# US 11,028,177 B2 · Effective targeting of primary human leukemia using anti-CD123 chimeric antigen receptor engineered T cells

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- Number: US 11,028,177 B2
- Document ID: US11028177B2
- Status: Granted
- Kind: B2
- Application number: 15/400,096
- Filed: 2017-01-06
- Published: 2017-06-29
- Granted: 2021-06-08
- Earliest claimed: 2013-02-20
- Assignees: Novartis AG; The Trustees of the University of Pennsylvania
- Applicants: Novartis AG; The Trustees of the University of Pennsylvania
- Inventors: Jennifer Brogdon; Saar Gill; Carl H. June; Michael D. Kalos; Andreas Loew; John Scholler
- Web page: https://patentgrep.com/patent/US11028177B2

## CPC

- C07K 16/2866: {against receptors for cytokines, lymphokines, interferons}
- A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
- A61K 38/1774: {Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)}
- A61K 39/3955: {against proteinaceous materials, e.g. enzymes, hormones, lymphokines}
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/31: Chimeric antigen receptors [CAR]
- A61K 40/4224: {Molecules with a "CD" designation not provided for elsewhere}
- A61P 35/00: Antineoplastic agents
- A61P 35/02: specific for leukemia
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 14/70517: {CD8}
- C07K 16/2803: {against the immunoglobulin superfamily}
- A61K 2039/505: {comprising antibodies}
- A61K 2039/507: {Comprising a combination of two or more separate antibodies}
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/53: Hinge
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2319/00: Fusion polypeptide
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

The invention provides compositions and methods for treating leukemia, for example, acute myeloid leukemia (AML) and B-cell acute lymphoid leukemia (B-ALL). The invention also relates to at least one chimeric antigen receptor (CAR) specific to CD123, vectors comprising the same, and recombinant T cells comprising the CD123 CAR. The invention also includes methods of administering a genetically modified T cell expressing a CAR that comprises a CD123 binding domain. The invention also includes methods of bone marrow ablation for use in treatments necessitating bone marrow reconditioning or transplant.

## Claims

### Claim 1

A method of eradicating at least a portion of existing CD123-expressing cells in the bone marrow in a subject, the method comprising administering to the subject an effective amount of T cells expressing a chimeric antigen receptor (CAR) molecule comprising an anti-CD123 binding domain, a transmembrane domain, and an intracellular signaling domain, thereby eradicating at least a portion of the existing CD123-expressing cells in the bone marrow in a subject, wherein said anti-CD123 binding domain comprises:
(a) a light chain variable region comprising:
a light chain complementarity determining region 1 (LC CDR1) comprising the sequence of SEQ ID NO: 20, a light chain complementarity determining region 2 (LC CDR2) comprising the sequence of SEQ ID NO: 21, and a light chain complementarity determining region 3 (LC CDR3) comprising the sequence of SEQ ID NO: 22, and
a heavy chain variable region comprising:
a heavy chain complementarity determining region 1 (HC CDR1) comprising the sequence of SEQ ID NO: 16, a heavy chain complementarity determining region 2 (HC CDR2) comprising the sequence of SEQ ID NO: 17, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the sequence of SEQ ID NO: 18; or
(b) a light chain variable region comprising:
a light chain complementarity determining region 1 (LC CDR1) comprising the sequence of SEQ ID NO: 28, a light chain complementarity determining region 2 (LC CDR2) comprising the sequence of SEQ ID NO: 29, and a light chain complementarity determining region 3 (LC CDR3) comprising the sequence of SEQ ID NO: 30, and
a heavy chain variable region comprising:
a heavy chain complementarity determining region 1 (HC CDR1) comprising the sequence of SEQ ID NO: 24, a heavy chain complementarity determining region 2 (HC CDR2) comprising the sequence of SEQ ID NO: 25, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the sequence of SEQ ID NO: 26; or
(c) a light chain variable region comprising:
a light chain complementarity determining region 1 (LC CDR1) comprising the sequence of SEQ ID NO: 87, a light chain complementarity determining region 2 (LC CDR2) comprising the sequence of SEQ ID NO: 88, and a light chain complementarity determining region 3 (LC CDR3) comprising the sequence of SEQ ID NO: 89, and
a heavy chain variable region comprising:
a heavy chain complementarity determining region 1 (HC CDR1) comprising the sequence of SEQ ID NO: 84, a heavy chain complementarity determining region 2 (HC CDR2) comprising the sequence of SEQ ID NO: 85, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the sequence of SEQ ID NO: 86.

