAR-T cells. The engineered cells may comprise a genetic modification in one or more of the HLa-a, HLa-B, TRAC, CIITA, TGFBR2, or CD70 genes.

Inventors

  • M. Ram
  • Y.ZHANG
  • LIU BIAO
  • ZHANG BONING
  • R. Oliveira
  • I. MILLER
  • R. S.V. Qi Wuku La
  • A. Prodius
  • O. Kilic
  • P. SHARMA
  • LIU DAI
  • N. N. Sheval
  • B. Schulters

Assignees

  • 因特利亚治疗公司

Dates

Publication Date
20260508
Application Date
20240813
Priority Date
20230814

Claims (20)

  1. 1. An anti-CD 70 Chimeric Antigen Receptor (CAR), the anti-CD 70 chimeric antigen receptor comprising: a. An antigen binding protein or fragment thereof that specifically binds to CD70, wherein the antigen binding protein comprises a heavy chain Variable (VH) region comprising complementarity determining region 1 (VH CDR 1), VH CDR2, and VH CDR3, and a light chain Variable (VL) region comprising complementarity determining region 1 (VL CDR 1), VL CDR2, and VL CDR3, and wherein I. the VH CDR1 comprises the amino acid sequence of any one of SEQ ID NOs 73, 67, 70, 76, 79 and 82; The VH CDR2 comprises the amino acid sequence of any one of SEQ ID NOs 74, 68, 71, 77, 80 and 83; The VH CDR3 comprises the amino acid sequence of any one of SEQ ID NOs 75, 69, 72, 78, 81 and 84; the VL CDR1 comprises the amino acid sequence of any one of SEQ ID NOs 55, 49, 52, 58, 61 and 64; v. the VL CDR2 comprises the amino acid sequence of any one of SEQ ID NOS 56, 50, 53, 59, 62 and 65, and The VL CDR3 comprises the amino acid sequence of any one of SEQ ID NOs 57, 51, 54, 60, 63 and 66; b. transmembrane domain, and C. an intracellular domain comprising a co-stimulatory domain comprising the amino acid sequence of SEQ ID NO 99 or 101.
  2. 2. The anti-CD 70 CAR of claim 1, wherein the VH CDR1, the VH CDR2, the VH CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 comprise the amino acid sequences of: (a) 73, 74, 75, 55, 56 and 57 respectively; (b) 67, 68, 69, 49, 50 and 51 respectively; (c) 70, 71, 72, 52, 53 and 54 respectively; (d) 76, 77, 78, 58, 59 and 60 respectively; (e) 79, 80, 81, 61, 62 and 63 respectively, or (F) SEQ ID NOS 82, 83, 84, 64, 65 and 66, respectively.
  3. 3. The anti-CD 70 CAR of claim 1 or 2 wherein said VH CDR1, said VH CDR2, said VH CDR3, said VL CDR1, said VL CDR2 and said VL CDR3 comprise the amino acid sequences SEQ ID NO 73, 74, 75, 55, 56 and 57, respectively.
  4. 4. The anti-CD 70 CAR of any one of claims 1-3, wherein the VH region comprises an amino acid sequence that is at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs 43-48.
  5. 5. The anti-CD 70 CAR of any one of claims 1-4, wherein the VL region comprises an amino acid sequence that is at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs 37-42.
  6. 6. The anti-CD 70 CAR of any one of claims 1-5, wherein: (a) The VH region comprises an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 45, and the VL region comprises an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 39; (b) The VH region comprises an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 43, and the VL region comprises an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 37; (c) The VH region comprises an amino acid sequence that is at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID No. 44, and the VL region comprises an amino acid sequence that is at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID No. 38; (d) The VH region comprises an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 46, and the VL region comprises an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 40; (e) The VH region comprises an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 47 and the VL region comprises an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 41, or (F) The VH region comprises an amino acid sequence that is at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID No. 48, and the VL region comprises an amino acid sequence that is at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID No. 42.
  7. 7. The anti-CD 70 CAR of any one of claims 1-6, wherein: the VH region comprises an amino acid sequence that is at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID No. 45, and the VL region comprises an amino acid sequence that is at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID No. 39.
