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T cell engagers (TCEs) are becoming an integral class of biological therapeutic owing to their highly potent ability to eradicate cancer cells. Nevertheless, the widespread utility of classical CD3-targeted TCEs has been limited by narrow therapeutic index (TI) linked to systemic CD4+ T cell activation and aberrant cytokine release. One attractive approach to circumvent the systemic activation of pan CD3+ T cells and reduce the risk of cytokine release syndrome is to redirect specific subsets of T cells. A promising strategy is the use of peptide-major histocompatibility class I bispecific antibodies (pMHC-IgGs), which have emerged as an intriguing modality of TCE, based on their ability to selectively redirect highly reactive viral-specific effector memory cytotoxic CD8+ T cells to eliminate cancer cells. However, the relatively low frequency of these effector memory cells in human peripheral blood mononuclear cells (PBMCs) may hamper their redirection as effector cells for clinical applications. To mitigate this potential limitation, we report here the generation of a pMHC-IgG derivative known as guided-pMHC-staging (GPS) carrying a covalent fusion of a monovalent interleukin-2 (IL-2) mutein (H16A, F42A). Using an anti-epidermal growth factor receptor (EGFR) arm as a proof-of-concept, tumor-associated antigen paired with a single-chain HLA-A *02:01/CMVpp65 pMHC fusion moiety, we demonstrate in vitro that the IL-2-armored GPS modality robustly expands CMVpp65-specific CD8+ effector memory T cells and induces potent cytotoxic activity against target cancer cells. Similar to GPS, IL-2-armored GPS molecules induce modulated T cell activation and reduced cytokine release profile compared to an analogous CD3-targeted TCE. In vivo we show that IL-2-armored GPS, but not the corresponding GPS, effectively expands grafted CMVpp65 CD8+ T cells from unstimulated human PBMCs in an NSG mouse model. Lastly, we demonstrate that the IL-2-armored GPS modality exhibits a favorable developability profile and monoclonal antibody-like pharmacokinetic properties in human neonatal Fc receptor transgenic mice. Overall, IL-2-armored GPS represents an attractive approach for treating cancer with the potential for inducing vaccine-like antiviral T cell expansion, immune cell redirection as a TCE, and significantly widened TI due to reduced cytokine release.

Summary: The CTLA-4 and PD-1 checkpoints control immune responses and are key targets in immunotherapy. Both pathways are connected via a cis interaction between CD80 and PD-L1, the ligands for CTLA-4 and PD-1, respectively. This cis interaction prevents PD-1-PD-L1 binding but is reversed by CTLA-4 trans-endocytosis of CD80. However, how CTLA-4 selectively removes CD80, but not PD-L1, is unclear. Here, we show CTLA-4-CD80 interactions are unimpeded by PD-L1 and that CTLA-4 binding with CD80 does not displace PD-L1 per se. Rather, both rigidity and bivalency of CTLA-4 molecules are required to orientate CD80 such that PD-L1 interactions are no longer permissible. Moreover, soluble CTLA-4 released PD-L1 only at specific expression levels of CD80 and PD-L1, whereas CTLA-4 trans-endocytosis released PD-L1 in all conditions. These data show that PD-L1 release from CD80 is driven by orientation and bivalent cross-linking of membrane proteins and that trans-endocytosis of CD80 efficiently promotes PD-L1 availability.

Abstract Tumour dendritic cells (DCs) internalise antigen and upregulate CCR7, which directs their migration to tumour-draining lymph nodes (dLN). CCR7 expression is coupled to an activation programme enriched in regulatory molecule expression, including PD-L1. However, the spatio-temporal dynamics of CCR7+ DCs in anti-tumour immune responses remain unclear. Here, we use photoconvertible mice to precisely track DC migration. We report that CCR7+ DCs are the dominant DC population that migrate to the dLN, but a subset remains tumour-resident despite CCR7 expression. These tumour-retained CCR7+ DCs are phenotypically and transcriptionally distinct from their dLN counterparts and heterogeneous. Moreover, they progressively downregulate the expression of antigen presentation and pro-inflammatory transcripts with more prolonged tumour dwell-time. Tumour-residing CCR7+ DCs co-localise with PD-1+CD8+ T cells in human and murine solid tumours, and following anti-PD-L1 treatment, upregulate stimulatory molecules including OX40L, thereby augmenting anti-tumour cytolytic activity. Altogether, these data uncover previously unappreciated heterogeneity in CCR7+ DCs that may underpin a variable capacity to support intratumoural cytotoxic T cells.

