Kevin G. Pinz

Efficient Repair of Abasic Sites in DNA by Mitochondrial Enzymes

ABSTRACT Mutations in mitochondrial DNA (mtDNA) cause a variety of relatively rare human diseases and may contribute to the pathogenesis of other, more common degenerative diseases. This stimulates interest in the capacity of mitochondria to repair damage to mtDNA. Several recent studies have shown that some types of damage to mtDNA may be repaired, particularly if the lesions can be processed through a base excision mechanism that employs an abasic site as a common intermediate. In this paper, we demonstrate that a combination of enzymes purified from Xenopus laevis mitochondria efficiently repairs abasic sites in DNA. This repair pathway employs a mitochondrial class II apurinic/apyrimidinic (AP) endonuclease to cleave the DNA backbone on the 5′ side of an abasic site. A deoxyribophosphodiesterase acts to remove the 5′ sugar-phosphate residue left by AP endonuclease. mtDNA polymerase γ fills the resulting 1-nucleotide gap. The remaining nick is sealed by an mtDNA ligase. We report the first extensive purification of mtDNA ligase as a 100-kDa enzyme that functions with an enzyme-adenylate intermediate and is capable of ligating oligo(dT) strands annealed to poly(rA). These properties together with preliminary immunological evidence suggest that mtDNA may be related to nuclear DNA ligase III.

DNA Binding Properties of Human pol γB

We have recently reported the crystal structure of the accessory subunit of mitochondrial DNA polymerase, pol γB, and identified a region of the protein involved in DNA binding. The DNA employed in previous studies was presumed to be single-stranded, because it was generated by single-sided PCR. Further characterization of this DNA indicated that, due to a strand transfer event during synthesis by single-sided PCR, the DNA adopts a double-stranded hairpin conformation under native conditions. We used a series of double- and single-stranded oligonucleotides of different lengths to confirm that human pol γB prefers to bind double-stranded DNA longer than 40 bp with little apparent sequence specificity. Site-specific deletion mutagenesis identified clusters of basic residues in two surface loops required for DNA binding located on opposite sides of the symmetrical pol γB dimer. A heterodimer of pol γB that contains one mutant and one wild-type DNA binding region was shown to be unable to bind double-stranded DNA, suggesting that a single DNA molecule must contact both DNA binding sites in the pol γB dimer. The ability to bind double-stranded DNA is not essential for pol γB stimulation of pol γA activity in vitro, but may play a role in DNA replication or repair.

Preclinical targeting of aggressive T-cell malignancies using anti-CD5 chimeric antigen receptor

The outlook for T-cell malignancies remain poor due to the lack of effective therapeutic options. Chimeric antigen receptor (CAR) immunotherapy has recently shown promise in clinical trials for B-cell malignancies, however, designing CARs for T-cell based disease remain a challenge due to the shared surface antigen pool between normal and malignant T-cells. Normal T-cells express CD5 but NK (natural killer) cells do not, positioning NK cells as attractive cytotoxicity cells for CD5CAR design. Additionally, CD5 is highly expressed in T-cell acute lymphoblastic leukemia (T-ALL) and peripheral T-cell lymphomas (PTCLs). Here, we report a robust anti-CD5 CAR (CD5CAR) transduced into a human NK cell line NK-92 that can undergo stable expansion ex vivo. We found that CD5CAR NK-92 cells possessed consistent, specific, and potent anti-tumor activity against a variety of T-cell leukemia and lymphoma cell lines as well as primary tumor cells. Furthermore, we were able to demonstrate significant inhibition and control of disease progression in xenograft mouse models of T-ALL. The data suggest that CAR redirected targeting for T-cell malignancies using NK cells may be a viable method for new and complementary therapeutic approaches that could improve the current outcome for patients.

