Safety, tumor trafficking and immunogenicity of chimeric antigen receptor (CAR)-T cells specific for TAG-72 in colorectal cancer

Abstract

Background: T cells engineered to express chimeric antigen receptors (CARs) have established efficacy in the treatment of B-cell malignancies, but their relevance in solid tumors remains undefined. Here we report results of the first human trials of CAR-T cells in the treatment of solid tumors performed in the 1990s.

Methods: Patients with metastatic colorectal cancer (CRC) were treated in two phase 1 trials with first-generation retroviral transduced CAR-T cells targeting tumor-associated glycoprotein (TAG)-72 and including a CD3-zeta intracellular signaling domain (CART72 cells). In trial C-9701 and C-9702, CART72 cells were administered in escalating doses up to 10¹⁰ total cells; in trial C-9701 CART72 cells were administered by intravenous infusion. In trial C-9702, CART72 cells were administered via direct hepatic artery infusion in patients with colorectal liver metastases. In both trials, a brief course of interferon-alpha (IFN-α) was given with each CART72 infusion to upregulate expression of TAG-72.

Results: Fourteen patients were enrolled in C-9701 and nine in C-9702. CART72 manufacturing success rate was 100% with an average transduction efficiency of 38%. Ten patients were treated in CC-9701 and 6 in CC-9702. Symptoms consistent with low-grade, cytokine release syndrome were observed in both trials without clear evidence of on target/off tumor toxicity. Detectable, but mostly short-term (≤14 weeks), persistence of CART72 cells was observed in blood; one patient had CART72 cells detectable at 48 weeks. Trafficking to tumor tissues was confirmed in a tumor biopsy from one of three patients. A subset of patients had ¹¹¹Indium-labeled CART72 cells injected, and trafficking could be detected to liver, but T cells appeared largely excluded from large metastatic deposits. Tumor biomarkers carcinoembryonic antigen (CEA) and TAG-72 were measured in serum; there was a precipitous decline of TAG-72, but not CEA, in some patients due to induction of an interfering antibody to the TAG-72 binding domain of humanized CC49, reflecting an anti-CAR immune response. No radiologic tumor responses were observed.

Conclusion: These findings demonstrate the relative safety of CART72 cells. The limited persistence supports the incorporation of co-stimulatory domains in the CAR design and the use of fully human CAR constructs to mitigate immunogenicity.

Fig. 1

CC49-ζ vector diagram. A gamma retroviral CAR construct consisting of a TAG-72 antigen-binding domain derived from the humanized CC49 single-chain antibody linked to the CD3-ζ signaling domain of the T-cell receptor via a human CD4 transmembrane domain. CAR, chimeric antigen receptor; Fv, variable fragment; (G₄-S)₃, (Gly₄, Ser)₃ peptide linker; IC, intracellular; TM, transmembrane; V_H, variable heavy chain; V_L, variable light chain; γ1, immunoglobulin γ1

Fig. 2

CART72 cell growth curves. Individual CART72 cell growth curves from trials C-9701 and C-9702 are shown. Lymphapheresis products were depleted of red blood cells and stimulated with CD3xCD28 beads on Day 1 in the presence of IL-2 (200 IU/mL) with removal of beads on Day 3. Spinoculation transduction with the CC49-ζ vector was performed on Days 5 and 7 followed by continued cell culture in serum-free media supplemented with IL-2 (200 IU/mL). Minimum harvest criteria of 3 x 10¹⁰ total T cells was achieved on all lots by Day 17

Fig. 3

Serum TAG-72 and CEA levels. Circulating sera levels of TAG-72 were measured by RIA in treated patients in: (a) Part A dose-escalation phase of IV CART72 cells in trial C-9701; (b) Part B expansion phase of trial C-9701; and (c) Intra-HA CART72 cells in trial C-9702. Circulating sera levels of CEA were measured in: (d) Part A of C-9701; (e) Part B of C-9701; and (f) C-9702. Seven of 8 evaluable patients treated with IV infusions of CART72 cells later demonstrated abrupt reductions (>80%) in serum TAG-72 levels (a, b). Two of 2 evaluable patients who received CART72 cells via HA infusion showed a marked decrease in measured serum TAG-72 at Week 8 (85% and 60%, respectively) (c). No associated reductions in sera CEA were observed, with an increase in the sera CEA levels over time observed in nine of 10 patients in trial C-9701 (d, e) and six of six patients in C-9702 (f). CEA, carcinoembryonic antigen; HA, hepatic artery; IV, intravenous

Fig. 4

CART72 persistence in blood. The CC49-ζ copy number measured by quantitative PCR and expressed per 10⁶ circulating PBMCs over time is shown in: (a) Part A dose escalation phase of CART72 cells administered by IV infusion in trial C-9701; (b) Part B expansion phase of trial C-9701; and (c) CART72 cells administered by HA infusion in trial C-9702. HA, hepatic artery; PBMC, peripheral blood mononuclear cell; PCR, polymerase chain reaction

Fig. 5

Tumor trafficking of ¹¹¹In-labeled CART72 cells. ¹¹¹In-labeled CART72 cells were infused in four patients and compared to infusion of ^99mTc-MAA and imaging was performed at 3–96 hours post-infusion. Representative images from one patient at 24 hours post infusion of 10¹⁰ CART72 cells are notable for decreased penetration of ¹¹¹In-labeled CART72 cells (b) into the center of a liver metastases compared with well distributed dissemination of ^99mTc-MAA (a). ¹¹¹In, ¹¹¹Indium; ^99mTc-MAA, technetium albumin aggregated particles