### Claim 2

A method of treating a subject having cancer, the method comprising
administering to the subject an amount of T cells expressing a chimeric antigen receptor (CAR) molecule effective to eradicate at least a portion of existing CD123-expressing cells in the bone marrow in a subject, wherein said CAR molecule comprises an anti-CD123 binding domain, a transmembrane domain, and an intracellular signaling domain,
thereby treating the subject having cancer, wherein said anti-CD123 binding domain comprises:
(a) a light chain variable region comprising:
a light chain complementarity determining region 1 (LC CDR1) comprising the sequence of SEQ ID NO: 20, a light chain complementarity determining region 2 (LC CDR2) comprising the sequence of SEQ ID NO: 21, and a light chain complementarity determining region 3 (LC CDR3) comprising the sequence of SEQ ID NO: 22, and
a heavy chain variable region comprising:
a heavy chain complementarity determining region 1 (HC CDR1) comprising the sequence of SEQ ID NO: 16, a heavy chain complementarity determining region 2 (HC CDR2) comprising the sequence of SEQ ID NO: 17, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the sequence of SEQ ID NO: 18; or
(b) a light chain variable region comprising:
a light chain complementarity determining region 1 (LC CDR1) comprising the sequence of SEQ ID NO: 28, a light chain complementarity determining region 2 (LC CDR2) comprising the sequence of SEQ ID NO: 29, and a light chain complementarity determining region 3 (LC CDR3) comprising the sequence of SEQ ID NO: 30, and
a heavy chain variable region comprising:
a heavy chain complementarity determining region 1 (HC CDR1) comprising the sequence of SEQ ID NO: 24, a heavy chain complementarity determining region 2 (HC CDR2) comprising the sequence of SEQ ID NO: 25, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the sequence of SEQ ID NO: 26; or
(c) a light chain variable region comprising:
a light chain complementarity determining region 1 (LC CDR1) comprising the sequence of SEQ ID NO: 87, a light chain complementarity determining region 2 (LC CDR2) comprising the sequence of SEQ ID NO: 88, and a light chain complementarity determining region 3 (LC CDR3) comprising the sequence of SEQ ID NO: 89, and
a heavy chain variable region comprising:
a heavy chain complementarity determining region 1 (HC CDR1) comprising the sequence of SEQ ID NO: 84, a heavy chain complementarity determining region 2 (HC CDR2) comprising the sequence of SEQ ID NO: 85, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the sequence of SEQ ID NO: 86.

### Claim 3

A method of treating a subject having a disease, disorder or condition that is treatable with a bone marrow transplant or bone marrow reconditioning, the method comprising
administering to the subject an amount of T cells expressing a chimeric antigen receptor (CAR) molecule effective to eradicate at least a portion of existing CD123-expressing cells in the bone marrow in a subject, wherein said CAR molecule comprises an anti-CD123 binding domain, a transmembrane domain, and an intracellular signaling domain,
thereby treating the subject having a disease, disorder or condition that is treatable with a bone marrow transplant or bone marrow reconditioning, wherein said anti-CD123 binding domain comprises:
(a) a light chain variable region comprising:
a light chain complementarity determining region 1 (LC CDR1) comprising the sequence of SEQ ID NO: 20, a light chain complementarity determining region 2 (LC CDR2) comprising the sequence of SEQ ID NO: 21, and a light chain complementarity determining region 3 (LC CDR3) comprising the sequence of SEQ ID NO: 22, and
a heavy chain variable region comprising:
a heavy chain complementarity determining region 1 (HC CDR1) comprising the sequence of SEQ ID NO: 16, a heavy chain complementarity determining region 2 (HC CDR2) comprising the sequence of SEQ ID NO: 17, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the sequence of SEQ ID NO: 18; or
(b) a light chain variable region comprising:
a light chain complementarity determining region 1 (LC CDR1) comprising the sequence of SEQ ID NO: 28, a light chain complementarity determining region 2 (LC CDR2) comprising the sequence of SEQ ID NO: 29, and a light chain complementarity determining region 3 (LC CDR3) comprising the sequence of SEQ ID NO: 30, and
a heavy chain variable region comprising:
a heavy chain complementarity determining region 1 (HC CDR1) comprising the sequence of SEQ ID NO: 24, a heavy chain complementarity determining region 2 (HC CDR2) comprising the sequence of SEQ ID NO: 25, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the sequence of SEQ ID NO: 26; or
(c) a light chain variable region comprising:
a light chain complementarity determining region 1 (LC CDR1) comprising the sequence of SEQ ID NO: 87, a light chain complementarity determining region 2 (LC CDR2) comprising the sequence of SEQ ID NO: 88, and a light chain complementarity determining region 3 (LC CDR3) comprising the sequence of SEQ ID NO: 89, and
a heavy chain variable region comprising:
a heavy chain complementarity determining region 1 (HC CDR1) comprising the sequence of SEQ ID NO: 84, a heavy chain complementarity determining region 2 (HC CDR2) comprising the sequence of SEQ ID NO: 85, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the sequence of SEQ ID NO: 86.

