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US-12624081-B2 - Tn-MUC1 chimeric antigen receptor (CAR) T cell therapy

US12624081B2US 12624081 B2US12624081 B2US 12624081B2US-12624081-B2

Abstract

Various TnMUC1-specific chimeric antigen receptors (CARs), nucleic acids encoding the same, and methods of using the same, are provided. Compositions and methods comprising a TnMUC1-specific CAR for treating MUC1-associated cancer in a subject in need thereof are provided.

Inventors

  • Avery D. Posey
  • Carl H. June

Assignees

  • THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA

Dates

Publication Date
20260512
Application Date
20210708

Claims (14)

  1. 1 . A modified immune cell comprising: (a) a chimeric antigen receptor (CAR) that specifically binds MUC1 on a target cell, wherein the CAR comprises a MUC1-specific antigen binding domain, a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain of CD3 zeta, and wherein the CAR comprises the amino acid sequence set forth in SEQ ID NO: 47; and (b) a switch receptor comprising an extracellular domain of a signaling protein associated with a negative signal, a transmembrane domain, and an intracellular domain of a signaling protein associated with a positive signal; wherein the extracellular domain and the intracellular domain are respectively selected from PD-1 and CD28, PD-1 A132L and CD28, PD-1 and 4-1BB, PD-1 A132L and 4-1BB, PD-1 and IL12Rβ1, PD-1 A132L and IL12Rβ1, PD-1 and IL12Rβ2, PD-1 A132L and IL12Rβ2, TGFβRII and IL12Rβ1, TGFβRII and IL12Rβ2, TGFβRII and CD28, or TIM3 and CD28; and wherein the switch receptor is able to switch a negative signal to a positive signal upon activation, thereby enhancing an immune response.
  2. 2 . The modified immune cell of claim 1 , wherein the switch receptor is PD1-CTM-CD28 or TGFβR-IL12Rβ1.
  3. 3 . The modified immune cell of claim 1 , wherein the modified cell is a modified natural killer (NK) cell, a modified natural killer T (NKT) cell, or a modified T cell.
  4. 4 . The modified immune cell of claim 3 , wherein the modified immune cell is a modified T cell.
  5. 5 . The modified immune cell of claim 1 , wherein: the switch receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 78, 80, 82, 84, 86, 88, 90, and 92.
  6. 6 . An isolated nucleic acid comprising: (a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) that specifically binds MUC1 on a target cell, wherein the CAR comprises a MUC1-specific antigen binding domain, a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain of CD3 zeta, and wherein the first nucleic acid sequence comprises SEQ ID NO: 46; and (b) a second nucleic acid sequence encoding a switch receptor comprising an extracellular domain of a signaling protein associated with a negative signal, a transmembrane domain, and an intracellular domain of a signaling protein associated with a positive signal; wherein the extracellular domain and the intracellular domain are respectively selected from PD-1 and CD28, PD-1 A132L and CD28, PD-1 and 4-1BB, PD-1 A132L and 4-1BB, PD-1 and IL12Rβ1, PD-1 A132L and IL12Rβ1, PD-1 and IL12Rβ2, PD-1 A132L and IL12Rβ2, TGFβRII and IL12Rβ1, TGFβRII and IL12Rβ2, TGFβRII and CD28, or TIM3 and CD28; wherein the switch receptor is able to switch the negative signal to the positive signal upon activation, thereby enhancing an immune response; and wherein the first and second nucleic acid sequences are separated by a nucleic acid sequence encoding a self-cleaving peptide.
  7. 7 . The isolated nucleic acid of claim 6 , wherein and the second nucleic acid sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 79, 81, 83, 85, 87, 89, 91, and 93.
  8. 8 . An expression construct comprising the isolated nucleic acid of claim 6 .
  9. 9 . A method for generating a modified immune cell, comprising introducing into an immune cell the isolated nucleic acid of claim 6 .
  10. 10 . A method of treating a MUC1 associated cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising the modified immune of claim 1 .
  11. 11 . The method of claim 10 , further comprising: (a) administering to the subject a lymphodepleting chemotherapy; and/or (b) administering to the subject a cytokine release syndrome (CRS) management regimen.
  12. 12 . The method of claim 11 , wherein the lymphodepleting chemotherapy comprises administering to the subject a therapeutically effective amount of cyclophosphamide, and a therapeutically effective amount of fludarabine.
  13. 13 . The method of claim 10 , wherein the MUC1-associated cancer is: (a) selected from the group consisting of multiple myeloma, non-small cell lung cancer, breast cancer, pancreatic adenocarcinoma, ovarian, and fallopian tube cancer; and/or (b) characterized by abnormal glycosylation of MUC1.
