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US-20260124299-A1 - BITE-ACTIVATED CAR-T CELLS

US20260124299A1US 20260124299 A1US20260124299 A1US 20260124299A1US-20260124299-A1

Abstract

The CAR-T cells described herein can provide highly effective therapies for diverse cancer types, e.g., solid cancers, hematological cancers, and metastatic forms thereof. Provided herein are methods of generating CAR-T cells, compositions comprising such CAR-T cells, methods of treatment using the cells, methods of identifying subjects susceptible to immune checkpoint immunotherapy treatment and methods of evaluating susceptibility of a subject to develop Cytokine-Release Syndrome.

Inventors

  • Daniel PRIMO RAMOS
  • Juan Antoinio BALLESTEROS NOBELL
  • Teresa Ann BENNETT
  • Julián GORROCHATEGUI GUILLÉN
  • Joaquin MARTÍNEZ LÓPEZ
  • Antonio VALERI LOZANO
  • Alejandra LEIVAS ALDEA

Assignees

  • VIVIA BIOTECH, S.L.

Dates

Publication Date
20260507
Application Date
20251218

Claims (19)

  1. 1 .- 180 . (canceled)
  2. 181 . An in vitro method of producing genetically engineered T cells expressing Chimeric Antigen Receptors (CAR-T cells): (a) providing a sample comprising T cells from a subject having a cancer; (b) providing a sample comprising cancer cells; (c) forming an ex vivo reaction mixture comprising the T cells, the cancer cells, and a bispecific T cell engager antibody or a multispecific antibody under conditions and for a period of time sufficient to activate the T cells and induce killing of the cancer cells, thereby producing activated T cells; (d) selecting the activated T cells, based on at least one parameter selected from the group consisting of increased cancer cell killing activity, reduced toxicity, reduced off-target effect, increased viability, increased proliferation, and an Effective E:T ratio; and (e) genetically engineering the activated T cells to produce Chimeric Antigen Receptors on the surface of the activated T cells, thereby producing CAR-T cells, wherein after activation of the T cells, the bispecific T cell engager antibody or the multispecific antibody is removed from the reaction mixture, and wherein said activated T cells kill cancer cells independently of the bispecific T cell engager antibody or of the multispecific antibody.
  3. 182 . The method of claim 181 , wherein the bispecific T cell engager antibody or the multispecific antibody comprises a first element having affinity for the T cells and a second element having affinity for the cancer cells.
  4. 183 . The method of claim 181 , wherein the selection of the activated T cells is based on the Effective E:T ratio, and wherein the activated T cells have an Effective E:T ratio higher than 1:5 between the number of activated T cells (E) and the number of target cancer cells (T) after exposure to the bispecific T cell engager antibody or to the multispecific antibody.
  5. 184 . The method of claim 181 , further comprising evaluating the activity of the CAR-T cells, wherein evaluating comprises: (a) providing CAR-T cells obtainable according to the method of claim 1 ; (b) providing a sample of cancer cells, wherein the cancer cells are from the same subject; (c) contacting the CAR-T cells with the cancer cells for a period of time sufficient to induce killing of the cancer cells by the CAR-T cells; and (d) determining the level of cancer cells after step (c), wherein an increased cell killing activity is assessed if there is a decrease in the level or amount of cancer cells relative to a reference level, or wherein a decreased cell killing activity is assessed if there is a reduction in the level or amount of cancer cells relative to a reference level.
  6. 185 . A composition comprising genetically engineered T cells expressing Chimeric Antigen Receptors (CAR-T cells) obtainable according to the method of claim 181 .
  7. 186 . The composition according to claim 185 , wherein the CAR-T cells exhibit cytotoxic activity toward cancer cells, and comprise at least one of: (a) 100 copies of a cancer cell surface marker; or (b) a detectable amount of one agent enhancing T cell activity or multiple agents enhancing T cell activity.
  8. 187 . A pharmaceutical composition comprising the composition of claim 185 and a pharmaceutically acceptable carrier.
  9. 188 . A method for treating a subject by Adoptive Cancer Therapy comprising administering the pharmaceutical composition according to claim 187 to the subject.
  10. 189 . A method for treating a subject having cancer comprising providing CAR-T cells obtainable according to the method of claim 181 , and administering an effective amount of the CAR-T cells, to the subject.
  11. 190 . The method of claim 189 , wherein the selection of the activated T cells is based on the parameter Effective E:T ratio, and wherein the activated T cells have an Effective E:T ratio higher than 1:5 between the number of activated T cells (E) and the number of target cancer cells (T) after exposure to the bispecific T cell engager antibody or to the multispecific antibody.
  12. 191 . The method of claim 181 , wherein the ex vivo reaction mixture further comprises one agent enhancing T cell activity or multiple agents enhancing T cell activity.
  13. 192 . The method of claim 191 , wherein the agent enhancing T cell activity is selected from the group consisting of a chemotherapy drug, a targeted anti-cancer therapy, an oncolytic drug, a cytotoxic agent, an immune-based therapy, a cytokine, an agonist of T cells, an agonistic antibody or fragment thereof, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, an immune checkpoint inhibitor, an immunomodulatory agent, a vaccine, and a cellular immunotherapy.
  14. 193 . The method of claim 192 , wherein the cytokine is selected from the group consisting of GM-CSF, IL-7, IL-12, IL-15, IL-18, and IL-21.
  15. 194 . The method of claim 189 , wherein the subject treated is different from the subject from whom the T cells and/or the cancer cells were obtained.
  16. 195 . The composition of claim 186 , wherein the agent enhancing T cell activity is selected from the group consisting of a chemotherapy drug, a targeted anti-cancer therapy, an oncolytic drug, a cytotoxic agent, an immune-based therapy, a cytokine, an agonist of T cells, an agonistic antibody or fragment thereof, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, an immune checkpoint inhibitor, an immunomodulatory agent, a vaccine, and a cellular immunotherapy.
  17. 196 . The composition of claim 195 , wherein the immune checkpoint inhibitor inhibits an immune checkpoint molecule selected from the group consisting of PDL-1, PDL-2, B7-1, B7-2, 4-1BBL, Galectin, ICOSL, GITRL, OX40L, CD155, B7-H3, PD1, CTLA-4, 4-1BB, TIM-3, ICOS, GITR, LAG-3, KIR, OX40, TIGIT, CD160, 2B4, B7-H4, HVEM, BTLA, KIR, MHC class I, MHC class II, GAL9, VISTA, LAIR1, and A2aR.
  18. 197 . The composition of claim 195 , wherein the cytokine is selected from the group consisting of GM-CSF, IL-7, IL-12, IL-15, IL-18, and IL-21.
  19. 198 . The pharmaceutical composition of claim 187 , wherein the pharmaceutical composition is substantially free of residual bispecific T cell engager antibody or a multispecific antibody.