Abstract Immune cell dysfunction within the tumor microenvironment (TME) undermines the control of cancer progression. Established tumors contain phenotypically distinct, tumor-specific natural killer (NK) cells; however, the temporal dynamics, mechanistic underpinning and functional significance of the NK cell compartment remains incompletely understood. Here, we use photo-labeling, combined with longitudinal transcriptomic and cellular analyses, to interrogate the fate of intratumoral NK cells. We reveal that NK cells rapidly lose effector functions and adopt a distinct phenotypic state with features associated with tissue residency. NK cell depletion from established tumors did not alter tumor growth, indicating that intratumoral NK cells cease to actively contribute to anti-tumor responses. IL-15 administration prevented loss of function and improved tumor control, generating intratumoral NK cells with both tissue-residency characteristics and enhanced effector function. Collectively, our data reveals the fate of NK cells after recruitment into tumors and provides insight into how their function may be revived.

IntroductionImmuno-oncology (IO) research relies heavily on murine syngeneic tumor models. However, whilst the average age for a cancer diagnosis is 60 years or older, for practical purposes the majority of preclinical studies are conducted in young mice, despite the fact that ageing has been shown to have a significant impact on the immune response.MethodsUsing aged (60-72 weeks old) mice bearing CT26 tumors, we investigated the impact of ageing on tumor growth as well as the immune composition of the tumor and peripheral lymphoid organs.ResultsWe found many differences in the immune cell composition of both the tumor and tumor-draining lymph node between aged and young mice, such as a reduction in the naïve T cell population and a decreased intratumoral CD8/Treg ratio in aged animals. We hypothesized that these differences may contribute to impaired anti-cancer immune responses in aged mice and therefore assessed the anti-tumor efficacy of different IO therapies in aged mice, including both co-stimulation (using an anti-OX40 antibody) and immune checkpoint blockade (using anti-PD-L1 and anti-CTLA-4 antibodies). Whilst aged mice retained the capacity to generate anti-tumor immune responses, these were significantly attenuated when compared to the responses observed in young mice.DiscussionThese differences highlight the importance of age-related immunological changes in assessing and refining the translational insights gained from preclinical mouse models.

IntroductionHumanized mice are emerging as valuable models to experimentally evaluate the impact of different immunotherapeutics on the human immune system. These immunodeficient mice are engrafted with human cells or tissues, that then mimic the human immune system, offering an alternative and potentially more predictive preclinical model. Immunodeficient NSG mice engrafted with human CD34+ cord blood stem cells develop human T cells educated against murine MHC. However, autoimmune graft versus host disease (GvHD), mediated by T cells, typically develops 1 year post engraftment.MethodsHere, we have used the development of GvHD in NSG mice, using donors with HLA alleles predisposed to autoimmunity (psoriasis) to weight in favor of GvHD, as an endpoint to evaluate the relative potency of monoclonal and BiSpecific antibodies targeting PD-1 and CTLA-4 to break immune tolerance.ResultsWe found that treatment with either a combination of anti-PD-1 & anti-CTLA-4 mAbs or a quadrivalent anti-PD-1/CTLA-4 BiSpecific (MEDI8500), had enhanced potency compared to treatment with anti-PD-1 or anti-CTLA-4 monotherapies, increasing T cell activity both in vitro and in vivo. This resulted in accelerated development of GvHD and shorter survival of the humanized mice in these treatment groups commensurate with their on target activity.DiscussionOur findings demonstrate the potential of humanized mouse models for preclinical evaluation of different immunotherapies and combinations, using acceleration of GvHD development as a surrogate of aggravated antigenic T-cell response against host.

