JP-7854975-B2 - Compositions and methods for inhibiting lineage-specific proteins

Inventors

• ボーレン，ジョゼフ
• ラドヴィク－モレノ，アレクサンダー，フィリップ
• ライダード，ジョン

Assignees

• ブイオーアール バイオファーマ インコーポレーテッド

Dates

Publication Date

20260507

Application Date

20231113

Priority Date

20170228

Claims (20)

1. (i) an effective amount of a cytotoxic agent comprising an antibody or antigen-binding fragment thereof that specifically binds to a non-essential epitope of CD33 ; and (ii) a population of hematopoietic stem cells for use in a method of treating a subject, comprising administering to a population of hematopoietic stem cells, wherein CD33 expressed by hematopoietic stem cells or their progeny lacks a non-essential epitope to which the cytotoxic agent binds, and the hematopoietic stem cells or their progeny are genetically modified in exon 2 of the gene encoding CD33 such that they have reduced binding to the cytotoxic agent.
2. A population of hematopoietic stem cells for use according to claim 1, wherein the gene modification encodes a mutation in one or more amino acid residues in a non-essential epitope to which a cytotoxic agent binds.
3. A population of hematopoietic stem cells for use according to claim 1 or 2, wherein the gene modification encodes the deletion of one or more amino acid residues in a non-essential epitope to which a cytotoxic agent binds.
4. A population of hematopoietic stem cells for use according to any one of claims 1 to 3, wherein the antigen-binding fragment is a single-chain antibody fragment (scFv) that specifically binds to a non-essential epitope of CD33 .
5. A population of hematopoietic stem cells for use according to any one of claims 1 to 3 , wherein the cytotoxic agent is an antibody or an antibody-drug conjugate (ADC).
6. A population of hematopoietic stem cells for use according to any one of claims 1 to 4, wherein the cytotoxic agent is an immune cell expressing a chimeric receptor containing an antigen-binding fragment.
7. A population of hematopoietic stem cells for use according to claim 6, wherein the immune cells are T cells.
8. A population of hematopoietic stem cells for use according to any one of claims 1 to 7, wherein the non-essential epitope comprises at least three amino acids.
9. A population of hematopoietic stem cells for use according to any one of claims 1 to 8, wherein the non-essential epitope consists of 6 to 10 amino acids.
10. A population of hematopoietic stem cells for use according to any one of claims 1 to 9 , wherein the gene modification is present in the sequence targeted by the sequence described in SEQ ID NO: 30.
11. A population of hematopoietic stem cells for use according to any one of claims 1 to 10 , wherein the hematopoietic stem cells are derived from bone marrow cells, umbilical cord blood cells, or peripheral blood mononuclear cells (PBMCs).
12. A population of hematopoietic stem cells for use according to any one of claims 1 to 11 , wherein the hematopoietic stem cells are autologous.
13. A population of hematopoietic stem cells for use according to any one of claims 1 to 11 , wherein the hematopoietic stem cells are allogeneic.
14. A population of hematopoietic stem cells for use according to any one of claims 1 to 13 , wherein the subject has a hematopoietic malignancy.
15. A population of hematopoietic stem cells for use according to any one of claims 1 to 14 , wherein the subject has Hodgkin lymphoma, non-Hodgkin lymphoma, leukemia, or multiple myeloma.
16. A population of hematopoietic stem cells for use according to any one of claims 1 to 15 , having leukemia, wherein the subject is acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, or chronic lymphoblastic leukemia.
17. Genetically modified hematopoietic cells containing the CD33 gene, which includes a gene modification present in the sequence targeted by the sequence described in Sequence ID No. 30.
18. A guide RNA (gRNA) containing the target sequence according to Sequence ID No. 30.
19. The gRNA according to claim 18 , wherein the gRNA is a single guide RNA (sgRNA) and/or the gRNA is chemically modified.
20. A method for producing genetically modified hematopoietic cells, The method comprising gene editing the CD33 gene in a population of hematopoietic cells using a CRISPR/Cas system comprising a Cas protein and the gRNA described in claim 18 or 19 , thereby producing genetically modified hematopoietic cells.

