Effects of Anti-CCR4 Antibody (KW-0761) on Regulatory T Cells and Natural Killer Cells in Patients with Cutaneous T-Cell Lymphoma

Abstract 444

The CC chemokine receptor 4 (CCR4) is expressed on malignant T-cells in cutaneous T-cell lymphoma (CTCL) and also on the surface of regulatory T cells (T-regs). T-regs also express the transcription factor, foxp3 and suppress effector immune cells, including natural killer (NK) cells. Thus, increased T-regs in the tumor microenvironment is associated with impaired anti-tumor immunity. KW-0761, a defucosylated, humanized monoclonal antibody, binds to CCR4 and induces effective antibody-dependent cellular cytotoxicity (ADCC) against CCR4+ malignant T-cells. The phase I/II clinical trial to determine the safety and efficacy of anti-CCR4 antibody (KW-0761) included a translational component to evaluate its effects on T-regs and NK cells in CTCL patients.

Peripheral blood mononuclear cells (PBMCs) were collected from 20 patients [10 with mycosis fungoides (MF) and 10 with Sézary syndrome(SS)] pre- and post-treatment at two centers for flow cytometry analysis of CD3+CD4+CD25+CD127- T-regs, CCR4+ T-regs, and CD3-CD56+CD16+ NK cell subsets. Total RNA was extracted from PBMCs, and foxp3 and CCR4 mRNA were quantified using real-time PCR. The standard 4-hour ⁵¹Cr release assay was used to assess the cytotoxicity of NK cells.

Fifteen of 20 patients (75.0%) had detectable CD3+CD4+CD25+CD127- T-regs (1.26±1.09 % or 33.79±46.88 /μl) at baseline with 60 –100 % of T-regs positive for CCR4. After 4–6 weeks of treatment with anti-CCR4 antibody (KW-0761), all 15 patients had a decrease in T-reg numbers (0.39±0.49 % or 5.65±8.95 /μl, *p<0.05, Figure 1 ). CCR4+ T-regs were significantly reduced from an average of 67.2% to 24.6% (p<0.01). In parallel, foxp3 and CCR4 mRNA levels were also significantly decreased from baseline levels (foxp3: from 0.57±0.91 to 0.07±0.08, **p<0.01, Figure 1 ; CCR4: from 23.40±33.50 to 1.73±2.35, p<0.05). The CD3-CD56+CD16+ NK cells were found at baseline in all 15 patients tested. Of 14 paired samples, 10 (71.4%) had increased NK cells after 4–16 weeks of treatment (pre-treatment: 16.02±15.86 % vs. post-treatment: 22.64±13.93 %, p=0.05, Figure 2 ) with reduced numbers of T-regs. Five of 6 patients studied also showed a dose dependent increase in NK cell cytotoxicity to target cells by ⁵¹Cr release assay (Figure 2). Ten patients with SS had a lower NK cells (13.37±18.48 %) and higher foxp3 (0.81±1.19) and CCR4 mRNA (34.60±39.90) at baseline compared to ten patients with MF (19.04±12.98%, 0.31±0.31; 10.94±20.07) respectively. Seven paired samples from SS patients all had increased NK cells post-treatment with a reduction of T-regs and foxp3 and CCR4 mRNA. Six of 7 SS patients had blood improvement with 3 complete and 3 partial blood responses.

Figure 1.

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Effect of KW-0761 on regulatory T cells in CTCL patients.

Figure 1.

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Effect of KW-0761 on regulatory T cells in CTCL patients.

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Figure 2.

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Effect of KW-0761 on natural killer cells in CTCL patients.

Figure 2.

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Effect of KW-0761 on natural killer cells in CTCL patients.

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Our results suggest that in addition to ADCC towards malignant T-cells, the anti-neoplastic activity of the anti-CCR4 antibody (KW-0761) may include a reduction in T-regs in most CTCL patients and a subsequent increase in NK numbers and function in some patients. Follow up studies need to be performed to confirm these findings.