The concept of exploiting tumor intrinsic deficiencies in DNA damage repair mechanisms by inhibiting compensatory DNA repair pathways is well established. For example, ATM-deficient cells show increased sensitivity to the ATR inhibitor ceralasertib. DNA damage response (DDR)-deficient cells are also more sensitive to DNA damaging agents like the DNA crosslinker pyrrolobenzodiazepine (PBD) SG-3199. However, additional antitumor benefits from targeting the DDR pathways, which could operate through the activation of the innate immune system are less well studied. DNA accumulation in the cytosol acts as an immunogenic danger signal, inducing the expression of type-I interferon (IFN) stimulated genes (ISGs) by the activation of the cGAS-STING pathway. Here, we demonstrate that ATM −/− FaDu tumor cells have higher basal expression of ISGs when compared to WT cells and respond to ceralasertib and PBD SG-3199 by inducing higher levels of ISGs in a cGAS-STING-dependent manner. We show that sensitive tumor cells treated with ceralasertib and PBD SG-3199 activate dendritic cells (DCs) via a type-I IFN-dependent mechanism. However, STING deficiency in tumor cells does not prevent DC activation, suggesting that transactivation of the STING pathway occurs within DCs. Furthermore, depletion of the cytosolic DNA exonuclease TREX1 in tumor cells increases DC activation in response to PBD SG-3199-treated tumor cells, indicating that an increase in tumor-derived cytosolic DNA may further enhance DC activation. In summary, in this study, we show that ceralasertib and PBD SG-3199 treatment not only intrinsically target tumor cells but also extrinsically increase tumor cell immunogenicity by inducing DC activation, which is enhanced in ATM-deficient cells.

Anti-CTLA-4 antibodies have pioneered the field of tumour immunotherapy. However, despite impressive clinical response data, the mechanism by which anti-CTLA-4 antibodies work is still controversial. Two major checkpoint antibodies (ipilimumab and tremelimumab) have been trialled clinically. Both have high affinity binding to CTLA-4 and occupy the ligand binding site, however recently it has been suggested that in some settings such antibodies may not block ligand-CTLA-4 interactions. Here we evaluated blocking capabilities of these antibodies in a variety of settings using both soluble and cell bound target proteins. We found that when ligands (CD80 or CD86) were expressed on cells, soluble CTLA-4-Ig bound in line with affinity expectations and that this interaction was effectively disrupted by both ipilimumab and tremelimumab antibodies. Similarly, cellular CTLA-4 binding to soluble ligands was comparably prevented. We further tested the ability of these antibodies to block transendocytosis, whereby CTLA-4 captures ligands from target cells during a cognate cell-cell interaction. Once again ipilimumab and tremelimumab were similar in preventing removal of ligand by transendocytosis. Furthermore, even once transendocytosis was ongoing and cell contact was fully established, the addition of these antibodies could prevent further ligand transfer. Together these data indicate that the above checkpoint inhibitors performed in-line with predictions based on affinity and binding site data and are capable of blocking CTLA-4-ligand interactions in a wide range of settings tested.

Lymphocytes are the central actors in adaptive immune responses. When challenged with antigen, a small number of B and T cells have a cognate receptor capable of recognising and responding to the insult. These cells proliferate, building an exponentially growing, differentiating clone army to fight off the threat, before ceasing to divide and dying over a period of weeks, leaving in their wake memory cells that are primed to rapidly respond to any repeated infection. Due to the non-linearity of lymphocyte population dynamics, mathematical models are needed to interrogate data from experimental studies. Due to lack of evidence to the contrary and appealing to arguments based on Occam’s Razor, in these models newly born progeny are typically assumed to behave independently of their predecessors. Recent experimental studies, however, challenge that assumption, making clear that there is substantial inheritance of timed fate changes from each cell by its offspring, calling for a revision to the existing mathematical modelling paradigms used for information extraction. By assessing long-term live-cell imaging of stimulated murine B and T cells in vitro, we distilled the key phenomena of these within-family inheritances and used them to develop a new mathematical model, Cyton2, that encapsulates them. We establish the model’s consistency with these newly observed fine-grained features. Two natural concerns for any model that includes familial correlations would be that it is overparameterised or computationally inefficient in data fitting, but neither is the case for Cyton2. We demonstrate Cyton2’s utility by challenging it with high-throughput flow cytometry data, which confirms the robustness of its parameter estimation as well as its ability to extract biological meaning from complex mixed stimulation experiments. Cyton2, therefore, offers an alternate mathematical model, one that is, more aligned to experimental observation, for drawing inferences on lymphocyte population dynamics.

