Transformative and Innovative CAR-T Cell Expansion for Cancer Care

Introduction

This groundbreaking CAR-T cell expansion technology provides a method to increase the production of antigen-specific CAR-T cells, offering a powerful tool for enhancing the effectiveness of CAR-T cell therapies in cancer treatment. By focusing on expanding specific CAR-T cells, this technology allows for more potent immune responses, improving targeting and efficacy against cancerous cells while minimizing off-target effects. For pharmaceutical and biotech companies, this technology represents an essential advancement in immunotherapy, providing a means to boost treatment outcomes, support patient recovery, and meet the growing demand for personalized cancer care.

The Challenge: Boosting Efficacy in CAR-T Therapies

As CAR-T therapies gain prominence in treating certain cancers, the ability to produce sufficient quantities of highly targeted, antigen-specific CAR-T cells remains a critical challenge. Current methods may limit the availability or potency of CAR-T cells, which can impact treatment efficacy, especially for patients with aggressive or resistant cancers. Healthcare providers and researchers need efficient methods to expand these cells to deliver robust, effective therapies, supporting long-term success in cancer management while minimizing adverse reactions.

Precise CAR-T Cell Expansion for Targeted Cancer Treatment

This technology offers a precise solution to these challenges by employing a method that promotes the rapid and targeted expansion of CAR-T cells specific to cancer antigens. By focusing on antigen-specific CAR-T cells, this method strengthens the body’s immune response against cancer cells while maintaining accuracy and reducing potential side effects. The technology provides a scalable and efficient means to increase CAR-T cell quantities, enabling clinicians to deliver potent, tailored treatments that address the unique requirements of each patient. This enhancement supports faster recoveries, improved survival rates, and more accessible CAR-T therapies.

Key Benefits for Pharmaceuticals and Immunotherapy Innovators

For pharmaceutical companies, this CAR-T cell expansion method opens up opportunities to develop advanced immunotherapies that offer higher efficacy with fewer side effects. Immunotherapy centers and oncology departments can integrate this technology into their treatment protocols, ensuring they can meet patient demands with powerful, scalable CAR-T therapies. The technology’s focus on antigen specificity and scalability aligns with the needs of personalized medicine, enabling providers to offer individualized care that maximizes therapeutic effectiveness while protecting healthy cells. This technology is poised to set new standards in immunotherapy, especially in areas where precision and potency are paramount.

Invest in the Future of Precision Cancer Immunotherapy

Licensing this CAR-T cell expansion technology positions your company as a leader in advanced cancer treatment solutions. By offering an effective, scalable approach to expanding antigen-specific CAR-T cells, your business can contribute to the future of immunotherapy, enhancing patient outcomes and supporting the broader goals of precision oncology. This technology is an invaluable asset for companies dedicated to transforming cancer care, promoting innovation in immunotherapy, and improving the lives of patients battling cancer.

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Provided herein are methods for preparing and characterizing CAR-T cell cultures and preparations.
1. A method of generating a composition comprising cytotoxic T cells that are specific for binding to a first antigen and that express a chimeric antigen receptor (CAR) specific for binding to a second antigen, said method comprising:

i) preparing a culture of cells enriched for CD3+ T cells, said CD3+ T cells being stimulated to recognize and bind to said first antigen through a T-cell receptor;
ii) transducing the CD3+ T cells of said culture with a viral vector comprising a nucleic acid sequence encoding a CAR that binds to said second antigen;
iii) culturing the CAR-transduced CD3+ T cells to allow proliferation of CAR-expressing, first-antigen-specific cytotoxic T cells; and
iv) harvesting the CAR-expressing, first-antigen-specific cytotoxic T cells.
2. A method of preparing an adoptive immunotherapy composition comprising cytotoxic T cells that are specific for binding to a first antigen and that express a chimeric antigen receptor (CAR) specific for binding to a second antigen, said method comprising:

i) preparing a culture of cells enriched for CD3+ T cells, said CD3+ T cells being stimulated to recognize and bind to said first antigen through a T-cell receptor;
ii) transducing the CD3+ T cells of said culture with a viral vector comprising a nucleic acid sequence encoding a CAR that binds to said second antigen;
iii) culturing the CAR-transduced CD3+ T cells to allow proliferation of CAR-expressing, first-antigen-specific cytotoxic T cells; and
iv) harvesting the CAR-expressing, first-antigen-specific cytotoxic T cells, thereby providing said adoptive immunotherapy composition.
3. A method for inducing proliferation of a population of cytotoxic T cells that are specific for binding to a first antigen and that express a chimeric antigen receptor (CAR) specific for binding to a second antigen, said method comprising:

