Targetedex vivoreduction of CD64-positive monocytes in chronic myelomonocytic leukemia and acute myelomonocytic leukemia using human granzyme B-based cytolytic fusion proteins

Abstract

CMML (chronic myelomonocytic leukemia) belongs to the group of myeloid neoplasms known as myelodysplastic and myeloproliferative diseases. In some patients with a history of CMML, the disease transforms to acute myelomonocytic leukemia (AMML). There are no specific treatment options for patients suffering from CMML except for supportive care and DNA methyltransferase inhibitors in patients with advanced disease. New treatment strategies are urgently required, so we have investigated the use of immunotherapeutic directed cytolytic fusion proteins (CFPs), which are chimeric proteins comprising a selective domain and a toxic component (preferably of human origin to avoid immunogenicity). The human serine protease granzyme B is a prominent candidate for tumor immunotherapy because it is expressed in cytotoxic T lymphocytes and natural killer cells. Here, we report the use of CD64 as a novel target for specific CMML and AMML therapy, and correlate CD64 expression with typical surface markers representing these diseases. We demonstrate that CD64-specific human CFPs kill CMML and AMML cells ex vivo, and that the mutant granzyme B protein R201K is more cytotoxic than the wild-type enzyme in the presence of the granzyme B inhibitor PI9. Besides, the human CFP based on the granzyme B mutant was also able to kill AMML or CMML probes resistant to Pseudomonas exotoxin A.The clonal hematopoietic stem cell disorder CMML (chronic myelomonocytic leukemia), is a highly aggressive and resistant form of leukemia. It is characterized by persistent monocytosis in the peripheral blood, at least one dysplasia component in the bone marrow, and <20% promonocytes and blasts in peripheral blood and bone marrow. 1 CMML can be classified further according to blast numbers, that is, CMML1 (<5% blasts in peripheral blood and <10% blasts in bone marrow) and CMML2 (<20% blasts in peripheral blood and 10-19% blasts in bone marrow).2,3 The disease occurs mainly in elderly people with a median survival of 15-20 months and leukemic transformation rates of 15-30%, both factors depending on the disease subtype. 4 Other diagnostic criteria include immunophenotyping based on the overlapping antigen expression patterns in CMML and acute myelomonocytic leukemia (AMML). The expression of CD56 combined with reductions in the levels of myeloid markers such as CD15 and CD13 is a unique signature of CMML monocytes.5 Furthermore, CD14 is expressed at higher levels on bone marrow monocytes in CMML compared with reactive monocytosis and normal marrow samples. 5 High levels of CD33 have also been reported. 6 Clonal cytogenetic abnormalities are found in 20-40% of CMML patients, but these do not appear to be specific. [7][8][9] The close relationship between CMML and other myelodysplastic (MDS)/MPNs makes correct diagnosis and treatment a significant challenge. Novel molecular markers described more recently include specific alleles of TET2 and CBL, but these

CD64-directed microtubule associated protein tau kills leukemic blastsex vivo

“…We have shown in previous studies that 200 nM is a sufficient dose of hCFP or IT to achieve the receptor-specific induction of apoptosis within 12 h [7, 8, 14, 15]. We therefore used the same dose for the constructs tested in this study.…”

“…The advantages of CD64 as a target for the delivery of cytotoxic effector molecules include its rapid internalization, which reduces the likelihood of off-target effects during hCFP therapy [8, 27, 28]. The therapeutic potential of H22(scFv)-MAP was recently demonstrated against inflammatory CD64 high macrophages (M1 subtype) in the context of chronic cutaneous inflammation [26].…”

“…In both cases, selectivity is achieved by an antibody derivative such as a single chain variable fragment (scFv). Conventional ITs use bacterial toxins to induce apoptosis, such as truncated Pseudomonas aeroginosa exotoxin A (ETA’) [13], whereas hCFPs are equipped with human pro-apoptotic effectors such as granzyme B, granzyme M or angiogenin [8, 14, 15]. The main disadvantages of ITs are the immunogenicity of the non-human effectors and off-target effects such as hepatotoxicity and vascular leakage, which have limited their performance in clinical trials [16].…”

Targeting CD64 mediates elimination of M1 but not M2 macrophages in vitro and in cutaneous inflammation in mice and patient biopsies

“…Several hCFPs have demonstrated specific activity and efficiency in vitro and in preclinical studies. 22,23 We show here for the first time that targeting CD64 on both murine (transgenic for hCD64) and human macrophages results in selective elimination of M1, but not M2 cells, although both subtypes express CD64. Compared to M2, M1 macrophages have reduced endosomal protease activity, which limits the proteolytic degradation of the immunotoxin after internalization through CD64.…”

Engineered Versions of Granzyme B and Angiogenin Overcome Intrinsic Resistance to Apoptosis Mediated by Human Cytolytic Fusion Proteins

“…The PI-9-resistant human granzyme B protein GrBR201K was fused to the CD64-specific H22(scFv) to generate the hCFP GrbBR201K-H22(scFv). Subsequent comparative ex vivo studies using CD64 + primary cells from AMML and 8 Engineered Versions of Granzyme B and Angiogenin … CMML patients treated with wild-type and mutant versions of granzyme B revealed that GrBR201K-H22(scFv) was much more cytotoxic than its wild-type counterpart in the presence of PI-9 as demonstrated by both the XTT-assay and the Annexin V/ propidium iodide (AV/PI) staining [167]. Western blot analysis of primary cell lysates showed that PI-9 was upregulated in cells from three of four CMML patients and one of three tested AMML patients after incubation for 14 h in RPMI medium containing 10 % fetal calf serum.…”