# US 10,369,172 B2 · Combination therapy for a stable and long term engraftment

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- Number: US 10,369,172 B2
- Document ID: US10369172B2
- Status: Granted
- Kind: B2
- Application number: 14/367,917
- Filed: 2012-12-20
- Published: 2014-12-11
- Granted: 2019-08-06
- Earliest claimed: 2011-12-22
- Assignees: Yeda Research and Development Co. Ltd.
- Applicants: Yeda Research and Development Co. Ltd.
- Inventors: Yair Reisner; Esther Bachar-Lustig
- Web page: https://patentgrep.com/patent/US10369172B2

## CPC

- A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
- A61K 31/198: Alpha-amino acids, e.g. alanine or edetic acid [EDTA] (betaine A61K31/205; proline A61K31/401; tryptophan A61K31/405; histidine A61K31/4172; peptides not degraded to individual amino acids A61K38/00)
- A61K 31/255: of sulfoxy acids or sulfur analogues thereof
- A61K 31/661: Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion {or mevinphos}
- A61K 31/675: having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
- A61K 31/7076: containing purines, e.g. adenosine, adenylic acid
- A61K 35/22: Urine; Urinary tract, e.g. kidney or bladder; Intraglomerular mesangial cells; Renal mesenchymal cells; Adrenal gland
- A61K 35/34: Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes (vascular smooth muscle A61K35/44)
- A61K 35/36: Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells (islets of Langerhans A61K35/39)
- A61K 35/38: Stomach; Intestine; Goblet cells; Oral mucosa; Saliva
- A61K 35/39: Pancreas; Islets of Langerhans (Langerhans cells of epidermis A61K35/36)
- A61K 35/407: Liver; Hepatocytes
- A61K 35/42: Respiratory system, e.g. lungs, bronchi or lung cells
- A61K 39/3955: {against proteinaceous materials, e.g. enzymes, hormones, lymphokines}
- A61P 1/16: for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 7/00: Drugs for disorders of the blood or the extracellular fluid
- A61P 7/04: Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
- A61P 7/06: Antianaemics
- A61P 17/00: Drugs for dermatological disorders
- A61P 17/06: Antipsoriatics
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 21/04: for myasthenia gravis
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 35/00: Antineoplastic agents
- A61P 35/02: specific for leukemia
- A61P 37/02: Immunomodulators
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- C12N 5/0087: {Purging against subsets of blood cells, e.g. purging alloreactive T cells}
- A61K 2035/124: {the cells being hematopoietic, bone marrow derived or blood cells}
- A61K 2039/507: {Comprising a combination of two or more separate antibodies}
- A61K 2039/515: {Animal cells}
- C12N 2501/06: Anti-neoplasic drugs, anti-retroviral drugs, e.g. azacytidine, cyclophosphamide
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A method of treating a subject in need of a non-syngeneic cell or tissue graft is disclosed. The method comprising: (a) transplanting into a subject a dose of T cell depleted immature hematopoietic cells, wherein the T cell depleted immature hematopoietic cells comprise less than 5×105 CD3+ T cells per kilogram body weight of the subject, and wherein the dose comprises at least about 5×106 CD34+ cells per kilogram body weight of the subject; and subsequently (b) administering to the subject a therapeutically effective amount of cyclophosphamide, wherein the therapeutically effective amount comprises 25-200 mg per kilogram body weight, thereby treating the subject.

## Claims

### Claim 1

A method of reducing graft rejection and/or graft versus host disease (GVHD), the method comprising:
(a) transplanting into a subject conditioned under reduced intensity conditioning, wherein said reduced intensity conditioning is a non-myeloablative conditioning comprising a total body irradiation (TBI) or a total lymphoid irradiation (TLI) at an irradiation dose within the range of 0.5-5 Gy, a dose of T cell depleted immature hematopoietic cells obtained from a non-syngeneic donor, wherein said dose of said T cell depleted immature hematopoietic cells comprises less than 5×105 CD3+ T cells per kilogram body weight of the subject, and wherein said dose comprises at least 5×106 CD34+ cells per kilogram body weight of the subject; and subsequently
(b) administering to the subject a therapeutically effective amount of cyclophosphamide, wherein said therapeutically effective amount comprises 25-200 mg cyclophosphamide per kilogram body weight of the subject, and wherein said cyclophosphamide is administered to the subject following transplantation, and wherein said subject is not treated with GVHD prophylaxis for more than 10 days post transplant, thereby reducing the graft rejection and/or the GVHD.

