For Advanced Heme Malignancies, CAR T Cells Overcome Exhaustion, Tumor Escape

December 28, 2019
Jennifer Gray Tomilov, PhD

Targeted Therapies in Oncology, December 2, Volume 8, Issue 18

Progress in the development of chimeric antigen receptor T-cell therapy and other cell-based therapies has led to new therapeutic options for advanced malignancies. CAR T-cell agents approved by the FDA in recent years include axicabtagene ciloleucel for diffuse large B-cell lymphoma and tisagenlecleucel for both DLBCL and acute lymphoblastic leukemia. 

The phase II JULIET trial evaluated the CD19-directed autologous T-cell immunotherapy tisagenlecleucel in patients with R/R DLBCL and DLBCL after transformation from follicular lymphoma.2The ORR in this study was 50%, and the CR rate was 32%. The estimated median response duration for patients with a PR was 3.4 months, but the DOR was not reached in patients achieving CR at a median follow-up of 9.4 months. Approval of tisagenlecleucel for B cell precursor ALL followed a study including patients with R/R pediatric precursor B-cell ALL. The confirmed overall remission rate was 82.5%, wherein 63% of patients achieved CR and 19% had CR with incomplete hematological recovery.3 The median remission duration was not reached in this study (range, 1.2-14.1+ months).

Advances in CAR T-Cell Therapies: Combating Tumor Escape and T-Cell Exhaustion

Although CD19-directed CAR T-cell therapies have shown initial efficacy in R/R hematologic malignancies, not all patients respond to this therapy, and long-term disease control is challenging because of the prevalence of CD19-neg-ative immune escape.4To address this issue, development of CAR T cells targeting the CD22 antigen is under way. Results from a recent phase I, first-in-human trial indicated the safety and efficacy of a CD22-targeted CAR (CD22-CAR) therapy in 21 children and adults with R/R B-ALL, including 17 patients previously treated with CD19-directed immunotherapy. All patients had received at least 1 prior hematopoietic stem cell transplantation (HSCT), and 2 had received prior HSCT twice.5Dosing was administered according to a standard 3 + 3 phase I dose-escalation design.

Dose-dependent anti-leukemic activity was observed, and CR was obtained in 11 of the 15 patients (73%) receiving ≥1 × _106 CD22-CAR T cells per kilogram of body weight, including 9 of 10 patients who had received prior CD19-directed immunotherapy and 5 of 5 patients with CD19-diminished or CD19-negative B-ALL.5

Median remission duration was 6 months. The primary adverse event (AE) was cytokine release syndrome, which occurred in 16 of 21 patients.

Despite clinical activity of CD22-CAR T cells in B-ALL and leukemia resistant to anti-CD19 immunotherapy, relapses were associated with diminished CD22 site density, which likely pro-moted tumor cell escape from killing by CD22- CAR T cells.5Specifically, of the 12 patients who obtained a CR, 3 remained in CR after 21, 9, and 6 months, but 8 patients relapsed after 1.5 to 12 months.

T-cell exhaustion is also being studied as a mechanism underlying CAR T cell therapy relapse.

“We are engineering exhaustion resistance in CAR T cells,” said Crystal Mackall, MD, of Stanford University School of Medicine in California, during a recent interview with Targeted Therapies in Oncology (TTO) prior to the Society for Immunotherapy of Cancer’s 34th Annual Meeting. Mackall explained the difference between exhausted and nonexhausted T cells is altered, and one of the ,major alterations is an imbalance between activating and inhibitory members of the AP-1/IRF family. By overexpressing c-Jun, a member of the AP-1 family, we can restore the balance, and our CAR T cells are much more functional and resistant to exhaustion.”

Researchers first coexpressedc-Junalongside a number of CAR T trans-genes in vitro and examined ifc-Juncould enhance the function of exhausted CAR T cells. Indeed,c-Junoverexpression (OE) contributed to reduced T-cell exhaustion in numerous CARs, including those with CD28 or 4-1BB costimulatory domains in the setting of exhaustion driven by either enforced long-term expansion or tonic signaling.6

