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Placenta-derived allogeneic car-t cells and uses thereof

A cell and placental technology, applied in embryonic cells, animal cells, receptors/cell surface antigens/cell surface determinants, etc., can solve problems such as reduced alloreactivity

Pending Publication Date: 2021-09-14
ANTHROGENESIS LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Inactivation of the T cell receptor gene reduces alloreactivity

Method used

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  • Placenta-derived allogeneic car-t cells and uses thereof
  • Placenta-derived allogeneic car-t cells and uses thereof
  • Placenta-derived allogeneic car-t cells and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0082] Example 1: Starting material, MNC isolation and T cell isolation

[0083] The starting material placental blood (which contains human umbilical cord blood (UCB) and / or human placental perfusate (HPP)) was collected by LifebankUSA with informed consent. Following collection, mononuclear cells (MNCs) were enriched in the starting material using Hetastarch RBC sedimentation or Ficoll-Paque density gradient cell separation. Next, MNCs undergo a forward selection process to deplete CD25+ T regulatory T cells (Treg) followed by forward selection against CD4+ and CD8+ T cells using the Militenyi Bead Cell Isolation Kit. Aliquots of isolated T cells were obtained for serological and sterility testing, as well as phenotypic analysis, prior to freezing the cells.

[0084] The phenotype of isolated P-T cells differs from peripheral blood mononuclear cells (PBMC). P-T cells contained >78% CD3+CD56- T cells and consisted primarily of CD3+CD45RA+CCR7+CD27+ naive T cells with a lo...

example 2

[0087] Example 2: T cell activation and expansion

[0088] Unmodified P-T cells:

[0089] The isolated P-T cells were thawed, subjected to CD25 depletion using Miltenyi anti-CD25 beads to remove CD4+CD25+CD127-Tregs (which can be included prior to the T cell isolation step), and treated with anti-CD3 / anti-CD28 Dynabeads from Invitrogen ( 1:1 bead:cell ratio) or activated with anti-CD3 / anti-CD28 nanoparticles Transact (1:100 volume dilution) from Miltenyi. Next, cells were expanded with 100 IU / mL IL-2, 10 ng / mL IL-7 + 10 ng / mL IL-15, or 100 IU / mL IL-2 + 10 ng / mL IL-7. Additional restimulations were done on days 12-14, and cells were expanded in Grex vessels until day 21 to maximize fold expansion.

[0090] When cultured to day 20, unmodified P-T cells can expand up to 600-fold on initial stimulation and up to 3,600-fold on restimulation (RS) on day 14.

[0091] Under various culture conditions, unmodified P-T expanded for 20 days exhibited an earlier differentiated phenoty...

example 3

[0102] Example 3: CD19 CAR and CD20 CAR in vitro activity

[0103] Day 15 Cytolytic activity of P-CD19 CAR-T cells against cancer cell lines

[0104] On days 2-4 of UCB-T culture, activated UCB-T cells were transduced with CD19 CAR retrovirus or lentivirus using centrifugation inoculation. CAR expression was detected using a FITC-labeled recombinant CD19-Fc fusion protein, or an anti-Myc PE antibody if the CAR vector contained a Myc tag. UCB-CAR-T activity was assessed using the following two assays.

[0105] CD19 CAR-transduced UCB-T cells specifically killed CD19+ Daudi cancer targets at levels comparable to PBMC CD19CAR T cells, but not CD19-K562 cells.

[0106] CD20 CAR-transduced UCB-T cells specifically killed CD20+ Daudi cancer targets at levels comparable to PBMC CD20 CAR T cells, but not CD20-Molp8 cells.

[0107] CD19 CAR-transduced UCB-T cells specifically secreted the proinflammatory cytokines IFN-g and GM-CSF and secreted the cytolytic effector protein pe...

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Abstract

The present invention discloses populations of T cells expressing a chimeric antigen receptor (CAR), wherein said T cells are placental T cells derived from cord blood, placental perfusate, or a mixture thereof. Such populations of cells are shown to be improved in a number of aspects over alternative populations of cells such as those derived from peripheral blood mono-nuclear cell T cells. It also discloses methods of treating cancer, such as a hematologic cancer, e.g., a B cell cancer, or a symptom thereof in a patient in need thereof. These methods comprise administering to the patient an amount of the population of T cells of any one of the invention effective to alleviate the cancer or symptom thereof in the patient.

Description

technical field [0001] The present invention relates, in part, to chimeric antigen receptor (CAR) cells and CAR therapy. Background technique [0002] CAR therapy has emerged as an extremely important tool against cancer. However, these therapies typically rely on the use of the patient's own cells, such as T cells derived from peripheral blood mononuclear cells (PBMCs), as effector cell populations. Because each patient's cells must be collected, tested, and made into a CAR therapeutic, CAR therapy is: 1) extremely expensive; and 2) available only in certain centers that are willing and / or able to perform the therapy. These shortcomings make CAR therapy largely unavailable to many groups in need. The present invention is directed, in part, to generating an off-the-shelf allogeneic CAR therapy aimed at alleviating these and other problems. [0003] Autologous CAR-T therapy has become part of the standard of care for blood cancer patients. The source of cells in CAR-T the...

Claims

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Application Information

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IPC IPC(8): A61K35/17C07K14/725C12N5/0783C12N15/63C12N5/073
CPCC12N5/0636C12N15/63C07K14/7051C12N5/0605A61K2239/38A61K39/4611A61K39/464424A61K2239/26A61K2239/48A61K39/46434A61K39/464412A61K2239/31A61K39/4631A61K39/4621A61P35/02C12N2510/00A61K35/17C12N15/86
Inventor R·J·哈里里K·卡拉谢维奇李天剑
Owner ANTHROGENESIS LLC
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