### Claim 4 (depends on 1)

The method of claim 1, wherein the method is a cellular conditioning method prior to bone marrow transplantation.

### Claim 5 (depends on 1)

The method of claim 1, wherein the portion of existing CD123-expressing cells in the bone marrow eradicated comprises normal CD123-expressing bone marrow cells.

### Claim 6 (depends on 1)

The method of claim 1, wherein the portion of existing CD123-epxressing cells in the bone marrow eradicated comprises CD123-expressing bone marrow myeloid progenitor cells.

### Claim 7 (depends on 1)

The method of claim 1, wherein all or essentially all of the existing CD123-expressing cells in the bone marrow in the subject is eradicated.

### Claim 8 (depends on 1)

The method of claim 1, wherein the number of CD123-expressing cells in bone marrow is reduced.

### Claim 9 (depends on 1)

The method of claim 1, wherein the subject is in need of a bone marrow transplant or bone marrow reconditioning.

### Claim 10 (depends on 1)

The method of claim 1, wherein the subject has a disease, disorder or condition that is treatable with a bone marrow transplant or bone marrow reconditioning.

### Claim 11 (depends on 10)

The method of claim 10, wherein the disease, disorder or condition is selected from the group consisting of a hematological cancer, a solid tumor, a hematological disease, a metabolic disorder, HIV, HTLV, a lysosomal storage disorder and an immunodeficiency.

### Claim 12 (depends on 11)

The method of claim 11, wherein the hematological cancer is a leukemia, lymphoma, or myeloma.

### Claim 13 (depends on 11)

The method of claim 11, wherein the hematological cancer acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphoid leukemia (CLL), chronic myelogenous leukemia (CML), Hodgkin's disease, Non-Hodgkin's lymphoma, or multiple myeloma.

### Claim 14 (depends on 1)

The method of claim 1, wherein the method is performed prior to a bone marrow transplant.

### Claim 15 (depends on 1)

The method of claim 1, further comprising administering a bone marrow transplant to the subject following the eradication of at least a portion of existing CD123-expressing cells in the bone marrow in the subject.

### Claim 16 (depends on 15)

The method of claim 15, wherein the bone marrow transplant comprises transplantation of a stem cell.

### Claim 17 (depends on 15)

The method of claim 15, wherein the bone marrow transplant comprises transplantation of autologous bone marrow cells or autologous stem cells.

### Claim 18 (depends on 15)

The method of claim 15, wherein the bone marrow transplant comprises transplantation of allogeneic bone marrow cells or allogeneic stem cells.

### Claim 19 (depends on 1)

The method of claim 1, wherein the subject has a hematological cancer or a solid tumor.

### Claim 20 (depends on 19)

The method of claim 19, further comprising administering one or more anti-cancer therapy.

### Claim 21 (depends on 20)

The method of claim 20, wherein the anti-cancer therapy is an anti-cancer CAR therapy, chemotherapy, or radiation.

### Claim 22 (depends on 1)

The method of claim 1, wherein the T cells expressing the CAR molecule are CD8+ T cells.

### Claim 23 (depends on 1)

The method of claim 1, wherein the T cells expressing the CAR molecule are human cells.

### Claim 24 (depends on 1)

The method of claim 1, wherein the T cells expressing the CAR molecule are allogeneic T cells.

### Claim 25 (depends on 1)

The method of claim 1, wherein the T cells expressing the CAR molecule are autologous T cells.

### Claim 26 (depends on 1)

The method of claim 1, wherein the anti-CD123 binding domain comprises an antibody molecule.

### Claim 27 (depends on 26)

The method of claim 26, wherein the antibody molecule comprises a scFv, a F(ab′), a F(ab′)2, a Fv fragment, an immunoglobulin single domain antibody (sdAb), a single light chain variable domain (VL), a single heavy chain variable domain (VH), or a nanobody.

### Claim 28 (depends on 26)

The method of claim 26, wherein the antibody molecule is a murine, human, or humanized antibody molecule.