Preclinical targeting of human T-cell malignancies using CD4-specific chimeric antigen receptor (CAR)-engineered T cells

Peripheral T-cell lymphomas (PTCLs) are aggressive lymphomas with no effective upfront standard treatment and ineffective options in relapsed disease, resulting in poorer clinical outcomes as compared with B-cell lymphomas. The adoptive transfer of T cells engineered to express chimeric antigen receptors (CARs) is a promising new approach for treatment of hematological malignancies. However, preclinical reports of targeting T-cell lymphoma with CARs are almost non-existent. Here we have designed a CAR, CD4CAR, which redirects the antigen specificity of CD8+ cytotoxic T cells to CD4-expressing cells. CD4CAR T cells derived from human peripheral blood mononuclear cells and cord blood effectively redirected T-cell specificity against CD4+ cells in vitro. CD4CAR T cells efficiently eliminated a CD4+ leukemic cell line and primary CD4+ PTCL patient samples in co-culture assays. Notably, CD4CAR T cells maintained a central memory stem cell-like phenotype (CD8+CD45RO+CD62L+) under standard culture conditions. Furthermore, in aggressive orthotropic T-cell lymphoma models, CD4CAR T cells efficiently suppressed the growth of lymphoma cells while also significantly prolonging mouse survival. Combined, these studies demonstrate that CD4CAR-expressing CD8+ T cells are efficacious in ablating malignant CD4+ populations, with potential use as a bridge to transplant or stand-alone therapy for the treatment of PTCLs.

Novel anti-CD3 chimeric antigen receptor targeting of aggressive T cell malignancies

Peripheral T-cell lymphomas (PTCLS) comprise a diverse group of difficult to treat, very aggressive non-Hodgkins lymphomas (NHLS) with poor prognoses and dismal patient outlook. Despite the fact that PTCLs comprise the majority of T-cell malignancies, the standard of care is poorly established. Chimeric antigen receptor (CAR) immunotherapy has shown in B-cell malignancies to be an effective curative option and this extends promise into treating T-cell malignancies. Because PTCLS frequently develop from mature T-cells, CD3 is similarly strongly and uniformly expressed in many PTCL malignancies, with expression specific to the hematological compartment thus making it an attractive target for CAR design. We engineered a robust 3rd generation anti-CD3 CAR construct (CD3CAR) into an NK cell line (NK-92). We found that CD3CAR NK-92 cells specifically and potently lysed diverse CD3+ human PTCL primary samples as well as T-cell leukemia cells lines ex vivo. Furthermore, CD3CAR NK-92 cells effectively controlled and suppressed Jurkat tumor cell growth in vivo and significantly prolonged survival. In this study, we present the CAR directed targeting of a novel target - CD3 using CAR modified NK-92 cells with an emphasis on efficacy, specificity, and potential for new therapeutic approaches that could improve the current standard of care for PTCLs.

Compound CAR T-cells as a double-pronged approach for treating acute myeloid leukemia

Acute myeloid leukemia (AML) bears heterogeneous cells that can consequently offset killing by single-CAR-based therapy, which results in disease relapse. Leukemic stem cells (LSCs) associated with CD123 expression comprise a rare population that also plays an important role in disease progression and relapse. Here, we report on the robust anti-tumor activity of a compound CAR (cCAR) T-cell possessing discrete scFv domains targeting two different AML antigens, CD123, and CD33, simultaneously. We determined that the resulting cCAR T-cells possessed consistent, potent, and directed cytotoxicity against each target antigen population. Using four leukemia mouse models, we found superior in vivo survival after cCAR treatment. We also designed an alemtuzumab safety-switch that allowed for rapid cCAR therapy termination in vivo. These findings indicate that targeting both CD123 and CD33 on AML cells may be an effective strategy for eliminating both AML bulk disease and LSCs, and potentially prevent relapse due to antigen escape or LSC persistence.

