SCMTHP: A New Approach for Identifying and Characterizing of Tumor-Homing Peptides Using Estimated Propensity Scores of Amino Acids

Tumor-homing peptides (THPs) are small peptides that can recognize and bind cancer cells specifically. To gain a better understanding of THPs’ functional mechanisms, the accurate identification and characterization of THPs is required. Although some computational methods for in silico THP identification have been proposed, a major drawback is their lack of model interpretability. In this study, we propose a new, simple and easily interpretable computational approach (called SCMTHP) for identifying and analyzing tumor-homing activities of peptides via the use of a scoring card method (SCM). To improve the predictability and interpretability of our predictor, we generated propensity scores of 20 amino acids as THPs. Finally, informative physicochemical properties were used for providing insights on characteristics giving rise to the bioactivity of THPs via the use of SCMTHP-derived propensity scores. Benchmarking experiments from independent test indicated that SCMTHP could achieve comparable performance to state-of-the-art method with accuracies of 0.827 and 0.798, respectively, when evaluated on two benchmark datasets consisting of Main and Small datasets. Furthermore, SCMTHP was found to outperform several well-known machine learning-based classifiers (e.g., decision tree, k-nearest neighbor, multi-layer perceptron, naive Bayes and partial least squares regression) as indicated by both 10-fold cross-validation and independent tests. Finally, the SCMTHP web server was established and made freely available online. SCMTHP is expected to be a useful tool for rapid and accurate identification of THPs and for providing better understanding on THP biophysical and biochemical properties.

A Dual-reporter Platform for Screening Tumor-targeted Extracellular Vesicles

Extracellular vesicle (EV)-mediated transfer of biomolecules plays an essential role in intercellular communication and may improve targeted drug delivery. In the past decade, various approaches to EV surface modification for targeting specific cells or tissues have been proposed, including genetic engineering of parental cells or postproduction EV engineering. However, due to technical limitations, targeting moieties of engineered EVs have not been thoroughly characterized. Here, we report the bioluminescence resonance energy transfer (BRET) EV reporter, PalmReNL-based dual-reporter platform for characterizing the cellular uptake of tumor homing peptide (THP)-engineered EVs, targeting PDL1, uPAR, or EGFR proteins expressed in MDA-MB-231 breast cancer cells, simultaneously by bioluminescence measurement and fluorescence microscopy. Bioluminescence analysis of cellular EV uptake revealed the highest binding efficiency of uPAR-targeted EVs, whereas PDL1-targeted EVs showed slower cellular uptake. EVs engineered with two known EGFR-binding peptides via lipid nanoprobes did not increase cellular uptake, indicating that designs of EGFR-binding peptide conjugation to the EV surface are critical for functional EV engineering. Fluorescence analysis of cellular EV uptake allowed us to track individual PalmReNL-EVs bearing THPs in recipient cells. These results demonstrate that the PalmReNL-based EV assay platform can be a foundation for high-throughput screening of tumor-targeted EVs.

Mesenchymal stem/stromal cells in cancer therapy

The multipotent mesenchymal stem/stromal cells (MSCs), initially discovered from bone marrow in 1976, have been identified in nearly all tissues of human body now. The multipotency of MSCs allows them to give rise to osteocytes, chondrocytes, adipocytes, and other lineages. Moreover, armed with the immunomodulation capacity and tumor-homing property, MSCs are of special relevance for cell-based therapies in the treatment of cancer. However, hampered by lack of knowledge about the controversial roles that MSC plays in the crosstalk with tumors, limited progress has been made with regard to translational medicine. Therefore, in this review, we discuss the prospects of MSC-associated anticancer strategies in light of therapeutic mechanisms and signal transduction pathways. In addition, the clinical trials designed to appraise the efficacy and safety of MSC-based anticancer therapies will be assessed according to published data.

CD30-Directed CAR-T Cells Co-Expressing CCR4 in Relapsed/Refractory Hodgkin Lymphoma and CD30+ Cutaneous T Cell Lymphoma