  8. 8. The anti-CD 70 CAR of any one of claims 1-7, wherein: (a) The VH region comprises the amino acid sequence of SEQ ID NO. 45 and the VL region comprises the amino acid sequence of SEQ ID NO. 39; (b) The VH region comprises the amino acid sequence of SEQ ID NO. 43 and the VL region comprises the amino acid sequence of SEQ ID NO. 37; (c) The VH region comprises the amino acid sequence of SEQ ID NO. 44 and the VL region comprises the amino acid sequence of SEQ ID NO. 38; (d) The VH region comprises the amino acid sequence of SEQ ID NO. 46 and the VL region comprises the amino acid sequence of SEQ ID NO. 40; (e) The VH region comprises the amino acid sequence of SEQ ID NO. 47 and the VL region comprises the amino acid sequence of SEQ ID NO. 41, or (F) The VH region comprises the amino acid sequence of SEQ ID NO. 48 and the VL region comprises the amino acid sequence of SEQ ID NO. 42.
  9. 9. The anti-CD 70 CAR of any one of claims 1-8, wherein: The VH region comprises the amino acid sequence of SEQ ID NO. 45 and the VL region comprises the amino acid sequence of SEQ ID NO. 39.
  10. 10. The anti-CD 70 CAR of any one of claims 1-9, wherein the antigen binding protein comprises the amino acid sequence of any one of SEQ ID NOs 25, 27, 29, 32, 34, and 36, or a sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 25, 27, 29, 32, 34, and 36.
  11. 11. The anti-CD 70 CAR of any one of claims 1-10, wherein the antigen binding protein comprises the amino acid sequence of SEQ ID No. 25 or 27.
  12. 12. The anti-CD 70 CAR of any one of claims 1-11, wherein the transmembrane domain comprises a CD8a transmembrane domain comprising the amino acid sequence of SEQ ID No. 95, or wherein the transmembrane domain comprises a CD28 transmembrane domain comprising the amino acid sequence of SEQ ID No. 93.
  13. 13. The anti-CD 70 CAR of any one of claims 1-12, further comprising a hinge domain between the antigen binding protein and the transmembrane domain, optionally wherein the hinge domain is a CD8a hinge domain comprising the amino acid sequence of SEQ ID No. 89 or a fragment thereof.
  14. 14. The anti-CD 70 CAR of any one of claims 1-13, wherein the intracellular domain further comprises an activation domain, optionally wherein the activation domain is a CD3z activation domain comprising the amino acid sequence of SEQ ID No. 103.
  15. 15. The anti-CD 70 CAR of any one of claims 1-11, wherein: a. the antigen binding protein comprises the sequence of SEQ ID NO. 32 and the costimulatory domain comprises the sequence of SEQ ID NO. 101; b. the antigen binding protein comprises the sequence of SEQ ID NO. 32 and the costimulatory domain comprises the sequence of SEQ ID NO. 99; c. The antigen binding protein comprises the sequence of SEQ ID NO. 25 and the costimulatory domain comprises the sequence of SEQ ID NO. 101; d. The antigen binding protein comprises the sequence of SEQ ID NO. 25 and the costimulatory domain comprises the sequence of SEQ ID NO. 99; e. The antigen binding protein comprises the sequence of SEQ ID NO. 27 and the costimulatory domain comprises the sequence of SEQ ID NO. 101; f. the antigen binding protein comprises the sequence of SEQ ID NO. 27 and the costimulatory domain comprises the sequence of SEQ ID NO. 99; g. The antigen binding protein comprises the sequence of SEQ ID NO. 36 and the costimulatory domain comprises the sequence of SEQ ID NO. 101, or H. the antigen binding protein comprises the sequence of SEQ ID NO. 36 and the costimulatory domain comprises the sequence of SEQ ID NO. 99.
  16. 16. The anti-CD 70 CAR of any one of claims 1-15, comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs 2,4, 6, 8, 14, 16, 20, and 22.
  17. 17. The anti-CD 70 CAR of any one of claims 1-16, comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID No. 2, 4, or 8.
  18. 18. The anti-CD 70 CAR of any one of claims 1-17, wherein the antigen binding protein is a scFv.
  19. 19. The anti-CD 70 CAR of claim 18, wherein the scFv comprises a linker between the VH region and the VL region, optionally wherein the scFv comprises a glycine-serine linker between the VH region and the VL region, and optionally the glycine-serine linker comprises the sequence of SEQ ID NO: 940.
  20. 20. A nucleic acid encoding the anti-CD 70 CAR of any one of claims 1-19.

