CAR'TCR-T cells co-expressing CD33-CAR and dNPM1-TCR as superior dual-targeting approach for AML treatment.

Acute myeloid leukemia (AML), a fast-progressing hematological malignancy affecting myeloid cells, is typically treated with chemotherapy or hematopoietic stem cell transplantation. However, approximately half of the patients face relapses and 5-year survival rates are poor. With the goal to facilitate dual-specificity, boosting anti-tumor activity, and minimizing the risk for antigen escape, this study focused on combining chimeric antigen receptor (CAR) and T cell receptor (TCR) technologies. CAR'TCR-T cells, co-expressing a CD33-CAR and a transgenic dNPM1-TCR, revealed increased and prolonged anti-tumor activity in vitro, particularly in case of low target antigen expression. The distinct transcriptomic profile suggested enhanced formation of immunological synapses, activation, and signaling. Complete elimination of AML xenografts in vivo was only achieved with a cell product containing CAR'TCR-T, CAR-T, and TCR-T cells, representing the outcome of co-transduction with two lentiviral vectors encoding either CAR or TCR. A mixture of CAR-T and TCR-T cells, without CAR'TCR-T cells, did not prevent progressive tumor outgrowth and was comparable to treatment with CAR-T and TCR-T cells individually. Overall, our data underscore the efficacy of co-expressing CAR and transgenic TCR in one T cell, and might open a novel therapeutic avenue not only for AML but also other malignancies.

Automated manufacture of ΔNPM1 TCR-engineered T cells for AML therapy.

Acute myeloid leukemia (AML) is a heterogeneous malignancy that requires further therapeutic improvement, especially for the elderly and for subgroups with poor prognosis. A recently discovered T cell receptor (TCR) targeting mutant nucleophosmin 1 (ΔNPM1) presents an attractive option for the development of a cancer antigen-targeted cellular therapy. Manufacturing of TCR-modified T cells, however, is still limited by a complex, time-consuming, and laborious procedure. Therefore, this study specifically addressed the requirements for a scaled manufacture of ΔNPM1-specific T cells in an automated, closed, and good manufacturing practice-compliant process. Starting from cryopreserved leukapheresis, 2E8 CD8-positive T cells were enriched, activated, lentivirally transduced, expanded, and finally formulated. By adjusting and optimizing culture conditions, we additionally reduced the manufacturing time from 12 to 8 days while still achieving a clinically relevant yield of up to 5.5E9 ΔNPM1 TCR-engineered T cells. The cellular product mainly consisted of highly viable CD8-positive T cells with an early memory phenotype. ΔNPM1 TCR CD8 T cells manufactured with the optimized process showed specific killing of AML in vitro and in vivo. The process has been implemented in an upcoming phase 1/2 clinical trial for the treatment of NPM1-mutated AML.

Combining CSPG4-CAR and CD20-CCR for treatment of metastatic melanoma.

The prognosis for patients with metastatic melanoma is poor and treatment options are limited. Genetically-engineered T cell therapy targeting chondroitin sulfate proteoglycan 4 (CSPG4), however, represents a promising treatment option, especially as both primary melanoma cells as well as metastases uniformly express CSPG4. Aiming to prevent off-tumor toxicity while maintaining a high cytolytic potential, we combined a chimeric co-stimulatory receptor (CCR) and a CSPG4-directed second-generation chimeric antigen receptor (CAR) with moderate potency. CCRs are artificial receptors similar to CARs, but lacking the CD3ζ activation element. Thus, T cells expressing solely a CCR, do not induce any cytolytic activity upon target cell binding, but are capable of boosting the CAR T cell response when both CAR and CCR engage their target antigens simultaneously. Here we demonstrate that co-expression of a CCR can significantly enhance the anti-tumor response of CSPG4-CAR T cells in vitro as well as in vivo. Importantly, this boosting effect was not dependent on co-expression of both CCR- and CAR-target on the very same tumor cell, but was also achieved upon trans activation. Finally, our data support the idea of using a CCR as a powerful tool to enhance the cytolytic potential of CAR T cells, which might open a novel therapeutic window for the treatment of metastatic melanoma.

The antimicrobial peptide snakin-2 is upregulated in the defense response of tomatoes (Solanum lycopersicum) as part of the jasmonate-dependent signaling pathway.