Progress in Tumor-Infiltrating Lymphocyte Adoptive Cell Therapy

Tumor-infiltrating lymphocytes (TILs) are nongenetically modified T cells used in 1 form of adoptive cell therapy (ACT).7 Although TIL therapy has long been an immuno-oncology treatment modality, culturing TILs has been a time-consuming process and has previously shown little efficacy in cancers other than melanoma.7 Notably, a recent study illustrated the application of TIL therapy for human papillomavirus (HPV)—associated epithelial cancers.8 The phase II clinical trial included 2 cohorts: those with cervical cancers and those with noncervical cancers. Cell infusion was preceded by a lymphocytedepleting regimen followed by systemic high-dose aldesleukin.8 TILs were preferentially selected according to HPV E6/E7 oncoprotein reactivity. Objective tumor responses occurred in 5 of 18 (28%) patients in the cervical cancer cohort and 2 of 11 (18%) in the noncervical cancer cohort. Two responses in the cervical cancer cohort were complete and ongoing at 67 and 53 months post treatment.8 The most common AEs were lymphopenia, neutropenia, and thrombo-cytopenia, which occurred in all 29 patients.

Recent work has focused on the utility of biomarkers and genetic alterations to guide improved patient outcomes with TIL therapy. One study indicated that functional and phenotypic changes occurred in CD8+ and CD4+ TILs taken from rare solid tumors after a single dose of the checkpoint inhibitor pembrolizumab (Keytruda).9 A similar approach examined TIL profiles in 74 patients with metastatic melanoma treated with autologous TILs. Researchers found that infusion with higher numbers of TILs having CD8 predominance and expression of B and T lymphocyte attenuator (BTLA) correlated with improved response in anti—CTLA-4–naïve patients but not in anti–CTLA-4–refractory patients.10 Further, baseline serum levels of interleukin (IL) 9 predicted response to TIL therapy, but TIL persistence, tumor recognition and tumor mutation burden did not correlate with clinical outcome.10

Characterizing Neoantigens for Targeted Cell-Based Therapies

Neoantigens are mutated proteins expressed by tumor cells that elicit T-cell activation and immune response.11T-cell receptors (TCRs) are antigen recognition structures expressed by all T cells that mediate recognition of epitopes derived from proteins within any subcellular compartment.12Identification of neoantigens has been a focus because these markers are exclusive to tumor cells and thus reduce the risk of off-target injury to healthy tissues.12Both “public” and “private” neoantigens have been evaluated as effective targets in cell-based therapies. “Public neoantigens are mutations in hot spot regions within genes that are found in potentially many different cancer types,” Ingunn Stromnes, PhD, of the University of Minnesota Medical School in Minneapolis, explained during an interview withTTO. “If researchers identify a T-cell receptor specific to [a public neoantigen], it could potentially work in multiple patients and be quite valuable,” Stromnes said, further illustrating the utility of public neoantigens.

To identify public neoantigens, 1 case study followed the impact of adoptive cell transfer of CD8+ T cells specifically targeting mutantKRASG12D obtained from TILs of a patient with met-astatic colorectal cancer.13Objective regression of 7 lung metastases was seen after infusion of approximately 1.11 × _1011 HLA-C*08:02- restricted TILs that were composed of 4 different T-cell clonotypes specifically targetingKRASG12D.13However, 1 lesion showed progression after 9 months of therapy, and lesion resection indicated loss of the chromosome 6 haplotype encoding the HLA-C*08:02 class I major histo-compatibility complex molecule.13Another patient achieved a complete response in stage IV acral melanoma after adoptive transfer of TILs targetingBRAFV600E. The patient’s tumor was sequenced and found to harbor rare CD4+ T cells specific forBRAFV600E and diverse CD8+ T cells reactive to nonmutated self-antigens.14These markers increased in the blood after TIL transfer and persisted long term, which suggested that the markers have a role in antitumor immune response.14Results from this study suggest that TCR-engineeredBRAFV600E-specific cell therapies may be efficacious in patients withBRAF-mutated cancers.

Although the characterization of public neoantigens has great potential to develop wide-reaching therapies, a recent study indicated that of 148 empirically identified neoantigens, just 2 (1.3%) were shared and 146 (98.7%) were patient specific.12These data indicate the current necessity of identifying and developing TILs enriched for private neoantigen reactivity.12One case study that described patient-specific neoantigens focused on a single patient with chemorefractory hormone receptor—positive metastatic breast cancer who underwent ACT with TILs specific for mutant versions of 4 proteins: SLC3A2, KIAA0368, CADPS2, and CTSB. Treatment in conjunction with IL-2 and checkpoint blockade conferred the complete, durable regression of metastatic breast cancer—ongoing for >22 months at time of study end—illustrating the application of a private neoantigen immunotherapy approach.15

