THERAPEUTIC COMPOSITIONS AND METHODS FOR ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION
Embodiments of the disclosure provide compositions, kits, and methods for allogeneic hematopoietic cell transplantation (alloHCT) to patients. In an embodiment, a therapeutic composition for alloHCT includes at least first and second populations of isolated CD45+ cells (ICC). At least a portion of the CD45+ cells in the first population may have an antibody bound to a marker on the cell surface which is used to separate CD34+ cells from a mixture of nucleated cells (MNC) from donor or invitro produced blood. The MNC may comprise various cell types in various amounts, for example, about 70% CD34+ cells, less than about 5% CD3+ cells and less about 20% granulocytes. The ICC's in the second population include regulatory T cells which are typically at least about 50% of the population. Embodiments of the disclosure are particularly useful for treatment of hematologic cancers (e.g., leukemia, lymphoma), sickle cell anemia, GVHD, autoimmune and other diseases.
1 . A method of treating a disease or condition in a human subject, the method comprising:
(a) obtaining isolated regulatory T cells (Tregs); wherein the obtaining Tregs comprises:
(i) contacting a donor cell sample comprising Tregs with an amount of an anti-human CD25 affinity reagent such that less than 85% of the CD25 polypeptides expressed on the surface of the Tregs in the donor cell sample are occupied by the anti-human CD25 affinity reagent;
(ii) isolating Tregs from (a)(i) occupied by the anti-human CD25 affinity reagent;
thereby obtaining the isolated Tregs; and
(b) administering the isolated Tregs to the human subject;
wherein the isolated Tregs exhibit pSTAT5 activity according to an in vitro assay.
2 . The method of claim 1 , wherein from 30% to 80% of the CD25 polypeptides on the Tregs are occupied by the anti-human CD25 affinity reagent.
3 . The method of claim 1 , wherein the isolated Tregs exhibit from 10% to 50% of the pSTAT5 activity in an in vitro assay as compared to Tregs that do not have CD25 polypeptides occupied by the anti-human CD25 affinity reagent.
4 . The method of claim 1 , wherein the method further comprises isolating hematopoietic stem cells (HSCs), hematopoietic stem and progenitor cells (HSPCs), or both HSCs and HSPCs from the donor cell sample.
5 . The method of claim 4 , wherein the isolating of the isolated HSCs or the isolated HSPCs comprises contacting the donor cell sample with an anti-human CD34 affinity reagent and isolating cells that have CD34s occupied by the anti-human CD34 affinity reagent, thereby obtaining the isolated HSCs, the isolated HSPCs, or both the isolated HSCs and the isolated HSPCs.
6 . The method of claim 4 , wherein the method further comprises administering the isolated HSCs, the HSPCs, or both the isolated HSCs and the isolated HSPCs to the human subject, wherein from 5×10 5 to 2×10 7 HSCs or from 5×10 5 to 2×10 7 HSPCs per kilogram of the human subject's actual body weight or ideal body weight are administered to the human subject.
7 . The method of claim 6 , wherein from 5×10 5 to 5×10 6 Tregs per kilogram of the human subject's actual body weight or ideal body weight are administered to the human subject.
8 . The method of claim 1 , further comprising contacting the donor cell sample with an anti-human CD3 affinity reagent and quantifying an amount of CD3 positive conventional T cells (Tcons) in the donor cell sample by identifying the presence of CD3 polypeptides on Tcons that are occupied by the anti-human CD3 affinity reagent.
9 . The method of claim 8 , wherein the human subject is further administered a cell population comprising from 5×10 5 to 5×10 6 Tcons per kilogram of actual body weight or ideal body weight of said human subject.
10 . The method of claim 1 , further comprising administering a single GVHD prophylactic agent to the human subject, wherein the single GVHD prophylactic agent is tacrolimus or sirolimus.
11 . The method of claim 1 , further comprising administering a conditioning regimen to the human subject.
12 . The method of claim 1 , wherein the donor cell sample is a blood sample.
13 . The method of claim 1 , wherein the contacting comprises determining that less than 85% of the CD25 polypeptides expressed on the surface of the Tregs in the donor cell sample are occupied by the anti-human CD25 affinity reagent according to an in vitro assay; wherein the in vitro assay comprises:
(1) measuring the amount of background fluorescence from a first sample of a cell population, wherein the background fluorescence is emitted at a wavelength corresponding to a first fluorophore and wherein a Mean Fluorescence Intensity Zero (MFI-0) value is assigned the measured amount of background fluorescence;
(2) contacting a second sample of a cell population with an anti-CD25 detection antibody which is conjugated to the first fluorophore, wherein the contacting occurs at a concentration, temperature, and time sufficient to achieve at least 90% saturation;
(3) measuring the amount of fluorescence emitted from the cells of step (2), wherein a Mean Fluorescence Intensity Saturation (MFI-sat) value is assigned the measured amount of fluorescence;
(4) contacting simultaneously a third sample of a cell population with the anti-CD25 detection antibody conjugated to the first fluorophore and the anti-human CD25 affinity reagent which lacks a fluorophore and under the contacting conditions used in step (2);
(5) measuring the amount of fluorescence emitted from the cells of step (4), wherein a Mean Fluorescence Intensity X (MFI-x) value is assigned the measured amount of fluorescence; and
(6) quantifying the percentage of receptor occupancy by the anti-human CD25 affinity reagent according to the following equation:
% Receptor Occupancy=100%−[(MFI-x−MFI-0)/(MFI-sat−MFI-0)×100%].