### Claim 29 (depends on 1)

The method of claim 1, wherein
(a) the anti-CD123 binding domain comprises a light chain variable region comprising an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a light chain variable region provided in SEQ ID NO: 36, SEQ ID NO: 42, SEQ ID NO: 48, SEQ ID NO: 54, SEQ ID NO: 60, SEQ ID NO: 66, SEQ ID NO: 72, or SEQ ID NO: 78, or a sequence with 95-99% identity to an amino acid sequence provided in SEQ ID NO: 36, SEQ ID NO: 42, SEQ ID NO: 48, SEQ ID NO: 54, SEQ ID NO: 60, SEQ ID NO: 66, SEQ ID NO: 72, or SEQ ID NO: 78;
(b) the anti-CD123 binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a heavy chain variable region provided in SEQ ID NO: 36, SEQ ID NO: 42, SEQ ID NO: 48, SEQ ID NO: 54, SEQ ID NO: 60, SEQ ID NO: 66, SEQ ID NO: 72, or SEQ ID NO: 78, or a sequence with 95-99% identity to an amino acid sequence provided in SEQ ID NO: 36, SEQ ID NO: 42, SEQ ID NO: 48, SEQ ID NO: 54, SEQ ID NO: 60, SEQ ID NO: 66, SEQ ID NO: 72, or SEQ ID NO: 78; or
(c) both (a) and (b).

### Claim 30 (depends on 1)

The method of claim 1, wherein the anti-CD123 binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 42, SEQ ID NO: 48, SEQ ID NO: 54, SEQ ID NO: 60, SEQ ID NO: 66, SEQ ID NO: 72, and SEQ ID NO: 78, or a sequence with 95-99% identity thereof.

### Claim 31 (depends on 1)

The method of claim 1, wherein the anti-CD123 binding domain comprises the amino acid sequence of SEQ ID NO: 101 or SEQ ID NO: 2, or an amino acid sequence with 95-99% identity thereto.

### Claim 32 (depends on 1)

The method of claim 1, wherein the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

### Claim 33 (depends on 1)

The method of claim 1, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 5; an amino acid sequence comprising at least one, two or three modifications but not more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO: 5; or an amino acid sequence with 95-99% identity to the amino acid sequence of SEQ ID NO:5.

### Claim 34 (depends on 1)

The method of claim 1, wherein the anti-CD123 binding domain is connected to the transmembrane domain by a hinge region.

### Claim 35 (depends on 34)

The method of claim 34, wherein the hinge region comprises the amino acid sequence of any of SEQ ID NO: 4, 104, 122 or 124, or a sequence with 95-99% identity thereof.

### Claim 36 (depends on 1)

The method of claim 1, wherein the intracellular signaling domain comprises a functional signaling domain of 4-1BB and a functional signaling domain of CD3 zeta.

### Claim 37 (depends on 1)

The method of claim 1, wherein the intracellular signaling domain comprises a costimulatory domain.

### Claim 38 (depends on 1)

The method of claim 1, wherein the intracellular signaling domain comprises a primary signaling domain.

### Claim 39 (depends on 1)

The method of claim 1, wherein the intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.

### Claim 40 (depends on 1)

The method of claim 1, wherein the intracellular signaling domain comprises:
(i) the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 23;
(ii) the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 98; or
(iii) both (i) and (ii).

### Claim 41 (depends on 37)

The method of claim 37, wherein the costimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and 4-1BB (CD137).

### Claim 42 (depends on 37)

The method of claim 37, wherein the costimulatory domain comprises
(i) the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 23; an amino acid sequence comprising at least one, two or three modifications but not more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 23; or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 23.

### Claim 43 (depends on 1)

The method of claim 1, wherein intracellular signaling domain comprises:
(i) a costimulatory domain comprising a functional signaling domain of a protein chosen from OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), or 4-1BB (CD137);
(ii) a primary signaling domain comprising a functional signaling domain derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278, CD66d, DAP10, or DAP12; or
(iii) both (i) and (ii).

### Claim 44 (depends on 39)

The method of claim 39, wherein the costimulatory domain comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 23; an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 23; or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 23, and the primary signaling domain comprises the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 98; an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 98; or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 98.

### Claim 45 (depends on 1)

The method of claim 1, wherein the CAR molecule comprises an amino acid sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:47, SEQ ID NO:53, SEQ ID NO:59, SEQ ID NO:65, SEQ ID NO:71, SEQ ID NO:77, or SEQ ID NO:83, or a sequence with 95-99% identity thereof.

### Claim 46 (depends on 1)

The method of claim 1, wherein the CAR molecule comprises an amino acid sequence of SEQ ID NO: 100, or a sequence with 95-99% identity thereof.

### Claim 47 (depends on 1)

The method of claim 1, wherein the CAR molecule comprises an amino acid sequence of SEQ ID NO: 97, or a sequence with 95-99% identity thereof.