Rationalizing the pathway to personalized neoadjuvant immunotherapy: the Lombard Street Approach

Neoadjuvant chemo(radio)therapy is part of the established standard of care in cancer treatment; neoadjuvant application of immunotherapy, however, is only performed within recent trials. Combination of programmed cell death protein 1 and cytotoxic T lymphocyte antigen 4 blockade shows promising results with high pathologic response rates in the neoadjuvant setting and a very low relapse rate in the responding patients. In addition, neoadjuvant administration allows direct determination of treatment efficacy within the individual patient, and offers easy access to paired tumor material, both pretherapy and post-therapy, thus facilitates the rational development of new combinations driven by preclinical analyses. Patient-derived human tumor explant systems such as a recently developed human patient-derived tumor fragment platform can provide an additional tool to further rationalize the development of new treatment combinations. We will discuss neoadjuvant immunotherapy as a unique opportunity for rational trial design, the development of immune signatures for non-responding patients to steer clinical trial development, and the use of patient-derived ex vivo models to identify new personalized immunotherapy combinations. In this context, we propose the 'Lombard Street Approach', a back and forth approach of characterizing non-responders on neoadjuvant immunotherapy combinations, identifying promising new combinations for this group in the tumor fragment platform, and performing subsequently signature-driven small proof-of-concept combination trials. Repeating this approach with smaller and smaller groups of non-responders will step by step increase the percentage of patients benefiting from neoadjuvant immunotherapy in a rational and fast manner.</p

A transcriptionally and functionally distinct PD-1⁺ CD8⁺ T cell pool with predictive potential in non-small-cell lung cancer treated with PD-1 blockade.

Evidence from mouse chronic viral infection models suggests that CD8 &lt;sup&gt;+&lt;/sup&gt; T cell subsets characterized by distinct expression levels of the receptor PD-1 diverge in their state of exhaustion and potential for reinvigoration by PD-1 blockade. However, it remains unknown whether T cells in human cancer adopt a similar spectrum of exhausted states based on PD-1 expression levels. We compared transcriptional, metabolic and functional signatures of intratumoral CD8 &lt;sup&gt;+&lt;/sup&gt; T lymphocyte populations with high (PD-1 &lt;sup&gt;T&lt;/sup&gt; ), intermediate (PD-1 &lt;sup&gt;N&lt;/sup&gt; ) and no PD-1 expression (PD-1 &lt;sup&gt;-&lt;/sup&gt; ) from non-small-cell lung cancer patients. PD-1 &lt;sup&gt;T&lt;/sup&gt; T cells showed a markedly different transcriptional and metabolic profile from PD-1 &lt;sup&gt;N&lt;/sup&gt; and PD-1 &lt;sup&gt;-&lt;/sup&gt; lymphocytes, as well as an intrinsically high capacity for tumor recognition. Furthermore, while PD-1 &lt;sup&gt;T&lt;/sup&gt; lymphocytes were impaired in classical effector cytokine production, they produced CXCL13, which mediates immune cell recruitment to tertiary lymphoid structures. Strikingly, the presence of PD-1 &lt;sup&gt;T&lt;/sup&gt; cells was strongly predictive for both response and survival in a small cohort of non-small-cell lung cancer patients treated with PD-1 blockade. The characterization of a distinct state of tumor-reactive, PD-1-bright lymphocytes in human cancer, which only partially resembles that seen in chronic infection, provides potential avenues for therapeutic intervention

Single-cell analysis of regions of interest (SCARI) using a photosensitive tag

The functional activity and differentiation potential of cells are determined by their interactions with surrounding cells. Approaches that allow unbiased characterization of cell states while at the same time providing spatial information are of major value to assess this environmental influence. However, most current techniques are hampered by a tradeoff between spatial resolution and cell profiling depth. Here, we develop a photocage-based technology that allows isolation and in-depth analysis of live cells from regions of interest in complex ex vivo systems, including primary human tissues. The use of a highly sensitive 4-nitrophenyl(benzofuran) cage coupled to a set of nanobodies allows high-resolution photo-uncaging of different cell types in areas of interest. Single-cell RNA-sequencing of spatially defined CD8+ T cells is used to exemplify the feasibility of identifying location-dependent cell states. The technology described here provides a valuable tool for the analysis of spatially defined cells in diverse biological systems, including clinical samples.ERC Cog KineTic grant 865175Bio-organic Synthesi