Description

CD70 CAR-T compositions and methods for cell-based therapies Cross reference to related applications The present application is based on the benefit of 35 USC 119 (e) claiming U.S. provisional application Ser. No. 63/519,551, U.S. provisional application Ser. No. 63/610,582, and U.S. provisional application Ser. No. 63/659,675, filed on 8, 14, 2023, 12, 15, and filed on 13, 2024, each of which is incorporated herein by reference in its entirety. Reference to electronic sequence Listing The present application contains a sequence table that has been electronically submitted in an XML file format and is hereby incorporated by reference in its entirety. The XML file was created at month 8 and 12 of 2024, named "01155-0059-00PCT. XML" and is 3,148,975 bytes in size. Disclosure of Invention The present disclosure generally describes Chimeric Antigen Receptor (CAR) reagents (e.g., cells and compositions) and methods. The present disclosure relates to the use of CAR technology in combination with a CRISPR system for providing CAR-transduced immune effector cells (e.g., T cells, NK cells). CD70 is a transmembrane protein that is typically transiently expressed on the surface of cd4+ and cd8+ T cells, regulatory T cells (tregs), B cells, antigen presenting cells (such as dendritic cells) and Natural Killer (NK) cells in response to immune activation. Although CD70 expression is tightly controlled in normal tissues, many cancer cell types exhibit high levels of CD70. For example, CD70 is overexpressed in solid and liquid cancer types, such as Renal Cell Carcinoma (RCC), acute Myelogenous Leukemia (AML), hodgkin's lymphoma, and non-Hodgkin's lymphoma, multiple myeloma, pancreatic cancer, ovarian cancer, and non-small cell lung cancer (NSCLC). Notably, many of these cancer types have proven difficult to treat, creating a significant unmet need for new therapeutic approaches. CD70 represents an attractive target for emerging therapies, including immunotherapy, due to its differential expression on the surface of healthy and cancerous cells. Despite the therapeutic promise, there are many logical and technical hurdles to anti-CD 70 CAR-T therapies. The current approach to CAR-T is based on autologous cell transfer. In this method, T cells recovered from a patient are genetically modified ex vivo and cultured in vitro, and then infused into the patient. This method of using patient's own lymphocytes reduces the risk of rejection. However, so-called autologous therapy is based on the availability of functional lymphocytes, which may be compromised by previous treatment lines. Furthermore, the autologous cell preparation of each patient is actually a new product, resulting in substantial changes in safety and efficacy. The "off-the-shelf" therapy using donor (allogeneic) cells eliminates the need to restore and modify the patient's own lymphocytes. However, allogeneic CAR-T cells may elicit an undesirable immune response or otherwise be transiently present. Typically, immune rejection of allogeneic cells is caused by mismatch of Major Histocompatibility Complex (MHC) molecules between the donor and recipient. For example, slight differences in MHC alleles between individuals can lead to T cell activation in the recipient. During T cell development, the T cell lineage of an individual is tolerant to self MHC molecules, but T cells that recognize another individual's MHC molecules may persist in the circulation and are referred to as alloreactive T cells. Alloreactive T cells may be activated, for example, by the presence of cells of another individual expressing MHC molecules in vivo, thereby causing, for example, graft versus host disease and graft rejection. Methods and compositions for reducing the susceptibility of allogeneic anti-CD 70 CAR-T cells to rejection or for improving anti-CD 70 CAR-T cell activity are of interest. Thus, there is a need for improved methods and compositions for modifying anti-CD 70 CAR-T cells to overcome the problem of immune rejection in recipients and to improve the activity of anti-CD 70 CAR-T cells. The present disclosure provides genome editing of CRISPR/Cas9 systems against CD70 CAR-T cells. The engineered cells contain genetic modifications in the HLA-A, HLa-B, TRAC, CIITA (class II major histocompatibility complex transactivator), TGFBR2 or CD70 genes, which can be used in cell therapies. The present disclosure further provides compositions and methods for reducing or eliminating the surface expression of endogenous T cell receptors, MHC class I or II proteins, CD70 or TGFBR2 in cells by genetically modifying HLA-A, HLa-B, TRAC, CIITA, TGFBR2 or CD70 loci. In some embodiments, a method of reducing surface expression of an HLA-A protein in an engineered cell relative to an unmodified cell is provided, the method comprising contacting the cell with a composition of any one of the embodiments provided herein. In some embodiments, a method of reducing surface expression of an HLA-B protein