Chimeric antigen receptor T cells targeting CD30 (CD30.CAR-T) have shown high response rates, including some durable remissions, in patients with relapsed/refractory (r/r) classical Hodgkin lymphoma (HL) (Ramos et al., JCO 2020). However, some patients are non-responders or eventually relapse after therapy. Because CD30 expression is retained in relapsing tumors, recurrence may be due to the insufficient persistence of CAR-Ts within the highly immunosuppressive tumor microenvironment of HL. We therefore reasoned that with enhanced trafficking to the tumor site, CD30.CAR-Ts would have increased opportunities to eliminate tumors before inhibitory mechanisms become predominant. This is especially relevant for HL, where Hodgkin Reed Sternberg (HRS) cells produce CCL17 (thymus and activation-regulated chemokine) to create a physical and inhibitory barrier to cytotoxic T cells. We have previously shown that CD30.CAR-Ts co-expressing the cognate receptor for CCL17, CCR4 (CCR4.CD30.CAR-Ts), have improved tumor homing and anti-lymphoma activity compared with CD30.CAR-Ts that do not express CCR4 (Di Stasi et al., Blood 2009). CCR4.CD30.CAR-Ts should also be more effective in CD30+ cutaneous T-cell lymphomas (CTCL) due to enhanced trafficking to the skin. We present the preliminary results of a clinical trial assessing the safety (primary objective) of this novel strategy and its efficacy compared to CD30.CAR-Ts lacking CCR4 in patients with r/r HL and CD30+ CTCL (NCT03602157). CCR4.CD30.CAR-Ts are infused in patients in a dose escalation fashion (DL1=2x10 7 CAR-Ts/m 2, DL3=5x10 7 CAR-Ts/m 2, DL5=1x10 8 CAR-Ts/m 2,). To provide definitive conclusions on the role of CAR-T tumor homing, after completion of each dose level, patients receive the dose of CCR4.CD30.CAR-Ts established to be safe in the prior DL, combined with a fixed dose of CD30.CAR-Ts (1x10 8 CAR-Ts/m 2) (DL2, DL4, DL6). All patients receive lymphodepletion with 3 days of bendamustine 70 mg/m 2 and fludarabine 30 mg/m 2. Key inclusion criteria are age ≥ 18 years and r/r HL or CTCL having failed ≥2 prior therapies. At the time of data cut off (8/1/2021), 6 patients were treated on DL1, 3 patients on DL2, and 3 patients on DL3. The median age is 43.5 (range 27-75) with a median of 5.5 prior lines of therapy (range 2-10). Ten patients had HL and 2 patients had CTCL. All patients had received prior brentuximab vedotin. Eleven patients received prior checkpoint inhibitors, 9 had prior autologous stem cell transplant, and 5 had prior allogeneic stem cell transplant. The treatment was well tolerated with no dose limiting toxicities observed. Two patients had grade 2 cytokine release syndrome (CRS) which resolved with tocilizumab, and 1 had self-limiting grade 1 CRS. None of the treated patients developed immune effector cell-associated neurotoxicity syndrome. All of the 8 patients with HL who have had disease assessment have responded with 6 complete responses (CR) (75%) and 2 partial responses (PR). Five patients are in remission to date, with one patient still in CR at 2.5 years post treatment. Two patients with HL have responses pending at time of data cut off. Among the 2 patients with CTCL, 1 patient had stable disease and went on to receive alternative therapy and 1 patient had progressive disease. At a median follow up of 12.7 months, the median progression free survival (PFS) for all 10 evaluable patients is 5.2 months and the median PFS for HL patients has not been reached. The median overall survival for all patients has not been reached. Plasma CCL17, a biomarker of disease response for HL, was reduced by 83±15% by week 2 post infusion in patients treated with CCR4.CD30.CAR-Ts as compared to 52±38% in patients on our previous trial that had received CD30.CAR-Ts lacking CCR4 (p=0.02). In a HL patient on DL1 biopsied 3 weeks post infusion, we found markedly enriched CAR-T signals at the tumor site (14.4 x10 5 copies/ug of DNA) as compared to the signals found at the same time point in the peripheral blood (4.3 x10 5 copies/ug of DNA). Our data confirm the safety of CCR4.CD30.CAR-Ts as well as their promising efficacy in patients with r/r HL. Interestingly, responses are already seen at the lowest dose level, suggesting that early tumor homing driven by CCR4 may allow more fitted cells to better exploit their antitumor potential. Our data serve as a proof of concept for future modifications of CAR-T cells to improve their localization to disease sites. Figure 1 Figure 1. Disclosures Grover: Kite: Other: Advisory Board; Tessa: Consultancy; ADC: Other: Advisory Board; Novartis: Consultancy; Genentech: Research Funding. Morrison: Vesselon: Consultancy. Dittus: BeiGene: Other: Advisory Board; Seattle Genetics: Research Funding; AstraZeneca: Research Funding; Genentech: Research Funding. Dotti: Tessa: Patents & Royalties: Approach for CD30.CAR-T Cells for Hodgkin Lymphoma. Serody: Tessa: Patents & Royalties: Approach for CD30.CAR-T Cells for Hodgkin Lymphoma. Savoldo: Tessa: Patents & Royalties: Approach for CD30.CAR-T Cells for Hodgkin Lymphoma.

Selective Display of a Chemoattractant Agonist on Cancer Cells Activates the Formyl Peptide Receptor 1 on Immune Cells

Current immunotherapeutics often work by directing components of the immune system to recognize biomarkers on the surface of cancer cells to generate an immune response. However, variable changes in biomarker distribution and expression can result in uneven patient response. The development of a more universal tumor-homing strategy has the potential to improve selectivity and extend therapy to cancers with decreased expression or absence of specific biomarkers. Here, we designed a bifunctional agent that exploits the inherent acidic microenvironment of most solid tumors to selectively graft the surface of cancer cells with a formyl peptide receptor ligand (FPRL). Our approach is based on the pH(Low) Insertion Peptide (pHLIP), a unique peptide that selectively targets tumors in vivo by anchoring onto cancer cells in a pHdependent manner. We establish that selectively remodeling cancer cells with a pHLIP-based FPRL activates formyl peptide receptors on recruited immune cells, potentially initiating an immune response towards tumors.