CD28 costimulation improves expansion and persistence of chimeric antigen receptor-modified T cells in lymphoma patients

Abstract

Targeted T cell immunotherapies using engineered T lymphocytes expressing tumor-directed chimeric antigen receptors (CARs) are designed to benefit patients with cancer. Although incorporation of costimulatory endodomains within these CARs increases the proliferation of CAR-redirected T lymphocytes, it has proven difficult to draw definitive conclusions about the specific effects of costimulatory endodomains on the expansion, persistence, and antitumor effectiveness of CAR-redirected T cells in human subjects, owing to the lack of side-by-side comparisons with T cells bearing only a single signaling domain. We therefore designed a study that allowed us to directly measure the consequences of adding a costimulatory endodomain to CAR-redirected T cells. Patients with B cell lymphomas were simultaneously infused with 2 autologous T cell products expressing CARs with the same specificity for the CD19 antigen, present on most B cell malignancies. One CAR encoded both the costimulatory CD28 and the ζ-endodomains, while the other encoded only the ζ-endodomain. CAR+ T cells containing the CD28 endodomain showed strikingly enhanced expansion and persistence compared with CAR+ T cells lacking this endodomain. These results demonstrate the superiority of CARs with dual signal domains and confirm a method of comparing CAR-modified T cells within individual patients, thereby avoiding patient-to-patient variability and accelerating the development of optimal T cell immunotherapies.

Figure 1. Transduction efficiency and phenotypic/function profile of T cell lines.

(A) FACS and Q-PCR analyses showing transduction efficiency with CAR.CD19ζ and CAR.CD19-28ζ vectors (left). Bars indicate mean values for peripheral blood samples from 6 patients (Supplemental Table 1). Each symbol represents an individual cell line. Representative histograms of T cells transduced with CAR.CD19ζ and CAR.CD19-28ζ vectors from patients number 1 and number 5 are also presented (right). Numbers represent the percentage of CAR⁺ cells. (B) Results of a 4-hour ⁵¹Cr-release assay at an effector/tumor cell (E/T) ratio of 20:1. Target cells were Raji (CD19⁺, CD80⁺, CD86^–), a Burkitt lymphoma cell line; HLDM-2 (CD19^–, CD80⁺, CD86⁺), a Hodgkin lymphoma cell line; and K562 (CD19^–, CD80^–, CD86^–), an erythroid leukemia cell line that is susceptible to natural killer cell activity. Both CAR.CD19ζ⁺ and CAR.CD19-28ζ⁺ T cells specifically targeted CD19⁺ tumors. Data are mean ± SD for the 6 T cell lines. (C) Phenotypic composition of CAR.CD19ζ⁺ or CAR.CD19-28ζ⁺ T cells. These products contained both CD8⁺ and CD4⁺ CAR-expressing T cells that are predominantly CD45RO⁺CD62L⁺, with a fraction of them expressing CD28. Each symbol represents an individual cell line, and horizontal bars denote mean group values.

Figure 2. In vivo expansion and persistence of infused CAR.CD19ζ⁺ versus CAR.CD19-28ζ⁺ T cell lines as assessed by Q-PCR in peripheral blood.

Data points represent critical postinfusion intervals after the first or second infusion of modified T cells. Patients number 1, number 3, and number 5, who had stable disease or clinical benefit at 6 weeks after the first T cell infusion, received a second infusion of CAR-modified T lymphocytes. Patient number 1 received only CAR.CD19-28ζ⁺ T cells (2 × 10⁷ cells/m², the same as for the first infusion), because this was the only product available. Patient number 3 received both CAR.CD19-28ζ⁺ and CAR.CD19ζ⁺ T cells, but the cell dose was 60% of their first dose (1 × 10⁸ cells/m²). Patient number 5 received both CAR.CD19-28ζ⁺ and CAR.CD19ζ⁺ T cells at the same dose administered during their first infusion (2 × 10⁸ cells/m²). Open arrows indicate the time of T cell infusion, and dashed arrows indicate the time when chemotherapy was initiated for disease progression. Pre, before the first infusion; Pre II, before the second infusion.

Figure 3. Detection of CAR⁺ T cells in a skin tumor biopsy.

(A) Immunohistochemical examination (diaminobenzidine with hematoxylin counterstaining) of a punch biopsy of a lymphoma skin lesion from patient number 5 at 2 weeks after T cell infusion showed that tumor cells were CD20⁺ (shown), CD10⁺, BCL2⁺, and BCL6⁺, consistent with involvement by follicular lymphoma with large cell transformation. Scattered CD3⁺ CD8⁺ cells infiltrated the tumor. Of note, the infused CAR.CD19-28ζ⁺ product consisted of 85% CD8⁺ cells. Scale bar: 100 μm. (B) FACS analysis of a cell suspension obtained from a fragment of the tumor biopsy. Viable cells represented approximately 45% of the preparation. The far left panel shows the gate on CD45⁺ cells, which represented 12% of the viable cells. The middle panel shows the CD3⁺ lymphocytes infiltrating the tumor, which accounted for 6.7% of CD45⁺ cells (0.8% of all viable cells). The far right panel illustrates that 20% of the gated CD3⁺ lymphocytes cells coexpressed the CAR, as assessed by the Fc-Cy5 monoclonal antibody, which binds to the IgG1-CH₂CH₃ spacer region of the CD19-specific CARs (~0.16% of all viable cells). SSC, side scatter.