Targeting T-cell malignancies using anti-CD4 CAR NK-92 cells

Peripheral T-cell lymphomas (PTCLs) are a group of very aggressive non-Hodgkins lymphomas (NHLs) with poor prognoses and account for a majority of T-cell malignancies. Overall, the standard of care for patients with T-cell malignancies is poorly established, and there is an urgent clinical need for a new approach. As demonstrated in B-cell malignancies, chimeric antigen receptor (CAR) immunotherapy provides great hope as a curative treatment regimen. Because PTCLs develop from mature T-cells, these NHLs are commonly CD4+, and CD4 is highly and uniformly expressed. Therefore, CD4 is an ideal target for PTCL CAR immunotherapy. To that effect, we created a robust third-generation anti-CD4 CAR construct (CD4CAR) and introduced it into clonal NK cells (NK-92). CD4CAR NK-92 cells specifically and robustly eliminated diverse CD4+ human T-cell leukemia and lymphoma cell lines (KARPAS-299, CCRF-CEM, and HL60) and patient samples ex vivo. Furthermore, CD4CAR NK-92 cells effectively targeted KARPAS-299 cells in vivo that modeled difficult-to-access lymphoma nodules, significantly prolonging survival. In our study, we present novel targeting of CD4 using CAR-modified NK cells, and demonstrate efficacy. Combined, our data support CD4CAR NK cell immunotherapy as a potential new avenue for the treatment of PTCLs and CD4+ T-cell malignancies.

647. Efficient Targeting of T Cell Malignancies In Vitro and In Vivo Using CD4-Specific Chimeric Antigen Receptor (CAR)-Engineered NK Cells

Chimeric antigen receptor (CAR) immunotherapy has shown exceptional promise in targeting otherwise untreatable hematologic and solid tumor malignancies, providing new hope to both pediatric and adult patients. Although remarkable progress has been achieved in clinical trials for patients with relapsed/refractory B cell malignancies, CAR immunotherapy for patients with T cell leukemias and lymphomas has not yet been developed, despite a generally poorer prognosis. In light of this unmet clinical need, we engineered natural killer (NK) cells to express a third-generation CAR directed against CD4. Indeed, most aggressive peripheral T-cell lymphomas are CD4-positive with uniform expression of this surface molecule. Therefore, CD4 is potentially an ideal target for CAR. Furthermore, in contrast to donor T cells, CAR NK cells have the advantage of mediating anti-cancer effects without the risk of inducing graft-versus-host disease (GvHD). Also, their shorter lifespan relative to T cells may limit off-target events and thus eliminate the need for a “suicide switch” that would ablate the modified cells in the event of off-target effects. Other potential advantages of CAR NK cells over CAR T cells include the opportunity to be an off-the-shelf therapy, and simpler manufacturing. We generated a third generation CD4-specific CAR (CD4CAR) containing CD28, 4-1BB and CD3zeta signaling domains. This CAR was introduced into the NK-92 cell line, which has used in multiple clinical studies, resulting in CD4CAR NK cells. When assayed in co-culture, these CD4CAR NK cells had a profound ability to kill CD4 positive tumor cells in vitro using both CD4+ cell lines (Karpas 299, HL60, and CCRF-CEM) and two primary patient samples from pediatric and adult T cell leukemia and lymphomas (Figure 1Figure 1). To address any potential CD4CAR NK cell impact on the hematopoietic compartments ability to repopulate, we also confirmed by CFU assay that CD34+ cells co-cultured with CD4CAR NK cells were able to differentiate into BFU-E and CFU-GM colonies at ratios statistically similar to CD34+ cells co-cultured with non-CAR NK cells. We then confirmed in vivo anti-CD4 positive tumor activity using xenogeneic mouse models. Together, our encouraging results of this preclinical study support the further development of anti-CD4 CAR-engineered NK cell immunotherapy for patients with T cell malignancies.Figure 1CD4CARNK cells kill both peripheral T cell lymphoma cell line and primary patient malignant cells at effector to target ratios of 2 to 1 and 5 to 1. Peripheral T cell lymphoma cell lines and primary patient T cell leukemia lymphoma samples were co-cultured for 24 hours with CD4CAR NK cells. The percent of malignant cell killing was determined by comparison to vector control transduced NK cells via flow cytometry analysis of cell survival.View Large Image | Download PowerPoint Slide