New Research in Off-the-Shelf Cell-Based Therapies

Despite great advances in autologous cell therapies, these specialized treatments remain expensive and clinically challenging to administer.16To this end, research has been under way to develop natural killer (NK) cells, innate effector lymphocytes with potent antitumor activity, as a reliable off-the-shelf cellular therapy. A proven safety profile and high availability make NK cells a viable new development in the allogeneic cell therapy setting.16

An ongoing phase I/II first-in-human clinical trial is testing the efficacy of off-the-shelf cord blood—derived NK cells engineered to express a CD19 CAR and IL-15 in advanced malignancies.16,17The dose-escalation study will examine CAR-engineered NK cells with lympho-depleting chemotherapy in patients with R/R B-lymphoid malignancies (NCT03056339). Another phase I/II trial is evaluating the efficacy of a GTB-3550 (CD16/IL-15/CD33) trispecific killer engager for the treatment of CD33-express-ing high-risk myelodysplastic syndromes, R/R acute myeloid leukemia, or advanced systemic mastocytosis (NCT03214666). This novel approach does not require patient-specific customization but, instead, may be developed for a disease broadly. Additionally, it will require less cost yet have similar or better efficacy than existing cell-based therapies.18

Gene editing has recently proved to be a promising addition to the engineering of T cells for adoptive cell therapies. It is performed in T cells removed from a patient and then infused back into the patient for therapeutic purposes. Most recently, CRISPR/Cas9-modulated gene knockout promoted the engineering of universal T cells and exhaustion-resistant T cells and reduction of on-target, off-tissue toxicity of redirected T cells.19One recent study illustrated the use of CRIS-PR to directly insert a CD19-CAR gene into the native T-cell receptor a constant (TRAC) locus of engineered T cells. This resulted in site-specific integration of the CAR gene, thus eliminating the possibility of disrupting an essential native gene. Additionally, site-specific CAR integration into the TRAC locus promoted TCR-like physiological regulation of CAR function, resulting in better antitumor activity than traditional CAR engineering.19,20Autologous T-cell therapies are associated with many barriers, including poor quality or quantity of T cells obtained from patients and considerable cost associated with generating patient-specific treatments.21Although allogeneic T cells may offer a solution to autologous T-cell therapy—related barriers, the endogenous TCR of allogeneic T cells may recognize alloantigens of the recipient and lead to graft-vs-host disease (GVHD). Additionally, HLA expression on the surface of allogeneic T cells leads to rejection by the host immune system.21CRISPR editing of allogenic T cells has shown great promise as a tool to overcome these hurdles and make allogenic cell therapies a viable option. In a recent study, CRISPR-mediated multiplex genome editing was performed to knock out the function of both TCR and B2M-modulated HLA-I complex expression. Double-ablated TCR/HLA-1 cells were infused into immunocompromised mice and compared with nonmanipulated cells for GVHD reactivity. Four of the 5 mice infused with nonmanipulated lymphocytes developed GVHD within 2 months after infusion, whereas none of the 5 mice that received the double-ablated TCR/HLA-1 cells developed GVHD.21Next, a TCR/B2M/PD1 triple-ablated CD19 CAR-T cell line was generated to test if the additional PD1 disruption improved the function of CAR T cells. The triple-ablated TCR/B2M/PD1 CD19 CAR T-cell line showed enhanced antitumor activity in a Nalm6—PD-L1 mouse leukemia tumor model versus the TCR/B2M double-ablated CD19 CAR T cells and nontransduced T cells.21

A landmark clinical trial is now ongoing for the first-in-human, phase I, 3-arm exploration of CRISPR-modified autologous T-cell therapy for patients with multiple myeloma, synovial sarcoma, and melanoma (NCT03399448). Following lymphodepletion, patients are to receive T cells transduced with a lentiviral vector to express the NY-ESO-1-targeted TCR and electroporated with CRISPR gRNA for the disruption of endogenous TCRα,_ _T_C_R_β, and PD-1 expression (NY-ESO-1-redirected CRIS-PR edited T cells). Primary outcomes are set to measure the patient safety and manufacturing feasibility of NYCE T-cell therapy, and secondary outcomes will measure the 5-year CR, PR, overall survival, and DOR of NYCE T-cell therapy.

Numerous developments in the fast-moving field of targeted immuno-oncology have recently provided therapeutic cell-based options for patients with advanced malignancies where once there were none. However, treatment modalities must meet the challenge of cellular and genetic heterogeneity of patients, where a one-size-fits-all approach is often not feasible. These challenges serve as a reminder that a balance between fundamental research and forward engineering is needed to bring effective targeted oncology solutions to all patients.


1. FDA approves axicabtagene ciloleucel for large B-cell lymphoma. FDA website. Updated October 25, 2017. Accessed October 5, 2019.

