The Capacity of CMV-Specific T-Cells to Prevent Outgrowth of Clonogenic Human Colon Carcinoma Cells Co-Expressing CMVpp65 in-Vivo and Durably Suppress Established CMVpp65⁺ Tumor Xenografts Is Dose Dependant and Can Be Augmented by IL-15/IL-15Rα

Abstract 4307

Adoptively transferred CMV-specific T cells (CMV-CTL) can effectively prevent CMV disease in HSCT recipients. However, data regarding the doses required to eradicate disease and the relative potency of T-cells (TC) specific for different epitopes are lacking. To test the capacity of T-cells specific for different CMV antigenic peptides to target and eliminate CMV⁺ cells in-vivo, and establish TC doses required for treatment we have developed an in-vivo model to assess specificity and efficacy of CMV-CTLs using human colon carcinoma cells transduced with CMVpp65 as a surrogate system.

HLA A0201⁺ colon carcinoma cells transduced to express CMVpp65 and a GFP-firefly luciferase transgene (cocapp65) were injected subcutaneously (s.c.) into NOD/Scid-IL2Rgc-KO/J mice (NSG) at doses ranging from 10⁴ – 10⁶ (4 mice /group) and tumor growth followed for 4 weeks using bio-luminescent imaging and tumor measurements. Consistent engraftment was observed with 3 × 10⁵ cells. Groups of NSG mice were then injected s.c with coca-pp65 (3 × 10⁵) mixed with HLA A0201-restricted CMVpp65 specific T-cells (A2-CMV-CTLs) at E:T ratios of 50:1, 10:1, 2:1, 0.1:1, 0.01:1 and 0.001:1. To ascertain antigen specificity of A2-CMV-CTLs, groups of 4 NSG mice were subsequently co-injected s.c. with 3 tumor-TC combinations (E:T= 1:1) as follows: (i) A2-CMV- CTLs + 3 × 10⁵ cocapp65 (ii) A2-CMV- CTLs + A2 tumor without CMVpp65 (iii) A2-Flu-CTLs + 3 × 10⁵ cocapp65. Control animals were injected with the cocapp65 without TC. IL-2 (2000 U i.p) was given 2 × a week to all groups. Tumor growth was measured by bioluminescence. No growth of cocapp65 was detected in any animal co-injected with A2-CMV-CTLs at E:T ratios ≥ 1:1. At E:T ratios of 0.1:1, 0.01:1 and 0.001:1, 1/4, 2/4 and 3/4 animals demonstrated tumor growth respectively. In contrast, consistent tumor growth was seen in all control animals over 4 weeks. Thus, tumors in condition (i) were completely inhibited while (ii) and (iii) continued to grow similar to controls, thus demonstrating antigen specific activity of the infused CMV-CTLs.

In subsequent experiments we assessed (1) the efficacy of titrated doses of intravenously administered CMV-CTLs in animals with established tumors using 20 × 10⁶, 7× 10⁶ or 2× 10⁶ T-cells per mouse (2) the contribution of IL-15/15Rα complex in augmenting the efficacy of CMV-CTLs by injecting each mouse i.p. with irradiated Baf-3 cells transduced to express human IL-15/15Rα complex.

Infusion of CMV-CTLs significantly suppressed growth of cocapp65 tumors at all doses tested. However after a period of 4 weeks, the tumors in animals receiving doses of 7 × 10⁶ and 2 × 10⁶ CMV-CTLs began to re-grow, while tumors in animals injected with 20 × 10⁶ remained suppressed till the end of the experiment at 6–8 weeks. At autopsy, human CD45[+] CD3[+] T-cells were detected in tumors expressing CMVpp65 as documented by IHC staining, but not in tumors lacking CMVpp65 or HLA A0201. In mice injected with the Baf-3 cells expressing IL-15/IL-15Rα, the anti-tumor activity of the transferred T-cells was sustained even in animals treated with only 2×10⁶ CMVpp65 specific CTLs. Differences in cytolytic capacity of CMV-CTLs reactive against dominant and subdominant epitopes are currently under study.

These studies demonstrate that (1) CMV-CTLs can prevent outgrowth of clonogenic human carcinoma cells co-expressing an immunogenic viral antigen in-vivo at E:T ratios that are significantly lower than those required in-vitro (2) These CMV-CTLs can also induce sustained suppression of established tumor xenografts following intravenous transfer (3) Lower doses of intravenously transferred CMV-CTLs are effective, but may require additional dosing to maintain disease control, and (4) In contrast, IL-15/IL-15Rα supplementation can augment and sustain the in-vivo activity of low doses of CMVpp65-specific CTLs. This model thus provides an in vivo system for estimating the concentrations of antigen-specific T-cells over time required for effective adoptive immunotherapy, for comparative evaluations of virus-specific T-cells differing in phenotype or specificity, and for testing the potential of cytokines and other agents to increase or sustain virus-specific T-cell activity.