Cannabinoid Receptors Are Overexpressed in CLL but of Limited Potential for Therapeutic Exploitation

<div><p>The cannabinoid receptors 1 and 2 (CNR1&2) are overexpressed in a variety of malignant diseases and cannabinoids can have noteworthy impact on tumor cell viability and tumor growth. Patients diagnosed with chronic lymphocytic leukemia (CLL) present with very heterogeneous disease characteristics translating into highly differential risk properties. To meet the urgent need for refinement in risk stratification at diagnosis and the search for novel therapies we studied CNR expression and response to cannabinoid treatment in CLL. Expression levels of CNR1&2 were determined in 107 CLL patients by real-time PCR and analyzed with regard to prognostic markers and survival. Cell viability of primary CLL cells was determined in suspension and co-culture after incubation in increasing cannabinoid concentrations under normal and reduced serum conditions and in combination with fludarabine. Impact of cannabinoids on migration of CLL cells towards CXCL12 was determined in transwell plates. We found CNR1&2 to be overexpressed in CLL compared to healthy B-cells. Discriminating between high and low expressing subgroups, only high CNR1 expression was associated with two established high risk markers and conferred significantly shorter overall and treatment free survival. Viability of CLL primary cells was reduced in a dose dependent fashion upon incubation with cannabinoids, however, healthy cells were similarly affected. Under serum reduced conditions, no significant differences were observed within suspension and co-culture, respectively, however, the feeder layer contributed significantly to the survival of CLL cells compared to suspension culture conditions. No significant differences were observed when treating CLL cells with cannabinoids in combination with fludarabine. Interestingly, biologic activity of cannabinoids was independent of both CNR1&2 expression. Finally, we did not observe an inhibition of CXCL12-induced migration by cannabinoids. In contrast to other tumor entities, our data suggest a limited usability of cannabinoids for CLL therapy. Nonetheless, we could define CNR1 mRNA expression as novel prognostic marker.</p></div

High CNR1 mRNA expression (≥ 1.52) confers significantly shorter survival in CLL patients (n = 107).

<p>(A) High expressing patients had a mean overall survival (OS) of 153 months compared to 277 months in low expressing patients (p = 0.001). (B) The mean treatment free survival (TFS) was 75 months in the CNR1 high group vs. 150 months in the CNR1 low group (p<0.0001).</p

Comparison of patient characteristics between CNR1 high and low mRNA expressing groups.

<p>Comparison of patient characteristics between CNR1 high and low mRNA expressing groups.</p

Impact of cannabinoids on CLL cell migration.

<p>B-cell enriched primary cells of 7 CLL patients (97.7% ± 1.04 CD19+CD5+) were incubated in transwell plates for 4h before the number of migrated cells was determined. Control experiments included CXCL12 alone (control), no CXCL12 (control w/o CXCL12), incubation with vehicle (DMSO, ethanol), and incubation with the CXCR4 inhibitor AMD3100. CLL cells were incubated either with agonist (ACEA, JWH133), antagonist (AM251, AM630), or a combination of antagonist plus agonist before migration (CB1: AMS251&ACEA; CB2: AM630&JWH133). Bars represent the mean values of migration indices + standard deviations, hatched lines indicate experimental blocks. *p = 0.0006; **p<0.0001.</p

Cytotoxic impact of cannabinoids on CLL primary cells.

<p>PBMC from CLL patients were incubated in triplicates both in suspension culture and in co-culture with M2-10B4 mouse fibroblast cells in increasing concentrations of compounds. Viability was determined after 48h, mean values and standard deviations are shown. (A) (R)-(+)-methanandamide (N = 10). (B) (-)-cannabidiol (N = 18). (C) ACEA (N = 16). (D) JWH133 (N = 16). (E) AM251 (N = 16). (F) AM630 (N = 16). For ACEA, JWH133, and AM251, the 50% reduction in viability required for IC₅₀ calculation could not be reached in co-culture. Note different scale on x-axis in A and D.</p

Cytotoxic impact of cannabinoids on primary cells from healthy individuals.