  14. 14 . The method of claim 13 , wherein the breast cancer is selected from the group consisting of a hormone receptor-positive breast cancer, a hormone receptor-negative breast cancer, an estrogen receptor-negative breast cancer, a progesterone receptor-negative breast cancer, triple negative breast cancer, and a Her2 receptor-negative breast cancer.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS The present application is a continuation of U.S. patent application Ser. No. 16/830,554, filed Mar. 26, 2020, now U.S. Pat. No. 11,090,336, which is entitled to priority under 35 U.S.C. § 119 (e) to U.S. Provisional Patent Application 62/824,532, filed Mar. 27, 2019 and to U.S. Provisional Patent Application No. 62/881,269, filed Jul. 31, 2019, which are hereby incorporated by reference in their entireties. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created Jul. 8, 2021, is named “046483-7229US2_Sequence_Listing.txt” and is 107,268 bytes in size. BACKGROUND OF THE INVENTION Chimeric antigen receptor (CAR) T cells are effector immune cells that are genetically-modified to recognize a specific tumor-associated antigen and subsequently kill the tumor cell. While success with CAR T therapy has led to approval for use in hematologic malignancy, the effectiveness of CAR T therapy in the treatment of solid tumors, such as breast cancer, remains uncertain. There are several obstacles to CAR T therapy in solid tumors. Foremost, most of the identified and best-studied cell-surface antigens expressed by tumors are also expressed by normal tissue, resulting in non-specific targeting by CAR T cells (on-target, off-tumor activity). Second, solid tumors have a generally immunosuppressive tumor microenvironment, which may inhibit CAR T cell activity once the cells reach the tumor and recognize the antigen. Third, the durability of anti-tumor responses is highly correlated with the persistence of the adoptively-transferred cells and optimal persistence for CAR T cells in solid tumors has yet to match the persistence observed in hematopoietic malignancies. Identifying tumor-specific antigens is essential in the continuing application of CAR T cell therapy to solid tumors. A need exists for novel compositions and methods that treat solid tumors, such as breast cancers. The present invention satisfies this need. SUMMARY Mucin 1 (MUC1) is a cell surface mucin that typically undergoes serial addition of glycans to form a hyperglycosylated protein (FIG. 1). The O-glycosylation process begins with GalNAc addition on serine and threonine residues. Elongation begins by addition of galactose by Core 1 synthase (composed of C1GalT1 and its chaperone C1GalT1C1 (Cosmc)) or GlcNAc addition by Core 3 synthase (B3GNT6). Aberrations in this serial glycosylation, such as epigenetic silencing of Cosmc, yield a hypoglycosylated product, Tn-MUC1, to which a sialic acid may be added by (ST6GALNAC-1) to form STn-MUC1. The present disclosure is based on the discovery that CAR T cells directed against Tn-MUC1 demonstrated potent cytolytic activity against various cancer cell lines in vitro and significant tumor eradication in vivo. In one aspect, a modified immune cell or precursor cell thereof, comprising a chimeric antigen receptor (CAR) that specifically binds MUC1, wherein the CAR comprises: a MUC1-specific antigen binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the heavy chain complementarity determining region (CDR) sequences set forth in SEQ ID NOs: 22, 23, and 24, and wherein the VL domain comprises the light chain complementarity determining region (CDR) sequences set forth in SEQ ID NOs: 19, 20, and 21; a transmembrane domain; a costimulatory signaling domain; and an intracellular signaling domain, is provided. In certain exemplary embodiments, the MUC1-specific antigen binding domain is specific for a glycoepitope of MUC1. In certain exemplary embodiments, the MUC1-specific antigen binding domain is specific for a truncated glycoepitope of MUC1. In certain exemplary embodiments, the VH domain comprises the amino acid sequence set forth in SEQ ID NO: 5. In certain exemplary embodiments, the VL domain comprises the amino acid sequence set forth in SEQ ID NO: 6. In certain exemplary embodiments, the VH domain comprises the amino acid sequence set forth in SEQ ID NO: 5, and the VL domain comprises the amino acid sequence set forth in SEQ ID NO: 6. In certain exemplary embodiments, the MUC1-specific antigen binding domain comprises the amino acid sequence set forth in SEQ ID NO: 4. In certain exemplary embodiments, the transmembrane domain comprises a transmembrane region of a protein selected from the group consisting of a type I transmembrane protein, an alpha, beta, or zeta chain of a T cell receptor, CD28, CD2, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In certain exemplary embodiments, the transmembrane domain comprises a CD8 transmembrane region. In certain exemplary