Description

Cross-reference of related applications This application claims the benefit of U.S. Provisional Application No. 62/464,975 filed on 28 February 2017 under 35 U.S.C § 119(e). The entire contents of the referenced application are incorporated herein by reference. Background to Disclosure: A major challenge in designing targeted therapies is the successful identification of proteins that are specifically expressed in cells that are appropriate to be removed therapeutically (e.g., abnormal, malignant, or other target cells) but not in cells that should not be removed (e.g., normal, healthy, or other non-target cells). For example, many cancer drugs struggle to effectively target cancer cells while leaving normal cells intact. Emerging alternative strategies include targeting entire cell lineages, including normal cells, cancer cells, and precancerous cells. For example, CD19-targeting chimeric antigen receptor T cells (CAR T cells) and anti-CD20 monoclonal antibodies (e.g., rituximab) target B cell lineage proteins (CD19 and CD20, respectively). While potentially effective for treating B-cell malignancies, the use of such therapies is limited due to the harmful effects of B cell elimination. Similarly, targeting lineage-specific proteins of other cell populations, such as myeloid cells (e.g., cancers arising from myeloblasts, monocytes, megakaryocytes, etc.), is impractical because these cell populations are essential for survival. Summary of Disclosure This disclosure is based, at least in part, on the identification of epitopes (e.g., non-essential epitopes) in lineage-specific cell surface proteins that can be targeted by cytotoxic agents, where the cytotoxic agent induces cell death in cells expressing the protein containing the epitope, but does not induce cell death in cells (e.g., hematopoietic stem cells) expressing the protein in which the epitope has been engineered (e.g., genetically) to reduce binding to the cytotoxic agent and consequently avoid cell death. Such methods are expected to provide a safe and effective treatment for hematopoietic malignancies. Therefore, one aspect of this disclosure provides a method for treating hematopoietic malignancies, the method comprising administering to a subject requiring it (i) an effective amount of a cytotoxic agent targeting cells expressing a lineage-specific cell surface protein, and (ii) a population of hematopoietic cells, wherein the hematopoietic cells or their progeny are engineered to not bind to the cytotoxic agent or to have reduced binding to the cytotoxic agent. In some embodiments, the cytotoxic agent comprises an antigen-binding fragment that specifically binds to an epitope of the lineage-specific cell surface protein. In some embodiments, the hematopoietic cells or their progeny express the lineage-specific cell surface protein and are genetically engineered so that the lineage-specific cell surface protein lacks an epitope to which the cytotoxic agent binds. In some embodiments, the hematopoietic cells are genetically engineered so that the lineage-specific cell surface protein expressed in the hematopoietic cells or their progeny has a mutant or variant epitope to which the cytotoxic agent has reduced binding activity or cannot bind. In any embodiment described herein, the epitope of the lineage-specific cell surface protein may be non-essential. Optionally, any of the methods provided herein may further include pre-conditioning the subject by administering, for example, one or more chemotherapeutic agents or other cancer therapies to the subject before administering the cytotoxic agent and/or hematopoietic cells. In some embodiments, the subject is pre-conditioned before administering the cytotoxic agent and/or hematopoietic cells. In other embodiments, any of the methods provided herein may further include administering one or more chemotherapeutic agents or one or more other cancer therapies to the subject in conjunction with the administration of the cytotoxic agent and/or hematopoietic cells. The chemotherapeutic agents or other cancer therapies may be administered before, simultaneously with, or after the administration of the cytotoxic agent and/or hematopoietic cells. Alternatively or additionally, any of the methods described herein may further include preparing hematopoietic cells lacking epitopes to which cytotoxic agents bind, for example, by genetic modification. A cytotoxic agent for use in any of the methods described herein comprises an antigen-binding fragment (e.g., a single-chain antibody fragment or scFv) that specifically binds to an epitope of a lineage-specific cell surface protein. In some embodiments, the cytotoxic agent is an antibody or an antibody-drug conjugate (ADC). In some embodiments, the cytotoxic agent may be an immune cell (e.g., a T cell) expressing a chimeric receptor containing the antigen-binding fragment. The immune cell may be allogeneic or autologous. The chimeric receptor may further include (a