### Claim 2 (depends on 1)

The method of claim 1, wherein said dose of said T cell depleted immature hematopoietic cells comprises 5-40×106 CD34+ cells per kilogram body weight of the subject.

### Claim 3 (depends on 1)

The method of claim 1, wherein said dose of said T cell depleted immature hematopoietic cells:
(i) comprises less than 0.5×106 CD8+ TCRα/β− cells per kilogram body weight of the subject; or
(ii) are obtained by T cell debulking.

### Claim 4 (depends on 1)

The method of claim 1, wherein said dose of said T cell depleted immature hematopoietic cells are treated by B cell debulking.

### Claim 5 (depends on 1)

The method of claim 1, wherein said reduced intensity conditioning
is effected 1-10 days prior to said transplanting.

### Claim 6 (depends on 1)

The method of claim 1, wherein:
(i) said TBI or said TLI comprises a single or fractionated irradiation dose within the range of 13.5 Gy; or
(ii) said non-myeloablative conditioning further comprises a chemotherapeutic agent comprising at least one of Busulfan, Fludarabine, Melphalan and Thiotepa; or
(iii) said non-myeloablative conditioning further comprises an antibody immunotherapy comprising at least one of an anti-CD52 antibody, an anti-thymocyte globulin (ATG) antibody and anti-CD3 (OKT3) antibody.

### Claim 7 (depends on 1)

The method of claim 1, wherein said amount of said cyclophosphamide is about 100-200 mg per kg body weight.

### Claim 8 (depends on 1)

The method of claim 1, wherein said cyclophosphamide is administered in a single dose or in two doses.

### Claim 9 (depends on 8)

The method of claim 8, wherein each of said two doses:
(i) comprises an amount of about 50 mg per kg body weight; or
(ii) is administered on days 3 and 4 following step (a).

### Claim 10 (depends on 1)

The method of claim 1, wherein the subject:
(i) is a human subject; or
(ii) has a malignant disease and optionally wherein said malignant disease is a hematopoietic cancer; or
(iii) has a non-malignant disease and optionally wherein said non-malignant disease is a genetic disease or disorder, a hematopoietic abnormality, an autoimmune disease or a metabolic disorder.

### Claim 11 (depends on 1)

The method of claim 1, wherein said subject is in need of a cell or tissue graft and wherein said cell or tissue graft is selected from the group consisting of immature hematopoietic cells, a liver, a pancreas, a spleen, a kidney, a heart, a lung, a skin, an intestine and a lymphoid tissue or organ.

### Claim 12 (depends on 11)

The method of claim 11,
(i) wherein said cell or tissue graft is transplanted into the subject prior to, concomitantly with or following said transplanting said dose of said T cell depleted immature hematopoietic cells into said subject; or
(ii) comprises a co-transplantation of several organs.

### Claim 13 (depends on 12)

The method of claim 12, wherein said cell or tissue graft and said dose of said T cell depleted immature hematopoietic cells are obtained from the same donor.

### Claim 14

A method of reducing graft rejection and/or graft versus host disease (GVHD), the method comprising:
(a) conditioning a subject under a reduced intensity conditioning, wherein said reduced intensity conditioning comprises a total body irradiation (TBI) or a total lymphoid irradiation (TLI) at an irradiation dose within the range of 0.5-5 Gy, and a chemotherapeutic agent;
(b) transplanting into the subject a dose of T cell depleted immature hematopoietic cells obtained from a non-syngeneic donor, wherein said dose of said T cell depleted immature hematopoietic cells comprises less than 5×105 CD3+ T cells per kilogram body weight of the subject, and wherein said dose comprises at least 5×106 CD34+ cells per kilogram body weight of the subject; and subsequently
(c) administering to the subject a therapeutically effective amount of cyclophosphamide, wherein said therapeutically effective amount comprises 25-200 mg cyclophosphamide per kilogram body weight of the subject, and wherein said cyclophosphamide is administered to the subject following transplantation, and wherein said subject is not treated with GVHD prophylaxis for more than 10 days post transplant,
thereby reducing graft rejection and/or GVHD.

