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EP-4739699-A1 - PROTEINS AND USES THEREOF

EP4739699A1EP 4739699 A1EP4739699 A1EP 4739699A1EP-4739699-A1

Abstract

Provided are Nef proteins and Nef fusion proteins, and methods of use thereof. Also provided are engineered cells (e.g., CAR-T cells) with reduced endogenous TCR levels, particularly engineered cells that overexpress the Nef protein or the Nef fusion protein.

Inventors

  • Zhao, Yuncheng
  • HUANG, XIN
  • WANG, BING
  • XU, Changmeng
  • ZHUANG, Qiuchuan

Assignees

  • Legend Biotech Ireland Limited
  • Legend Biotech USA Inc.

Dates

Publication Date
20260513
Application Date
20240705

Claims (20)

  1. A non-naturally occurring negative regulatory factor (Nef) protein, comprising a deletion of amino acids 207-223 as numbered in SEQ ID NO: 1, SEQ ID NO: 2 or corresponding amino acids thereof.
  2. The non-naturally occurring Nef protein of claim 1, comprising a deletion of amino acids 50-91 as numbered in SEQ ID NO: 1, SEQ ID NO: 2 or corresponding amino acids thereof.
  3. The non-naturally occurring Nef protein of claim 1 or claim 2, which down-regulates cell surface expression of an endogenous T cell receptor (TCR) .
  4. The non-naturally occurring Nef protein of any one of claims 1-3, consisting of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5.
  5. A fusion protein comprising (1) a non-naturally occurring Nef protein and (2) an E3 ubiquitin ligase or fragment thereof.
  6. The fusion protein of claim 5, wherein the non-naturally occurring Nef protein comprises a deletion of amino acids 207-223 as numbered in SEQ ID NO: 1, SEQ ID NO: 2 or corresponding amino acids thereof.
  7. The fusion protein of claim 5 or claim 6, wherein the non-naturally occurring Nef protein comprises a deletion of amino acids 50-91 as numbered in SEQ ID NO: 1, SEQ ID NO: 2 or corresponding amino acids thereof.
  8. The fusion protein of any one of claims 5-7, wherein the non-naturally occurring Nef protein consists of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.
  9. The fusion protein of any one of claims 5-8, wherein the E3 ubiquitin ligase or fragment thereof is derived from a Tripartite Motif Containing 21 (TRIM21) protein.
  10. The fusion protein of claim 9, wherein the TRIM21 protein is truncated; optionally wherein the TRIM21 protein comprises a RING domain.
  11. The fusion protein of claim 9 or claim 10, wherein the TRIM21 protein comprises the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12; optionally wherein the amino acid sequence of the TRIM21 protein is set forth in SEQ ID NO: 12.
  12. The fusion protein of any one of claims 5-11, wherein the non-naturally occurring Nef protein and the E3 ubiquitin ligase or fragment thereof are connected via a linker; optionally wherein the linker comprises the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence of SEQ ID NO: 25.
  13. The fusion protein of any one of claims 5-11, wherein the non-naturally occurring Nef protein and the E3 ubiquitin ligase or fragment thereof are directly connected to each other without a linker.
  14. The fusion protein of any one of claims 5-13, wherein the non-naturally occurring Nef protein is N-terminal to the E3 ubiquitin ligase or fragment thereof or C-terminal to the E3 ubiquitin ligase or fragment thereof.
  15. The fusion protein of any one of claims 5-14, wherein the fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 6-10, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence of any one of SEQ ID NOs: 6-10.
  16. The fusion protein of claim 15, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence of SEQ ID NO: 10.
  17. The fusion protein of any one of claims 5-16, which downregulates and/or degrades an endogenous CD3ζ.
  18. A nucleic acid comprising a first nucleic acid sequence encoding the non-naturally occurring Nef protein of any one of claims 1-4, or the fusion protein of any one of claims 5-17.
  19. The nucleic acid of claim 18, wherein the nucleic acid further comprises a second nucleic acid sequence encoding an engineered receptor.
  20. The nucleic acid of claim 19, wherein the engineered receptor is a chimeric antigen receptor (CAR) ; optionally wherein the CAR comprises the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14.

