Novel CD7-specific Nanobody-based Immunotoxins Potently Mediate Apoptosis Of CD7-positive Malignant Cells

In the last few decades, the treatment outcome of patients with leukemia and lymphoma has significantly improved. Nonetheless, only a minority of patients with T-cell acute lymphoblastic leukemia(T-ALL) or peripheral T-cell lymphoma achieve long term tumor-free survival. Conventional cytotoxic therapy in these diseases is usually associated with substantial side effects and limited response. As we all know, once a patient with leukemia or lymphoma experiences chemoresistance or a relapse, treatment options are limited, and the survival duration is short. Thus, effective novel therapies with distinctive mechanisms that can avoid multi-drug resistance and have favorable specificity and toxicity profiles are needed to treat human T-cell malignancies. In the development of new drugs, immunotoxins consisting of peptide toxins fused with tumor cell-selective antibody fragments or ligands are believed to be effective against and remain sensitive to chemoresistant T-cell disorders.One of the key points in using immunotoxins is to choose appropriate target receptors in tumor cells. Many studies indicated that CD7 is expressed mainly on T-cell lymphoma and leukemia cell but absent from at least a small portion of normal T lymphocytes. Another key property of CD7 for therapeutic applications in cancer cases is its rapid internalization after binding to an antibody or antibody derivative, which makes it well suited for drug delivery. Because of the above advantages, various immunotoxins specific for CD7 have been generated and tested for their anti-leukemic effects. However, most studies focused on plant-derived toxins such as ricin, saporin and derivatives. Unfortunately, none of those immunotoxins was approved clinically because of lacking enough efficacy and safety. Another toxin, a truncated form of Pseudomonas exotoxin A(ETA’ or PE38), fused to a CD7 scFv fragment caused only approximately 20% cell death of primary leukemia-derived cells, and without further examination in in vivo model, implying that T-lineage leukemia cells may not be sensitive to PE38, or further improvement for the reported CD7 scFv is needed. Indeed, anti-CD22 variable domain formed immunotoxin with PE38 showed impressive 46% complete remission without obvious dose-limited toxicity(DLT) in the clinical trial for hairy cell leukemia patients, suggesting PE38 is a potent toxin for at least some lymphocytes. Therefore, novel anti-CD7 variable fragments may provide us a new option to improve the immunotoxin efficacy on T-cell lymphomas and leukemias.To develop novel anti-CD7 antibody, nanobody is selected as our development strategy for these reasons: nanobody is an antibody fragment consisting of a single monomeric variable antibody domain derived from camelidae heavy-chain antibodies that was discovered by Hamers-Casterman et al. The outstanding biochemical and physical properties of nanobodies make them exceptional candidates for targeted delivery of biologically active drugs. Investigators have shown that nanobodies can be coupled with toxins and other functional molecules, and then used to deliver conjugates to target cells for the treatment of cancer and other diseases.Purpose: To screen CD7 specific nanobodies, conststruct novel CD7 nanobody-based immunotoxins, test their effects on T-ALL cell lines and patient-derived primary T-ALL and AML cells in vitroand evaluated their anti-leukemic potential in vivo.Methods: We construct a phage nanobody library constructed using the immunoglobulin repertoire of peripheral blood lymphocytes obtained from llamas immunized with CD7-positive Jurkat cells and then obtain nanobodies against CD7 by biopaning. The binding affinity and specificity of VHH6 for CD7 and specificity of PG001(VHH6-PE38) and PG002(dVHH6-PE38) for CD7 were measured by flow cytometry. In vitro cytotoxicity of PG001 and PG002 was measured by inhibition of protein synthesis and apoptosis in human CD7 positive Jurkat, CEM cell line and CD7 negative RPMI8226, H460 cell line. To evaluate the cytotoxic potential of PG001 and PG002 in patient-derived primary leukemia cells by annexin V and 7-AAD staining. In vivo antitumor efficacy of PG001 and PG002 was evaluated using CEM cells xenotransplant tumor models.Results: We identified a nanobody against CD7, VHH6, with high affinity(15 nM) and specificity. The purified monovalent and bivalent VHH6-based immunotoxins PG001(16 nM) and PG002(4 nM) exhibit superior specificity for CD7-positive cells. These two immunotoxins efficiently promoted antigen-restricted apoptosis of the CD7-positive leukemia cell lines Jurkat and CEM, but had little impact on proliferation of the CD7-negative human myeloma cell line RPMI8226 and human lung cell line H460. In particular, the half-maximal effective concentrations(EC50) of PG002 in Jurkat and CEM cells were about 30 pM and 23 pM, respectively, suggesting that PG002 has great potential as a therapeutic for CD7-positive leukemia. Moreover, PG002 effectively mediated apoptosis of freshly collected patient-derived T-cell acute lymphoblastic leukemia and acute myeloid leukemia cells at single doses as low as 100 ng/mL. In xenotransplant tumor models, PG001 and PG002 prevented engraftment of the CEM cells and markedly prolonged mouse survival. Moreover, PG002 was also much more effective than PG001 in tumor models.Conclusions: We constructed monovalent(PG001) and bivalent(PG002) CD7 nanobody-based immunotoxins which can effectively eliminate CD7-positive T-ALL and fresh patient-derived T-ALL and AML cells with antigen-specificity at low concentrations. Based on the observations that PG001 and PG002 effectively killed human leukemia cells and significantly prolonged the survival of treated mice, PG001 and PG002 may deserve further evaluation of their potential for anti-leukemia in preclinical studies and clinical trials.