Immunotherapy using modified Chimeric Antigen Receptor (CAR) T-cells has greatly improved survival rates for relapsed and recurrent pediatric leukemia and lymphomas, but not brain cancers and solid tumors. Scientists from St. Jude Children’s Research Hospital investigated why this is happening and if improvements can be made.

The results of the study supported by grants from the National Institutes of Health (NIH), National Cancer Institute, ChadTough Defeat DIPG Foundation, American Brain Tumor Association (ABTA), Humor to Fight the Tumor and ALSAC, the fundraising and awareness organization of St. Jude, have been published in Cell Reports Medicine. [1]

The findings confirm that Glucose-Regulated Protein 78 (GRP78; also known as HSPA5, a member of the heat shock protein 70 (HSP70) family), a chaperone protein localized primarily in the endoplasmic reticulum (ER) lumen, where it helps in proper protein folding by targeting misfolded proteins and facilitating protein assembly, is a promising but complex target for CAR T–cell therapy for brain and solid tumors.  The conclusions of the study may be helpful in moving the field of cancer immunotherapy one step closer to treating broad range of difficult-to-treat brain and solid tumors expressing GRP78.

Validating a target
Scientists at St. Jude validated a cellular immunotherapy target called GRP78 in proof-of-principle experiments. The group also discovered a resistance mechanism whereby some tumors trick the cancer-killing immune cells into expressing GRP78, thereby turning off the immune cells or causing them to be killed, too.

GRP78 is the master of the unfolded protein response (UBR) in the Endoplasmic Reticulum (ER) in normal, healthy cells.[2][3] However, in response to elevated ER stress, GRP78 is overexpressed and translocated to the cancer cell surface in several malignancies, including solid tumors. [3]

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In earlier studies, the research team has shown that reprogramming a patient’s immune cells to target cancer using GRP78-CAR T-cells, has been successful against acute myeloid leukemia (AML), resulting in robust anti-tumor activity, recognizing and killing GRP78-positive AML cells without toxicity to Hematopoietic Progenitor Cells (HPCs), in vivo, [4]  Its has, however not been the case in the treatment of glioblastoma or solid tumors.

CAR T-cell therapy
These reprogrammed cells CAR T-cells, target a specific protein expressed on cancer cells but not healthy ones. This targeting enables CAR T–cell immunotherapy to selectively kill the tumor while leaving healthy tissues unharmed. One difficulty that has stymied the success of CAR T-cells in brain and solid tumors is the challenge of identifying a good target for these cancers.

“We found GRP78 is a great CAR T–cell target,” noted senior co-corresponding author Giedre Krenciute, Ph.D., St. Jude Department of Bone Marrow Transplantation and Cellular Therapy.

“We saw high GRP78 expression in a multitude of brain and solid tumor types, including adult glioblastoma, diffuse intrinsic pontine glioma (DIPG), osteosarcoma, triple-negative breast cancer and Ewing sarcoma, but our therapeutic efficacy was variable.” Krenciute said.

No relationship
The researchers created GRP78-targeted CAR T-cells that successfully killed many types of cancers in both cell and mouse models, though with significant variation. The researchers expected that higher levels of GRP78 (more protein to target) would make it easier for the CAR T-cells to locate and destroy the cancer; however, that was not the case. The scientists found no relationship between the amount of GRP78 and the ability of the CAR T-cells to kill cancer.

“We showed the conventional approach of targeting expression doesn’t mean an equal response,” said co-corresponding author Paulina Velasquez, M.D., St. Jude Department of Bone Marrow Transplantation and Cellular Therapy.

“GRP78 seems to be a special target that did not react as we expected, making it a promising but complicated candidate,” she added.

Tumors trick CAR T cells
“We expected two different tumors with the exact same level of antigen [GRP78] expression to be affected by the CAR T–cell therapy in the same way, but they aren’t,” said first author Jorge Ibanez, Ph.D., St. Jude Department of Bone Marrow Transplantation and Cellular Therapy.

“Instead, we found certain tumor cell types were altering T-cell activation and T-cell GRP78 expression,” Ibanez added

Ibanez found that resistant tumor cell types were altering the CAR T-cells. The tumor cells caused the GRP78-targeted CAR T-cells to express GRP78 on the CAR T-cells’ surface. The more GRP78 on the T-cells, the less active they became, reducing their cancer-killing activity. In addition, CAR T cells that remained active targeted and killed their counterparts expressing GRP78 on their surface.

In effect, the resistant tumors were conning the CAR T-cells. These tumors raised the flag of GRP78, saying, “I’m here,” and then convinced the approaching T-cells to raise their own GRP78 flag. This tricked the CAR T-cells into killing each other or giving up, leaving the tumor relatively unscathed.

A tantalizing target
Through these experiments, the St. Jude group unveiled the tricky biology of GRP78. The protein remains a tantalizing target, given its presence on many difficult-to-treat tumor types. Findings show scientists will need to expand their understanding of this newfound interaction with T cells to make viable GRP78-targeted immunotherapies. Still, if they can, these CAR T-cells may be broadly applicable across a broad range of tumor cell types.

“We always need to find new targets to improve cancer treatment,” Krenciute said.

“What we found from a biological perspective is that GRP78 has potential but is different from previous cancer-associated molecules. We showed that as scientists develop the next generation of CAR T–cell therapies, we need to recognize that not all targets are equal,” she concluded.

Limitations of the study
While the results of the study are indeed interesting, the authors recognized that one of the limitations of the study is that their research only focused on testing a second-generation GRP78-CAR with CD28.ζ signaling. They recognize that the questions about the role of other co-stimulatory domains, and the anti-tumor efficacy of GRP78-CAR T-cells remain unanswered and need to be incorporated into future studies.

Reference
[1] Ibanez J, Hebbar N, Thanekar U, Chiang J, Velasquez MP, Krenciute G, et al GRP78-CAR T cell effector function against solid and brain tumors is controlled by GRP78 expression on T cells. Cell Reports Medicine Volume 4, Issue 11, November 21, 2023 DOI: 10.1016/j.xcrm.2023.101297
[2] Ibrahim IM, Abdelmalek DH, Elfiky AA. GRP78: A cell’s response to stress. Life Sci. 2019 Jun 1;226:156-163. doi: 10.1016/j.lfs.2019.04.022. Epub 2019 Apr 9. PMID: 30978349; PMCID: PMC7094232.
[3] Bertolotti A, Zhang Y, Hendershot LM, Harding HP, Ron D. Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nat Cell Biol. 2000 Jun;2(6):326-32. doi: 10.1038/35014014. PMID: 10854322.
[4] Hebbar N, Epperly R, Vaidya A, Thanekar U, Moore SE, Umeda M, Ma J, Patil SL, Langfitt D, Huang S, Cheng C, Klco JM, Gottschalk S, Velasquez MP. CAR T cells redirected to cell surface GRP78 display robust anti-acute myeloid leukemia activity and do not target hematopoietic progenitor cells. Nat Commun. 2022 Jan 31;13(1):587. doi: 10.1038/s41467-022-28243-6. PMID: 35102167; PMCID: PMC8803836.

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