Third-generation CAR-CD30 T-cell therapy for CD30-positive lymphomas

The two currently approved chimeric antigen receptor (CAR) T-cell therapies for the treatment of relapsed/refractory (R/R) large B-cell lymphoma (LBCL) — axicabtagene ciloleucel and tisagenlecleucel — have demonstrated significant clinical benefit in patients with R/R disease.^1,2 These CAR T-cell therapies are CD19-directed, but other types of lymphoma, such as classical Hodgkin lymphoma (cHL) and anaplastic large cell lymphoma (ALCL), lack CD19 expression and so alternative CAR T-cell-based therapies are required.³

CD30 is a cell membrane protein and a member of the tumor necrosis factor receptor superfamily which has been identified as an attractive target for CAR T-cell therapy. This is due to its expression on most cHLs and ALCLs, as well as in a proportion of other lymphomas, such as cutaneous T-cell lymphomas (CTCLs) and diffuse LBCLs (DLBCLs).⁴

CD30 is a target of a number of antibody-based therapies. Most notably, positive results have been achieved with brentuximab vedotin (BV), an antibody drug conjugate targeting CD30⁺ cells, which is approved by the EMA for the treatment of cHL, ALCL, and CTCL.⁵ However, there are limitations associated with BV treatment, such as inadequate persistence and tumor penetration. Consequently, anti-CD30 CAR T cells are being investigated for the treatment of CD30⁺ lymphomas.⁴

Thus far, two phase I/II clinical trials (NCT01316146 and NCT02259556) have investigated the potential use of anti-CD30 CAR T-cell therapy in R/R cHL or ALCL. Despite small cohort sizes, the two trials reported tolerable CD30 CAR T-cell regimens, however clinical benefit was sub-optimal.^6–8 Preclinical advances of a third-generation, anti-CD30 CAR T-cell therapy were presented by Biagio De Angelis, Bambino Gesù Pediatric Hospital, IRCCS, Rome, IT, at the 2^nd European CAR T Cell Meeting.³ The data are based on results previously shown at the  61^st ASH Annual Meeting & Exposition in Orlando, US.⁸

Study design⁹

• A comparative preclinical study evaluating the stability and anti-tumor activity of two CD30 CAR-T constructs

CAR design³

• A third-generation CAR construct containing an anti-CD30 single-chain variable-fragment cassette linked to CD3ζ by the signaling domains of either:
  • CD28.4-1BB

or

• • CD28.OX40
• The vector incorporates an inducible caspase-9 (iCasp9) suicide gene
• A CD34 antibody recognition domain was incorporated in the hinge region of all vectors as a selection marker

Methods⁹

• In vitro anti-tumor efficacy was evaluated using non-Hodgkin lymphoma (Karpas299) and cHL (L428) cell lines, a short-term cytotoxic assay (51Cr release assay), and long-term co-cultures
  • Supernatant from co-culture experiments was analyzed by enzyme-linked immunosorbent assays (ELISA)
• In vivo NSG mouse models were engrafted i.v. with lymphoma FF-luciferase cell lines, Karpas299 or L428
• In vivo tumor growth was monitored using bioluminescent imaging
• Tumor re-challenging was initiated by i.v. infusion of 0.2x106 Karpas299 cells into mice that survived until Day +140 and were observed for an additional 110 days

Results^8,9

• High transduction rates were obtained with all constructs (80–90%)
• Insertion of a CD34 antigen allowed for convenient selection and monitoring of transduced cells
• Introduction of the iCasp9 suicide gene allowed controlled apoptosis of CD30 CAR T cells, irrespective of their co-stimulatory domains
• Incorporation of a CD28.OX40 vs CD28.4-1BB costimulatory domain into a CD30-CAR resulted in a more stable CAR expression 30 days after transduction (84.72 ± 5.30% vs 63.98 ± 11.51; p = 0.002)
• CAR CD30-CD28.OX40 elicited greater anti-lymphoma properties in comparison to CAR CD30-CD28.4-1BB in vitro
  • The level of IFNγ (pg/ml/1e6) T cells was significantly higher in CD30-CD28.OX40 vs CD30-CD28.4-1BB transfected T cells
  • In long-term co-culture experiments with various cell lines, CD28.OX40 CD30 CAR T cells showed significantly higher anti-lymphoma activity compared to CD28.41BB CAR T cells when challenged at high tumor/effector ratio
  • Secretion of Th1 cytokines (IFNγ, IL-2, and TNFα) after antigen stimulation was significantly higher in CD30-CD28.OX40 vs CD30-CD28.4-1BB transfected T cells (p = 0.040, p = 0.008, and p = 0.02, respectively)
• At the experimental endpoint (Day +165), overall survival was significantly improved in CD30-CD28.OX40 vs CD30-CD28.4-1BB CAR T-cell-treated mice (60% vs 0%), p = 0.0021
• Persistence of CD28.OX40 CAR T-cell established long-term immunological memory as demonstrated by second tumor challenge experiments in mouse tumor models

Conclusions³

• The iCasp9 system stands as a promising approach to decrease toxicities across CAR T-cell therapies
• The CD30-CD28.OX40 CAR construct could overcome the limited efficacy of previously investigated CD30 CAR T-cell therapies in CD30⁺ lymphomas if the preclinical data translates to clinical studies