14 . The method of claim 1 , wherein the in vitro assay to assess pSTAT5 activity comprises an intracellular cytokine staining comprising:
(1) obtaining a cell having been exposed to the anti-human CD25 affinity reagent;
(2) contacting the cell with IL-2 at a temperature and time sufficient for the IL-2 to interact with its cognate receptor on the cell and activate IL-2 signaling by the cell;
(3) contacting the cell with each of:
(a) a first antibody conjugated with a first fluorophore and directed against CD3,
(b) a second antibody conjugated with a second fluorophore and directed against CD4,
(c) a third antibody conjugated with a third fluorophore and directed against CD25,
(d) a fourth antibody conjugated with a fourth fluorophore and directed against CD127, and
(e) a fifth antibody conjugated with a fifth fluorophore and directed against pSTAT5, wherein each of the first, second, third, fourth, and fifth fluorophore are different fluorophores;
(4) subjecting the cell of step (3) to flow cytometry which is gated for CD3 + CD4 + CD25 + CD127− and collecting the CD3 + CD4 + CD25 + CD127− cells:
(5) detecting the amount of fluorescence from the fifth fluorophores and detecting the amount of florescence from the first, the second, and/or the third fluorophores in the collected cells of step (5); and
(6) calculating the percentage of the collected cells which have fluorescence from the first, the second, and/or the third fluorophores and which have fluorescence from the fifth fluorophore, wherein this percentage represents the fraction of cells having receptors occupied by the anti-human CD25 affinity reagent.
15 . The method of claim 14 , wherein the fifth antibody directed against pSTAT5 is directed against pSTAT5 phosphorylated at Tyrosine 694.
16 . The method of claim 1 , wherein the pSTAT5 activity is assessed by determining MFI and/or percentage positive of the isolated Treg bound by an antibody directed against STAT5 phosphorylated at Tyrosine 694 as quantified by flow cytometry.
17 . The method of claim 1 , wherein the in vitro assay to assess pSTAT5 activity comprises determining the IL2 signaling capacity of the isolated Tregs.
18 . The method of claim 1 , wherein the anti-human CD25 affinity reagent is capable of blocking the pSTAT5 activity and/or blocking IL2 signaling in the isolated Tregs.
19 . A method of treating a disease or condition in a human subject, the method comprising:
(a) obtaining isolated regulatory T cells (Tregs); wherein the obtaining Tregs comprises:
(i) contacting a donor cell sample comprising Tregs with an amount of an anti-human CD25 affinity reagent such that more than 85% of the CD25 polypeptides expressed on the surface of the Tregs in the donor cell sample are occupied by the anti-human CD25 affinity reagent;
(ii) isolating Tregs from (a)(i) obtained by the anti-human CD25 affinity reagent;
thereby obtaining the isolated Tregs; and
(b) administering the isolated Tregs to the human subject;
wherein the isolated Tregs exhibit pSTAT5 activity according to an in vitro assay;
wherein the in vitro assay to assess pSTAT5 activity comprises determining the IL2 signaling capacity of the isolated Tregs; and
wherein the anti-human CD25 affinity reagent does not block IL2 signaling in the Tregs.
20 . A kit for use in preparation of a therapeutic composition, the kit comprising:
(a) an anti-human CD25 affinity reagent; and
(b) instructions for use of (a) to isolate regulatory T cells (Tregs) from a donor cell sample;
wherein the instructions include directions to isolate the Tregs which have the anti-human CD25 affinity reagent such that less than 85% of CD25 polypeptides expressed on the surface of the Tregs in the donor cell sample are occupied by the anti-human CD25 affinity reagent, and
optionally wherein the kit further comprises an anti-human CD34 affinity reagent and instructions to isolate a cell population of CD34 positive hematopoietic stem and progenitor cells (HSPCs); a means to isolate CD25 positive cells and/or CD34 positive cells; and an anti-human CD3 affinity reagent and instructions for quantifying an amount of conventional T cells (Tcons) in the donor cell sample, optionally wherein the means comprises a sorting column, optionally wherein the sorting column is a magnetized column, and optionally wherein the instructions include directions to detect pSTAT5 activity in the Tregs in an in vitro assay.