2. FDA approves tisagenlecleucel for adults with relapsed or refractory large B-cell lymphoma. FDA website Updated May 3, 2018. Accessed October 5, 2019.

3. FDA approves tisagenlecleucel for B-cell ALL and tocilizumab for cyto-kine release syndrome. FDA website. Updated September 7, 2017. Accessed October 5, 2019.

4. Two is better than one: arming the T cells with dual cancer-targeting an-tibodies. Standford medicine website. Published 2019. Accessed October 4, 2019.

5. Fry TJ, Shah NN, Orentas RJ, et al. CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immu-notherapy.Nat Med. 2018;24(1):20-28. doi: 10.1038/nm.4441.

6. Lynn RC, Weber EW, Gennert D, et al. c-Jun overexpressing CAR-T cells are exhaustion-resistant and mediate enhanced antitumor activity [pub-lished online May 29, 2019]. bioRxiv. doi: 10.1101/653725.

7. Garber K. Pursuit of tumor-infiltrating lymphocyte immunotherapy speeds up. Nat Biotechnol. 2019;37:969-971. doi: 10.1038/d41587-019-00023-6.

8. Stevanović S, Helman SR, Wunderlick JR, et al. A phase II study of tumor-infiltrating lymphocyte therapy for human papillomavirus-as-sociated epithelial cancers.Clin Cancer Res. 2019;25(5):1486-1493. doi: 10.1158/1078-0432.CCR-18-2722.

9. Creasy CA, Forget MA, Singh G, et al. Exposure to anti-PD-1 causes functional differences in tumor-infiltrating lymphocytes in rare solid tu-mors.Eur J Immunol. 2019;00:1-7. doi: 10.1002/eji.201948217.

10. Forget MA, Haymaker C, Hess KR, et al. Prospective analysis of adop-tive TIL therapy in patients with metastatic melanoma: response, impact of anti-CTLA4, and biomarkers to predict clinical outcome.Clin Cancer Res. 2018;24(18):4416-4428. doi: 10.1158/1078-0432.CCR-17-3649.

11. Society for Immunotherapy of Cancer. Immunotherapy: the path to a cancer cure (for clinicians) [video]. YouTube website. Published June 4, 2017. Accessed October 9, 2019.

12. Chandran SS, Klebanoff CA. T cell receptor-based cancer immuno-therapy: emerging efficacy and pathways of resistance.Immunol Rev. 2019;290(1):127-147. doi: 10.1111/imr.12772.

13. Tran E, Robbins PF, Lu YC, et al. T-cell transfer therapy targeting mutant KRAS in cancer.N Engl J Med. 2016;375(23):2255-2263. doi: 10.1056/NEJMoa1609279.

14. Veatch JR, Lee SM, Fitzgibbon M, et al. Tumor-infiltrating BRAFV600E-specific CD4+ T cells correlated with complete clinical response in melanoma.J Clin Invest. 2018;128(4):1563-1568. doi: 10.1172/JCI98689.

15. Zacharakis N, Chinnasamy H, Black M, et al. Immune recognition of somatic mutations leading to complete durable regression in metastatic breast cancer.Nat Med.2018;24(6):724-730. doi: 10.1038/s41591-018- 0040-8.

16. Rezvani K. Adoptive cell therapy using engineered natural killer cells. Bone Marrow Transplant. 2019;54(suppl 2):785-788. doi: 10.1038/ s41409-019-0601-6.

17. André P, Denis C, Soulas C, et al. Anti-NKG2A mAB is a checkpoint inhibitor that promotes antitumor immunity by unleashing both T and NK cells. Cell. 2018;175(7):1731-1743. doi: 10.1016/j.cell.2018.10.014.

18. University of Minnesota discovery is now a first-in-human clinical trial for leukemia. WBOC website. Published September 19, 2019. Accessed October 10, 2019.

19. Singh N, Shi J, June CH, Ruella M. Genome-editing technologies in adoptive T cell immunotherapy for cancer.Curr Hematol Malig Rep.2017;12(6):522-529. doi: 10.1007/s11899-017-0417-7.

20. Eyquem J, Mansilla-Soto J, Giavridis T, et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection.Nature. 2017;543(7643):113-117. doi: 10.1038/nature21405.

21. Ren J, Liu X, Fang C, Jiang S, June CH, Zhao Y. Multiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition.Clin Cancer Res.2017;23(9):2255-2266. doi: 10.1158/1078-0432.CCR-16-1300.