Description

PROTEINS AND USES THEREOF CROSS REFERENCE TO RELATED APPLICATIONS This application claims benefit of priority of International Patent Application No. PCT/CN2023/106325 filed on July 7, 2023, the content of which is incorporated herein by reference in its entirety. SUBMISSION OF SEQUENCE LISTING ON XML FILE The content of the following submission on XML file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: IEC240352PCT SEQUENCE LISTING. XML, date recorded: July 4, 2024, size: 38, 569 KB) . TECHNICAL FIELD This disclosure relates to proteins for downmodulation of a target and uses thereof. The disclosure also relates to engineered cells (e.g., CAR-T cells) expressing the proteins. BACKGROUND Chimeric antigen receptor (CAR) -T cell therapy utilizes genetically modified T cells carrying an engineered receptor specifically recognizing a target tumor antigen to direct T cells to tumor site. It has shown promising results in treating hematological cancer and multiple myeloma (MM) . Nevertheless, due to individual differences, autologous CAR-T or TCR-T therapy (using patient’s own T cells) presents significant challenges in manufacturing and standardization, with extremely expensive cost for manufacturing and treatment. Furthermore, cancer patients usually have lower immune function, with lymphocytes having reduced number, lower immune activity, and hard to expand in vitro. Universal allogeneic CAR-T or TCR-T therapy is considered as an ideal model, with T cells derived from healthy donors. However, the key challenge is how to effectively eliminate graft-versus-host disease (GvHD) during treatment due to histoincompatibility. TCR is a cell surface receptor involved in T cell activation in response to antigen presentation. 95%of T cells  in human have TCR consisting of an alpha (α) chain and a beta (β) chain. TCRα and TCRβ chains combine to form a heterodimer and associate with CD3 subunits to form a TCR complex present on the cell surface. GvHD happens when donor’s T cells recognize non-self major histocompatibility complex (MHC) molecules via TCR and perceive host (transplant recipient) tissues as antigenically foreign and attack them. In order to eliminate endogenous TCR from donor T cells thereby preventing GvHD, people have been using gene editing technologies such as Zinc Finger Nuclease (ZFN) , transcription activator-like effector nucleases (TALEN) , and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -CRISPR associated (Cas) (CRISPR/Cas) for endogenous TCRα or TCRβ gene knockout (KO) , then enriching TCR-negative T cells for allogeneic CAR-T or TCR-T production. Nef (Negative Regulatory Factor) is a small 27-35 kDa myristoylated protein encoded by primate lentiviruses. Nef (e.g., SIV Nef) can mediate downmodulation of endogenous T cell receptor (TCR) and can dampen inflammation. SUMMARY This disclosure relates to Nef proteins and Nef fusion proteins, and methods of making and use thereof. This disclosure also relates to engineered cells with reduced endogenous TCR levels, particularly engineered cells that overexpress a Nef protein (e.g., a truncated Nef protein) or a Nef fusion protein (e.g., a Nef protein fused to a TRIM21 protein) . The engineered cells may elicit no or reduced graft-versus-host disease (GvHD) response in a histoincompatible individual as compared to the GvHD response elicited by a primary T cell isolated from the donor of the precursor T cell from which the engineered cell is derived. In one aspect, the disclosure is related to a non-naturally occurring negative regulatory factor (Nef) protein, comprising a deletion of amino acids 207-223 as numbered in SEQ ID NO: 1, SEQ ID NO: 2 or corresponding amino acids thereof. In some embodiments, the non-naturally occurring Nef protein comprises a deletion of amino acids 50-91 as numbered in SEQ ID NO: 1, SEQ ID NO: 2 or corresponding amino acids thereof. In some embodiments, the non-naturally occurring Nef protein down-regulates cell surface expression of an endogenous T cell receptor (TCR) . In some embodiments, the non-naturally occurring Nef protein consists of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In one aspect, the disclosure is related to a fusion protein comprising (1) a non-naturally occurring Nef protein and (2) an E3 ubiquitin ligase or fragment thereof. In some embodiments, the non-naturally occurring Nef protein comprises a deletion of amino acids 207-223 as numbered in SEQ ID NO: 1, SEQ ID NO: 2 or corresponding amino acids thereof. In some embodiments, the non-naturally occurring Nef protein comprises a deletion of amino acids 50-91 as numbered in SEQ ID NO: 1, SEQ ID NO: 2 or corresponding amino acids thereof. In some embodiments, the non-naturally occurring Nef protein consists of the ami