Optimization of a protein’s pharmaceutical properties is usually carried out by rational design and/or directed evolution. Here we test an alternative approach based on ancestral sequence reconstruction. Using available genomic sequence data on coagulation factor VIII and predictive models of molecular evolution, we engineer protein variants with improved activity, stability. biosynthesis potential, and reduced inhibition by clinical anti-drug antibodies. In principle, this approach can be applied to any protein drug based on a conserved gene sequence.

Relapsed T-cell malignancies have poor outcomes when treated with chemotherapy, but survival after allogeneic bone marrow transplantation (BMT) approaches 50%. A limitation to BMT is the difficulty of achieving remission prior to transplant. Chimeric antigen receptor (CAR) T-cell therapy has shown successes in B-cell malignancies. This approach is difficult to adapt for the treatment of T-cell disease due to lack of a T-lymphoblast specific antigen and the fratricide of CAR T cells that occurs with T-cell antigen targeting. To circumvent this problem two approaches were investigated. First, a natural killer (NK) cell line, which does not express CD5, was used for CAR expression. Second, CRISPR-Cas9 genome editing technology was used to knockout CD5 expression in CD5-positive Jurkat T cells and in primary T cells, allowing for the use of CD5-negative T cells for CAR expression. Two structurally distinct anti-CD5 sequences were also tested, i) a traditional immunoglobulin-based single chain variable fragment (scFv) and ii) a lamprey-derived variable lymphocyte receptor (VLR), which we previously showed can be used for CAR-based recognition. Our results show i) both CARs yield comparable T-cell activation and NK cell-based cytotoxicity when targeting CD5-positive cells, ii) CD5-edited CAR-modified Jurkat T cells have reduced self-activation compared to that of CD5-positive CAR-modified T cells, iii) CD5-edited CAR-modified Jurkat T cells have increased activation in the presence of CD5-positive target cells compared to that of CD5-positive CAR-modified T cells, and iv) although modest effects were seen, a mouse model using the CAR-expressing NK cell line showed the scFv-CAR was superior to the VLR-CAR in delaying disease progression.

Immune responses to coagulation factors VIII (FVIII) and IX (FIX) represent primary obstacles to hemophilia treatment. Previously, we showed that hematopoietic stem cell (HSC) retroviral gene therapy induces immune nonresponsiveness to FVIII in both naive and preimmunized murine hemophilia A settings. Liver-directed adeno-associated viral (AAV)-FIX vector gene transfer achieved similar results in preclinical hemophilia B models. However, as clinical immune responses to FVIII and FIX differ, we investigated the ability of liver-directed AAV-FVIII gene therapy to affect FVIII immunity in hemophilia A mice. Both FVIII naive and preimmunized mice were administered recombinant AAV8 encoding a liver-directed bioengineered FVIII expression cassette. Naive animals receiving high or mid-doses subsequently achieved near normal FVIII activity levels. However, challenge with adjuvant-free recombinant FVIII induced loss of FVIII activity and anti-FVIII antibodies in mid-dose, but not high-dose AAV or HSC lentiviral (LV) vector gene therapy cohorts. Furthermore, unlike what was shown previously for FIX gene transfer, AAV-FVIII administration to hemophilia A inhibitor mice conferred no effect on anti-FVIII antibody or inhibitory titers. These data suggest that functional differences exist in the immune modulation achieved to FVIII or FIX in hemophilia mice by gene therapy approaches incorporating liver-directed AAV vectors or HSC-directed LV.

An effective therapy regimen for relapsed/refractory high-risk neuroblastoma (NB) includes the anti-GD2 monoclonal antibody, dinutuximab, in combination with temozolomide and irinotecan, supporting a role for chemo-immunotherapy in NB. γδ T cells are an attractive anti-tumor immunotherapy because of their direct cytotoxic activity mediated through cell surface receptors NKG2D and CD16. NKG2D facilitates the innate recognition of stress-induced ligands whereas CD16 recognizes antibody bound to tumors and activates mechanisms of antibody-dependent cellular cytotoxicity (ADCC). This study demonstrates an efficient method for expanding and storing γδ T cells from NB patient-derived apheresis products at clinically relevant amounts. The expanded patient-derived γδ T cells were cytotoxic against the K562 cell line and multiple NB cell lines. Combining γδ T cells with dinutuximab led to a 30% increase in tumor cell lysis compared to γδ T cells alone. Furthermore, low-dose temozolomide in combination with expanded γδ T cells and dinutuximab resulted in increased IFNγ secretion and increased γδ T-cell surface expression of FasL and CD107a. IMR5 NB cell line xenografts established subcutaneously in NSG mice were treated with a regimen of dinutuximab, temozolomide, and γδ T cells. This combination caused targeted killing of NB xenografts in vivo, reducing tumor burden and prolonging survival. These data support the continued preclinical testing of dinutuximab and temozolomide in conjunction with γδ T-cell immunotherapy for patients with recurrent/refractory NB.

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Orthologous proteins contain sequence disparity guided by natural selection. In certain cases, species-specific protein functionality predicts pharmacological enhancement, such as greater specific activity or stability. However, immunological barriers generally preclude use of nonhuman proteins as therapeutics, and difficulty exists in the identification of individual sequence determinants among the overall sequence disparity. Ancestral sequence reconstruction (ASR) represents a platform for the prediction and resurrection of ancient gene and protein sequences. Recently, we demonstrated that ASR can be used as a platform to facilitate the identification of therapeutic protein variants with enhanced properties. Specifically, we identified coagulation factor VIII (FVIII) variants with improved specific activity, biosynthesis, stability, and resistance to anti-human FVIII antibody–based inhibition. In the current study, we resurrected a panel of ancient mammalian coagulation factor IX (FIX) variants with the goal of identifying improved pharmaceutical candidates. One variant (An96) demonstrated 12-fold greater FIX activity production than human FIX. Addition of the R338L Padua substitution further increased An96 activity, suggesting independent but additive mechanisms. after adeno-associated virus 2 (AAV2)/8-FIX gene therapy, 10-fold greater plasma FIX activity was observed in hemophilia B mice administered AAV2/8-An96–Padua as compared with AAV2/8-human FIX–Padua. Furthermore, phenotypic correction conferred by the ancestral variant was confirmed using a saphenous vein bleeding challenge and thromboelastography. Collectively, these findings validate the ASR drug discovery platform as well as identify an ancient FIX candidate for pharmaceutical development.

Hematopoietic stem and progenitor cell (HSPC) lentiviral gene therapy is a promising strategy toward a lifelong cure for hemophilia A (HA). The primary risks associated with this approach center on the requirement for pre-transplantation conditioning necessary to make space for, and provide immune suppression against, stem cells and blood coagulation factor VIII, respectively. Traditional conditioning agents utilize genotoxic mechanisms of action, such as DNA alkylation, that increase risk of sterility, infection, and developing secondary malignancies. In the current study, we describe a non-genotoxic conditioning protocol using an immunotoxin targeting CD117 (c-kit) to achieve endogenous hematopoietic stem cell depletion and a cocktail of monoclonal antibodies to provide transient immune suppression against the transgene product in a murine HA gene therapy model. This strategy provides high-level engraftment of hematopoietic stem cells genetically modified ex vivo using recombinant lentiviral vector (LV) encoding a bioengineered high-expression factor VIII variant, termed ET3. Factor VIII procoagulant activity levels were durably elevated into the normal range and phenotypic correction achieved. Furthermore, no immunological rejection or development of anti-ET3 immunity was observed. These preclinical data support clinical translation of non-genotoxic antibody-based conditioning in HSPC LV gene therapy for HA.