Addition of interleukin-2 overcomes resistance to neoadjuvant CTLA4 and PD1 blockade in ex vivo patient tumors

Neoadjuvant immunotherapy with anti-cytotoxic T lymphocyte-associated protein 4 (CTLA4) + anti-programmed cell death protein 1 (PD1) monoclonal antibodies has demonstrated remarkable pathological responses and relapse-free survival in -80% of patients with clinically detectable stage III melanoma. However, about 20% of the treated patients do not respond. In pretreatment biopsies of patients with melanoma, we found that resistance to neoadjuvant CTLA4 + PD1 blockade was associated with a low CD4/interleukin-2 (IL-2) gene signature. Ex vivo, addition of IL-2 to CTLA4 + PD1 blockade induced T cell activation and deep immunological responses in anti-CTLA4 + anti-PD1-resistant human tumor specimens. In the 4T1.2 breast cancer mouse model of neoadjuvant immunotherapy, triple combination of anti-CTLA4 + anti-PD1 + IL-2 cured almost twice as many mice as compared with dual checkpoint inhibitor therapy. This improved efficacy was due to the expansion of tumor-specific CD8(+) T cells and improved proinflammatory cytokine polyfunctionality of both CD4(+) and CD8(+) T effector cells and regulatory T cells. Depletion studies suggested that CD4(+) T cells were critical for priming of CD8(+) T cell immunity against 4T1.2 and helped in the expansion of tumor-specific CD8(+) T cells early after neoadjuvant triple immunotherapy. Our results suggest that the addition of IL-2 can overcome resistance to neoadjuvant anti-CTLA4 +anti-PD1, providing the rationale for testing this combination as a neoadjuvant therapy in patients with early-stage cancer

PD-1T TILs as a Predictive Biomarker for Clinical Benefit to PD-1 Blockade in Patients with Advanced NSCLC

Purpose: Durable clinical benefit to PD-1 blockade in non- small cell lung cancer (NSCLC) is currently limited to a small fraction of patients, underlining the need for predictive biomar-kers. We recently identified a tumor-reactive tumor-infiltrating T lymphocyte (TIL) pool, termed PD-1T TILs, with predictive potential in NSCLC. Here, we examined PD-1T TILs as biomarker in NSCLC.Experimental Design: PD-1T TILs were digitally quantified in 120 baseline samples from advanced NSCLC patients treated with PD-1 blockade. Primary outcome was disease control (DC) at 6 months. Secondary outcomes were DC at 12 months and survival. Exploratory analyses addressed the impact of lesion-specific responses, tissue sample properties, and combination with other biomarkers on the predictive value of PD-1T TILs.Results: PD-1T TILs as a biomarker reached 77% sensitivity and 67% specificity at 6 months, and 93% and 65% at 12 months,respectively. Particularly, a patient group without clinical benefit was reliably identified, indicated by a high negative predictive value (NPV) (88% at 6 months, 98% at 12 months). High PD-1T TILs related to significantly longer progression-free (HR 0.39, 95% CI, 0.24-0.63, P < 0.0001) and overall survival (HR 0.46, 95% CI, 0.28-0.76, P < 0.01). Predictive performance was increased when lesion-specific responses and samples obtained immediately before treatment were assessed. Notably, the pre-dictive performance of PD-1T TILs was superior to PD-L1 and tertiary lymphoid structures in the same cohort.Conclusions: This study established PD-1T TILs as predictive biomarker for clinical benefit to PD-1 blockade in patients with advanced NSCLC. Most importantly, the high NPV demon-strates an accurate identification of a patient group without benefit