Antimicrobial peptides (AMPs) are produced by all living organisms and play an important role in innate immunity because they are readily available and non-specific against invading pathogenic microorganisms. Snakin-2 (SN2) from tomato is a short, cationic peptide that forms lethal pores in biomembranes of microbes. In plant cells, SN2 is produced as a prepeptide with a signal sequence for ER targeting and an acidic region to decrease toxicity in the producing organism. Gene expression analysis by qRT-PCR in tomato plants demonstrated that SN2 is constitutively expressed, mostly in leaves and flowers. After fungal infection, wounding, or external application of phytohormones (such as methyl jasmonate, MeJa) operating in the JA-dependent defense response, a systemic reaction with an elevated expression of the SN2 gene is triggered in all parts of tomato plants. Abiotic stress factors like extreme temperatures or dehydration do not affect SN2 expression. Upon wounding, the expression of SN2 and LoxD are strongly enhanced in tomato fruits. Furthermore, we provide evidence that the protein level of bioactive SN2 is also increased upon application of methyl jasmonate in tomato seedlings.

Mode of action and membrane specificity of the antimicrobial peptide snakin-2.

Antimicrobial peptides (AMPs) are a diverse group of short, cationic peptides which are naturally occurring molecules in the first-line defense of most living organisms. They represent promising candidates for the treatment of pathogenic microorganisms. Snakin-2 (SN2) from tomato (Solanum lycopersicum) is stabilized through six intramolecular disulphide bridges; it shows broad-spectrum antimicrobial activity against bacteria and fungi, and it agglomerates single cells prior to killing. In this study, we further characterized SN2 by providing time-kill curves and corresponding growth inhibition analysis of model organisms, such as E. coli or B. subtilis. SN2 was produced recombinantly in E. coli with thioredoxin as fusion protein, which was removed after affinity purification by proteolytic digestion. Furthermore, the target specificity of SN2 was investigated by means of hemolysis and hemagglutination assays; its effect on plant cell membranes of isolated protoplasts was investigated by microscopy. SN2 shows a non-specific pore-forming effect in all tested membranes. We suggest that SN2 could be useful as a preservative agent to protect food, pharmaceuticals, or cosmetics from decomposition by microbes.

Recombinant Production of Snakin-2 (an Antimicrobial Peptide from Tomato) in E. coli and Analysis of Its Bioactivity.

Antimicrobial peptides (AMPs) represent a diverse group of biologically active molecules that are part of the innate immune systems of a variety of organisms. Their primary function consists of protecting the host organism against invading microorganisms, including pathogens. AMPs show a broad spectrum of secondary structures, which are essential for antimicrobial activity. In this study, we produced snakin-2 (SN2), a 66-amino-acid-(aa)-long AMP from Solanum lycopersicum as a recombinant protein in E. coli. This AMP belongs to the GASA/GAST protein family and possesses a highly conserved 60-aa-long domain with six disulfide bonds in the C-terminus of the peptide. Because of the toxicity of SN2 against its producing E. coli strain, the AMP was attached to an N-terminal fusion protein (thioredoxin A), which was removed after affinity chromatography purification. The total yield of recombinant SN2 was approximately 1 mg/L. The membrane-active SN2 showed a bactericidal and fungicidal bioactivity, which can be explained by perforation of biomembranes of bacteria and fungi.

Lifetimes of Arabidopsis cryptochrome signaling states in vivo.

One crucial component in light signaling is the quantity of photoreceptor present in the active signaling state. The lifetime of the signaling state of a photoreceptor is limited because of thermal or otherwise back reversion of the chromophore to the ground state, and/or degradation of the photoreceptor in the light-activated state. It was previously shown that the lit state of plant cryptochromes contains flavin-neutral semiquinone, and that the half-lives of the lit state were in the range of 3-4 min in vitro. However, it was unknown how long-lived the signaling states of plant cryptochromes are in situ. Based on the loss of degradation of cry2 after prolonged dark incubation and loss of reversibility of photoactivated cry1 by a pulse of green light, we estimate the in vivo half-lives of the signaling states of cry1 and cry2 to be in the range of 5 and 16 min, respectively. Based on electron paramagnetic resonance measurements, the lifetime of the Arabidopsis cry1 lit state in insect cells was found to be ~6 min, and thus very similar to the lifetime of the signaling state in planta. Thus, the signaling state lifetimes of plant cryptochromes are not, or are only moderately, stabilized in planta.