Description

FIELD OF THE INVENTION The disclosure relates to three novel approaches using bispecific antibodies (BiTE)-activated T Cells. One is to generate chimeric antigen receptor (CAR) T cells using these BiTE-activated T cells as the source of T cells. These new CAR-T cells may be a better cellular therapy treatment for cancer patients. A second approach is a method to identify which immune check point inhibitors are responsible for resistance to these BiTE-activated T cells. This can be helpful to personalize immunotherapy treatments to cancer patients. This may also be helpful for other immunotherapy treatments, such as CAR-T cells, independently of the BiTE-activated T cells. A third approach is to identify patients less susceptible to suffer Cytokine-Release Syndrome. This can also be helpful to personalize immunotherapy treatments to cancer patients. This may also be helpful for other immunotherapy treatments, such as CAR-T cells, independently of the BiTE-activated T cells. BACKGROUND OF THE INVENTION Adoptive cell therapy (ACT) is a process involving collection of immune cells from a patient, expansion of the cells, and reintroduction of the cells into the same patient or a different patient. For example, ACT of donor-derived, ex vivo expanded human cytotoxic T lymphocytes (CTLs) has emerged as a promising approach to treat cancer. Examples of ACT include cultured tumor infiltrating lymphocytes (TILs), isolated and expanded T cell clones, and genetically engineered lymphocytes (e.g., T cells) that express conventional T cell receptors or chimeric antigen receptors. The genetically engineered lymphocytes are designed to eliminate cancer cells expressing specific antigen(s) and are expanded and delivered to a patient. Another example of an ACT is the isolation and use of T cells from a patient's blood after administration of a cancer vaccine. ACT can provide tumor specific lymphocytes (e.g., T cells) that lead to a reduction in tumor cells in a patient. Despite the clinical efficacy of Chimeric Antigen Receptor (CAR)-T cells for cancer treatment, they still have major limitations related to toxicity or immune mechanism of resistance that could be overcome through the integration of these different approaches with the Cancer-Killing T Cells. Standard CAR-T cells are generated using peripheral blood naïve T cells. A limitation of these standard CAR-T cells is that they can only recognize the tumor antigen of the CAR construct. However, tumor cells can be heterogeneous with some clones not expressing the CAR antigen leading to resistance to such CAR-T cells. Relapsed patients treated with CAR-T cells are showing this resistance mechanisms. Document Borrello I et al., 2016 discloses utilization of marrow-infiltrating lymphocytes (MILs) for adoptive T-cell therapy. The document discloses activation of MILs with anti CD3/CD28 beads. Also disclosed in this document is the suggestion that MILs could potentially serve a better source of T-cells for CAR-based adoptive T-cell therapy. However, no experimental results are provided in the document supporting this hypothesis. The method of producing CAR-T cells, often by transducing a CAR with a lentivirus, generates an heterogenous population of T Cells. The CAR construct may insert at different positions into the genome, resulting in different activity of the ensuing CAR-T cells; e.g. different levels of expression could affect activity, or disrupting different genes. Furthermore, the different types of T cells present in the mixed T cell population used as a source for producing CAR-T cells may result in different activities; e.g. memory T cells versus naïve T cells, highly proliferating versus terminally proliferating T cells. It has been recently reported that expansion of a single CAR T-cell clone inside a patient with CLL resulted in complete remission (Fraietta et al. Nature. 2018 June; 558(7709):307-312). This document discloses that at the peak of the response, 94% of CAR T cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene disrupted the methylcytosine dioxygenase TET2 gene. This genetic disruption was validated to confer an advantage to T cells for CAR-T expansion. Therefore, there are likely different, maybe thousands of different CAR-T cell clones produced when producing a CAR-T, which is in reality an heterogenous mixture of CAR-T clones. Methods to identify the best CAR-T clones would be beneficial to enhance the CAR-T activity and hence patient clinical responses. Cytokine Storm, also called Cytokine Release Syndrome, has been recognized as a major toxicity challenge for CAR-T treatments (Park et al. N Engl J Med. 2018 Feb. 1; 378(5):449-459). It also a major toxicity for bispecific antibodies. However, there are no methods to identify patient most likely to suffer this toxicity when treated with CAR-T cells. A key immunotherapy treatment often combined with CAR-T and BiTE treatments are immune check poin