i) preparing a culture of cells enriched for CD3+ T cells, said CD3+ T cells being stimulated to recognize and bind to said first antigen through a T-cell receptor;
ii) transducing the CD3+ T cells of said culture with a viral vector comprising a nucleic acid sequence encoding a CAR that binds to said second antigen; and
iii) culturing the CAR-transduced CD3+ T cells to allow proliferation of CAR-expressing, first-antigen-specific cytotoxic T cells.
4. The method of claim 1, further comprising incubating the culture of steps i), ii), iii), or any combination thereof, with one or more cytokines.
5. The method of claim 1, wherein the culture of cells enriched for CD3+ cells comprises a sample of peripheral blood mononuclear cells (PBMCs) depleted of red blood cells, platelets, monocytes, and granulocytes, or comprises CD3+ cells positively selected from a sample of PBMCs.
6. (canceled)
7. The method of claim 1, wherein the step of preparing a culture of cells enriched for CD3+ T cells stimulated to recognize and bind to said first antigen through a T-cell receptor, comprises co-culturing a responder population of CD3+ cells with stimulator cells that present said first antigen on their cell surface; and optionally re-culturing said responder population of CD3+ cells with stimulator cells that present said first antigen on their surface prior to CAR transduction.
8. The method of claim 7, wherein said co-culturing is initiated at a responder cells:stimulator cells ratio of 1:4.
9. (canceled)
10. The method of claim 7, wherein a first re-culture step is initiated at least 7 days after initiation of the co-culture step.
11. The method of claim 7, wherein the responder and stimulator cells are derived from the same donor.
12–13. (canceled)
14. The method of claim 7, wherein the stimulator cells are infected with a native and/or wild-type virus comprising at least one immunogenic peptide antigen comprising a T cell epitope or are engineered to express at least one immunogenic peptide antigen comprising a T cell epitope.
15. (canceled)
16. The method of claim 1, wherein the first antigen is a viral antigen selected from an Epstein-Bar virus (EBV), cytomegalovirus (CMV), human papilloma virus (HPV), BK virus (BKV), John-Cunningham virus (JCV), Merkel cell virus (MCV), human T-lymphotropic virus (HTLV) or human immunodeficiency virus (HIV) antigen.
17. (canceled)
18. The method of claim 16, wherein the viral antigen comprises an EBV LMP1 peptide or fragment thereof, an EBV LMP2A peptide or fragment thereof, or an EBV EBNA1 peptide or fragment thereof.
19. The method of claim 7, wherein the stimulator cells further express said second antigen, said second antigen being optionally selected from a CD19 antigen, a CD20 antigen or a mesothelin antigen.
20. (canceled)
21. The method of claim 1, optionally comprising freezing and storing the culture of cells enriched for CD3+ T cells for transduction at a future date.
22. The method of claim 1, optionally comprising freezing and storing the culture of CAR-expressing, first-antigen-specific cytotoxic T cells for use at a future date or for administering to a patient in need at a future date.
23. (canceled)
24. The method of claim 1, comprising maintaining the culture of cells enriched for CD3+ T cells for at least 2 days to at least 28 days prior to CAR transduction.
25–28. (canceled)
29. The method of claim 1, comprising culturing the CAR-transduced CD3+ T cells for at least 2 days to at least 17 days prior to said step of harvesting.
30–32. (canceled)
33. The method of claim 1, optionally comprising co-culturing the CAR-transduced CD3+ T cells with stimulator cells that express said first antigen at least once.
34. The method of claim 1, wherein the viral vector comprising the nucleic acid sequence encoding the CAR is a replication-incompetent lentiviral or retroviral vector.
35. (canceled)
36. The method of claim 1, wherein the CAR comprises one or more signal transduction domains, wherein the one or more signal transduction domains comprise a CD28 signaling domain, a 4-1BB signaling domain, a CD3 signaling domain, or a variant or fragment thereof.
37–38. (canceled)
39. The method of claim 36, wherein the CAR comprises a variant CD3ζ domain lacking at least one functional ITAM region.
40. The method of claim 36, wherein the CAR comprises a Mut06 domain.
41. The method of claim 1, wherein the viral vector comprising the nucleic acid sequence encoding the CAR further comprises a nucleic acid sequence encoding a selectable marker, e.g., wherein the selectable marker confers resistance to the antibiotic blasticidin.
42–51. (canceled)
52. An ex vivo method for generating a composition comprising cytotoxic T cells that are specific for binding to a first antigen and that express a chimeric antigen receptor (CAR) specific for binding to a second antigen, said method comprising:

i) obtaining a sample of cells from a subject, the sample of cell comprising CD3+ T cells;
ii) enriching said sample of cells for CD3+ T cells to provide an enriched sample of cells;
iii) contacting said enriched sample of cells with antigen-presenting stimulator cells that present said first antigen on their cell surface to provide a first-antigen-stimulated sample of cells;
iv) transducing said first-antigen-stimulated sample of cells with a viral vector comprising a nucleic acid sequence encoding a CAR that binds to said second antigen;
v) culturing the CAR-transduced cells to allow proliferation of CAR-expressing, first-antigen-specific cytotoxic T cells; and
vi) harvesting the CAR-expressing, first-antigen-specific cellular composition.
53. An ex vivo method for generating a composition comprising cytotoxic T cells that are specific for binding to a first antigen and that express a chimeric antigen receptor (CAR) specific for binding to a second antigen, said method comprising:

i) obtaining a sample of cells from a subject, the sample of cell comprising CD3+ T cells;
ii) enriching said sample of cells for CD3+ T cells to provide an enriched sample of cells;
iii) transducing said enriched sample of cells with a viral vector comprising a nucleic acid sequence encoding a CAR that binds to said second antigen;
iv) contacting the CAR-transduced cells with antigen presenting stimulator cells that present said first antigen on their cell surface; and
v) harvesting the CAR-expressing, first-antigen-specific cellular composition.
54–56. (canceled)
57. A method of treating a disorder associated with expression of a peptide antigen in a patient in need thereof, said method comprising administering to said patient the CAR-expressing, first-antigen-specific cytotoxic T cells obtained by the method of claim 1.
58. A T cell composition prepared by the method of claim 1.
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Title

Methods for expanding antigen-specific car-t cells, compositions and uses related thereto

Inventor(s)

Blake T. AftabChristina PhamRhine ShenMichelle Wu

Assignee(s)

Atara Biotherapeutics Inc

Patent #

20210301255

Patent Date

September 30, 2021

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