### Claim 15 (depends on 14)

The method of claim 14, wherein said TBI:
(i) comprises a single or fractionated irradiation dose of 2-3 Gy; or
(ii) is effected in a single dose 2 days prior to step (b).

### Claim 16 (depends on 14)

The method of claim 14, wherein said chemotherapeutic agent comprises Fludarabine, and optionally wherein said Fludarabine:
(i) is effected at a dose of 30 mg/m2/day; or
(ii) is administered daily on days 3 to 7 prior to step (b).

### Claim 17 (depends on 14)

The method of claim 14, wherein said dose of said T cell depleted immature hematopoietic cells:
(i) comprises T cell depleted G-CSF mobilized peripheral blood progenitor cells; or
(ii) are obtained by T cell debulking.

### Claim 18 (depends on 14)

The method of claim 14, wherein said dose of said T cell depleted immature hematopoietic cells are treated by B cell debulking.

### Claim 19 (depends on 14)

The method of claim 14, wherein said cyclophosphamide is administered in two doses.

### Claim 20 (depends on 19)

The method of claim 19, wherein each of said two doses:
(i) comprises an amount of about 50 mg per kg body weight; or
(ii) is administered on days 3 and 4 following step (b).

### Claim 21

A method of inducing donor specific tolerance in a subject, the method comprising:
(a) transplanting into a subject conditioned under reduced intensity conditioning, wherein said reduced intensity conditioning is a non-myeloablative conditioning comprising a total body irradiation (TBI) or a total lymphoid irradiation (TLI) at an irradiation dose within the range of 0.5-5 Gy, a dose of T cell depleted immature hematopoietic cells obtained from a non-syngeneic donor, wherein said dose of said T cell depleted immature hematopoietic cells comprises less than 5×105 CD3+ T cells per kilogram body weight of the subject, and wherein said dose comprises at least 5×106 CD34+ cells per kilogram body weight of the subject; and subsequently
(b) administering to the subject a therapeutically effective amount of cyclophosphamide, wherein said therapeutically effective amount comprises 25-200 mg cyclophosphamide per kilogram body weight of the subject, and wherein said cyclophosphamide is administered to the subject following transplantation, and wherein said subject is not treated with GVHD prophylaxis for more than 10 days post transplant,
thereby inducing donor specific tolerance in the subject.

### Claim 22 (depends on 21)

The method of claim 21, wherein said subject is in need of a cell or tissue graft and wherein said cell or tissue graft is selected from the group consisting of immature hematopoietic cells, a liver, a pancreas, a spleen, a kidney, a heart, a lung, a skin, an intestine and a lymphoid tissue or organ.

### Claim 23 (depends on 22)

The method of claim 22,
(i) wherein said cell or tissue graft is transplanted into the subject prior to, concomitantly with or following said transplanting said dose of said T cell depleted immature hematopoietic cells into said subject; or
(ii) comprises a co-transplantation of several organs.

### Claim 24 (depends on 22)

The method of claim 22, wherein said cell or tissue graft and said dose of said T cell depleted immature hematopoietic cells are obtained from the same donor.

### Claim 25 (depends on 21)

The method of claim 21, wherein said dose of said T cell depleted immature hematopoietic cells comprises less than 0.5×106 CD8+ TCRα/β negative cells per kilogram body weight of the subject.

### Claim 26 (depends on 21)

The method of claim 21, wherein said reduced intensity conditioning is effected 1-10 days prior to said transplanting.

### Claim 27 (depends on 21)

The method of claim 21, wherein:
(i) said TBI or said TLI comprises a single or fractionated irradiation dose within the range of 1-3.5 Gy; or
(ii) said non-myeloablative conditioning further comprises a chemotherapeutic agent comprising at least one of Busulfan, Fludarabine, Melphalan and Thiotepa; or
(iii) said non-myeloablative conditioning further comprises an antibody immunotherapy comprising at least one of an anti-CD52 antibody, an anti-thymocyte globulin (ATG) antibody and anti-CD3 (OKT3) antibody.

### Claim 28 (depends on 27)

The method of claim 27, wherein:
(i) said TBI or said TLI comprises a single or fractionated irradiation dose within the range of 2-3 Gy.

### Claim 29 (depends on 6)

The method of claim 6, wherein:
(i) said TBI or said TLI comprises a single or fractionated irradiation dose within the range of 2-3 Gy.