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Evaluation of chimeric antigen receptor of humanized rabbit-derived T cell receptor-like antibody.

ABSTRACT: T-cell receptor (TCR)-like Abs that specifically recognize antigenic peptides presented on MHC molecules have been developed for next-generation cancer immunotherapy. Recently, we reported a rapid and efficient method to generate TCR-like Abs using a rabbit system. We humanized previously generated rabbit-derived TCR-like Abs reacting Epstein-Barr virus peptide (BRLF1p, TYPVLEEMF) in the context of HLA-A24 molecules, produced chimeric antigen receptor (CAR)-T cells, and evaluated their antitumor effects using in vitro and in vivo tumor models. Humanization of the rabbit-derived TCR-like Abs using the complementarity-determining region grafting technology maintained their specificity and affinity. We prepared a second-generation CAR using single-chain variable fragment of the humanized TCR-like Abs and then transduced them into human T cells. The CAR-T cells specifically recognized BRLF1p/MHC molecules and lysed the target cells in an antigen-specific manner in vitro. They also demonstrated antitumor activity in a mouse xenograft model. We report the generation of CAR-T cells using humanized rabbit-derived TCR-like Abs. Together with our established and efficient generation procedure for TCR-like Abs using rabbits, our platform for the clinical application of humanized rabbit-derived TCR-like Abs to CAR-T cells will help improve next-generation cancer immunotherapy.

SUBMITTER: Nakamura T

PROVIDER: S-EPMC10243493 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Evaluation of chimeric antigen receptor of humanized rabbit-derived T cell receptor-like antibody.

Nakamura Tomoko T Kobayashi Eiji E Hamana Hiroshi H Hayakawa Yoshihiro Y Muraguchi Atsushi A Hayashi Atsushi A Ozawa Tatsuhiko T Kishi Hiroyuki H

Cancer science 20220731 10

T-cell receptor (TCR)-like Abs that specifically recognize antigenic peptides presented on MHC molecules have been developed for next-generation cancer immunotherapy. Recently, we reported a rapid and efficient method to generate TCR-like Abs using a rabbit system. We humanized previously generated rabbit-derived TCR-like Abs reacting Epstein-Barr virus peptide (BRLF1p, TYPVLEEMF) in the context of HLA-A24 molecules, produced chimeric antigen receptor (CAR)-T cells, and evaluated their antitumor ...[more]

PMID: 35766417

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Project description:IntroductionThe human epidermal growth factor receptor 2 (HER2) represents one of the most studied tumor-associated antigens (TAAs) for cancer immunotherapy. The monoclonal antibody (mAb) trastuzumab has improved the outcomes of patients with HER2+ breast cancer. However, a large number of HER2+ tumors are not responsive to, or become resistant to, trastuzumab-based therapy, and thus more effective therapies targeting HER2 are needed.MethodsHER2-specific T cells were generated by the transfer of genes that encode chimeric antigen receptor (CAR). Using a multistep overlap extension PCR method, we constructed a novel, humanized HER2 CAR-containing, chA21 single-chain variable fragment (scFv) region of antigen-specific mAb and T-cell intracellular signaling chains made up of CD28 and CD3ζ. An interferon γ and interleukin 2 enzyme-linked immunosorbent assay and a chromium-51 release assay were used to evaluate the antitumor immune response of CAR T cells in coculture with tumor cells. Furthermore, SKBR3 tumor-bearing nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were treated with HER2 CAR T cells to evaluate antitumor activity. Human CD3+ T cell accumulation in tumor xenograft was detected by immunohistochemistry.ResultschA21-28z CAR was successfully constructed, and both CD4+ and CD8+ T cells were transduced. The expanded HER2 CAR T cells expressed a central memory phenotype and specifically reacted against HER2+ tumor cell lines. Furthermore, the SKBR3 tumor xenograft model revealed that HER2 CAR T cells significantly inhibited tumor growth in vivo. Immunohistochemical analysis showed robust accumulation of human CD3+ T cells in regressing SKBR3 lesions.ConclusionsThe results of this study show that novel chA21 scFv-based, HER2-specific CAR T cells not only recognized and killed HER2+ breast and ovarian cancer cells ex vivo but also induced regression of experimental breast cancer in vivo. Our data support further exploration of the HER2 CAR T-cell therapy for HER2-expressing cancers.

Project description:Outcomes for post-chimeric antigen receptor (CAR) T cell therapy (CART) relapse are poor. The utilization of a unique CAR T cell construct for post-CART failure is increasing, but this approach is not well described. In this study, with CART-A the first unique CAR T cell construct received and CART-B the second, the primary objective was to characterize outcomes following CART-B. Secondary objectives included evaluating safety and toxicity with sequential CART infusions; investigating the impact of potential factors, such as antigen modulation and interval therapy, on CART-B response; and characterizing long-term outcomes in patients receiving multiple CARTs. This was a retrospective review (NCT03827343) of children and young adults with B cell acute lymphoblastic leukemia (B-ALL) undergoing CART therapy who received at least 2 unique CART constructs, excluding interim CART reinfusions of the same product. Of 135 patients, 61 (45.1%) received 2 unique CART constructs, including 13 who received >2 CARTs over time. Patients included in this analysis received 14 distinct CARTs targeting CD19 and/or CD22. The median age at CART-A was 12.6 years (range, 3.3 to 30.4 years). The median time from CART-A to CART-B was 302 days (range, 53 to 1183 days). CART-B targeted a different antigen than CART-A in 48 patients (78.7%), owing primarily to loss of CART-A antigen target. The rate of complete remission (CR) was lower with CART-B (65.5%; 40 of 61) than with CART-A (88.5%; 54 of 61; P = .0043); 35 of 40 (87.5%) CART-B responders had CART-B targeting a different antigen than CART-A. Among the 21 patients with a partial response or nonresponse to CART-B, 8 (38.1%) received CART-B with the same antigen target as CART-A. Of 40 patients with CART-B complete response (CR), 29 (72.5%) relapsed. For the 21 patients with evaluable data, the relapse immunophenotype was antigennegative in 3 (14.3%), antigendim in 7 (33.3%), antigenpositive in 10 (47.6%), and lineage switch in 1 (4.8%). The median relapse-free survival following CART-B CR was 9.4 months (95% confidence interval [CI], 6.1 to 13.2 months), and overall survival was 15.0 months (95% CI, 13.0 to 22.7 months). Given the limited salvage options for post-CART relapse, identifying optimizing strategies for CART-B is critical. We raise awareness about the emerging use of CART for post-CART failure and highlight clinical implications accompanying this paradigm shift.

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Evaluation of chimeric antigen receptor of humanized rabbit-derived T cell receptor-like antibody.

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Evaluation of chimeric antigen receptor of humanized rabbit-derived T cell receptor-like antibody.

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