<p>PBMC from 3 healthy donors were incubated in triplicates in suspension culture in increasing concentrations of compounds. Viability was determined after 48h, mean values and standard deviations are shown. (A) (R)-(+)-methanandamide. (B) (-)-cannabidiol. (C) ACEA. (D) JWH133. (E) AM251. (F) AM630. Note different scale on x-axis in A and D, note different scale on y-axis in D.</p

P-values of the pairwise comparison of IC₅₀ values between suspension and co-culture in serum reduced experiments.

<p>P-values of the pairwise comparison of IC₅₀ values between suspension and co-culture in serum reduced experiments.</p

DataSheet_1_A chimeric antigen receptor-based cellular safeguard mechanism for selective in vivo depletion of engineered T cells.pdf

Adoptive immunotherapy based on chimeric antigen receptor (CAR)-engineered T cells has exhibited impressive clinical efficacy in treating B-cell malignancies. However, the potency of CAR-T cells carriethe potential for significant on-target/off-tumor toxicities when target antigens are shared with healthy cells, necessitating the development of complementary safety measures. In this context, there is a need to selectively eliminate therapeutically administered CAR-T cells, especially to revert long-term CAR-T cell-related side effects. To address this, we have developed an effective cellular-based safety mechanism to specifically target and eliminate the transferred CAR-T cells. As proof-of-principle, we have designed a secondary CAR (anti-CAR CAR) capable of recognizing a short peptide sequence (Strep-tag II) incorporated into the hinge domain of an anti-CD19 CAR. In in vitro experiments, these anti-CAR CAR-T cells have demonstrated antigen-specific cytokine release and cytotoxicity when co-cultured with anti-CD19 CAR-T cells. Moreover, in both immunocompromised and immunocompetent mice, we observed the successful depletion of anti-CD19 CAR-T cells when administered concurrently with anti-CAR CAR-T cells. We have also demonstrated the efficacy of this safeguard mechanism in a clinically relevant animal model of B-cell aplasia induced by CD19 CAR treatment, where this side effect was reversed upon anti-CAR CAR-T cells infusion. Notably, efficient B-cell recovery occurred even in the absence of any pre-conditioning regimens prior anti-CAR CAR-T cells transfer, thus enhancing its practical applicability. In summary, we developed a robust cellular safeguard system for selective in vivo elimination of engineered T cells, offering a promising solution to address CAR-T cell-related on-target/off-tumor toxicities.</p

Ex Vivo <i>trans</i>-V(D)J Recombination Assay

<div><p>(A) Schematic representation of <i>trans</i>-V(D)J recombination between an ESJ (donor) and a 12-RSS (target). The expected breakpoints (one SJ and one ΨHJ) are shown. The various donor/target combinations assayed are boxed. PCR primers are depicted by arrows. Nested IR800-labeled PE primers are indicated by an asterisk.</p> <p>(B) Typical example of PCR/PE assays obtained from the (Dβ1Δ) ESJ × Jβ2.7 12-RSS combination. The expected sizes of the PE products are indicated. As the resolution of the PE assay is to the base, ΨHJ patterns typically show multiple bands (corresponding to the spectra of nucleotide addition and deletion), while SJ patterns are typically centered on a major band (corresponding to no or limited nucleotide processing). PE assays shown were performed on 0.75 μl PCR. Five independent PCRs from two independent transfections performed with (T3–T4) or without RAG-1/2 (T1–T2) are shown for each junction.</p></div

Breakpoint Sequences of Ex Vivo <i>trans</i>-V(D)J Recombinants

<p>SJ (primer combination 3 + 2). Top and bottom lanes depict sequences before recombination (heptamers, spacers and nonamers are specified). Recombined clones are depicted between top and bottom lanes, with homology to the unrecombined sequences indicated by vertical lines. Lower cases represent reactive RSS involved in the <i>trans</i>-V(D)J recombination reactions (specified as donor RSS and target RSS). Upper cases represent coding segments, or bystander RSSs in the ESJ behaving like coding segments (specified as bystander RSSs). Italics indicate potential P nucleotides; bold type, N nucleotides; heptamers and nonamers are underlined; identical sequences shown on separate lanes are issued from distinct transfections and represent therefore independent junctions. Since identical sequences were often obtained in a given transfection, this representation might in some cases bias the representation towards processed junctions (because they acquire distinguishing features).</p