Abstract Background Immune checkpoint blockade (ICB) promotes adaptive immunity and tumor regression in some cancer patients. However, in patients with immunologically “cold” tumors, tumor-resident innate immune cell activation may be required to prime an adaptive immune response and so exploit the full potential of ICB. Whilst Toll-like receptor (TLR) agonists have been used topically to successfully treat some superficial skin tumors, systemic TLR agonists have not been well-tolerated. Methods The response of human immune cells to TLR7 and 8 agonism was measured in primary human immune cell assays. MEDI9197 (3M-052) was designed as a novel lipophilic TLR7/8 agonist that is retained at the injection site, limiting systemic exposure. Retention of the TLR7/8 agonist at the site of injection was demonstrated using quantitative whole-body autoradiography, HPLC-UV, and MALDI mass spectrometry imaging. Pharmacodynamic changes on T cells from TLR7/8 agonist treated B16-OVA tumors was assessed by histology, quantitative real time PCR, and flow cytometry. Combination activity of TLR7/8 agonism with immunotherapies was assessed in vitro by human DC-T cell MLR assay, and in vivo using multiple syngeneic mouse tumor models. Results Targeting both TLR7 and 8 triggers an innate and adaptive immune response in primary human immune cells, exemplified by secretion of IFNα, IL-12 and IFNγ. In contrast, a STING or a TLR9 agonist primarily induces release of IFNα. We demonstrate that the TLR7/8 agonist, MEDI9197, is retained at the sight of injection with limited systemic exposure. This localized TLR7/8 agonism leads to Th1 polarization, enrichment and activation of natural killer (NK) and CD8+ T cells, and inhibition of tumor growth in multiple syngeneic models. The anti-tumor activity of this TLR7/8 agonist is enhanced when combined with T cell-targeted immunotherapies in pre-clinical models. Conclusion Localized TLR7/8 agonism can enhance recruitment and activation of immune cells in tumors and polarize anti-tumor immunity towards a Th1 response. Moreover, we demonstrate that the anti-tumor effects of this TLR7/8 agonist can be enhanced through combination with checkpoint inhibitors and co-stimulatory agonists.

In vitro assays that evaluate CD8+ T cell-mediated cytotoxicity are important to aid in the development of novel therapeutic approaches to enhance anti-tumor immune responses. Here, we describe a novel cytotoxicity co-culture assay that circumvents the problem of highly variable allogeneic responses and obviates the constraints of HLA-restriction between effector and target cells. We show that this assay can be easily applied to a panel of tumor cell lines to provide additional insights into intrinsic drivers of sensitivity/resistance to T cell-mediated killing, and to evaluate the impact of targeted therapies on both tumor and T cell compartments.

Abstract Background Numerous oncology combination therapies involving modulators of the cancer immune cycle are being developed, yet quantitative simulation models predictive of outcome are lacking. We here present a model-based analysis of tumor size dynamics and immune markers, which integrates experimental data from multiple studies and provides a validated simulation framework predictive of biomarkers and anti-tumor response rates, for untested dosing sequences and schedules of combined radiation (RT) and anti PD-(L)1 therapies. Methods A quantitative systems pharmacology model, which includes key elements of the cancer immunity cycle and the tumor microenvironment, tumor growth, as well as dose-exposure-target modulation features, was developed to reproduce experimental data of CT26 tumor size dynamics upon administration of RT and/or a pharmacological IO treatment such as an anti-PD-L1 agent. Variability in individual tumor size dynamics was taken into account using a mixed-effects model at the level of tumor-infiltrating T cell influx. Results The model allowed for a detailed quantitative understanding of the synergistic kinetic effects underlying immune cell interactions as linked to tumor size modulation, under these treatments. The model showed that the ability of T cells to infiltrate tumor tissue is a primary determinant of variability in individual tumor size dynamics and tumor response. The model was further used as an in silico evaluation tool to quantitatively predict, prospectively, untested treatment combination schedules and sequences. We demonstrate that anti-PD-L1 administration prior to, or concurrently with RT reveal further synergistic effects, which, according to the model, may materialize due to more favorable dynamics between RT-induced immuno-modulation and reduced immuno-suppression of T cells through anti-PD-L1. Conclusions This study provides quantitative mechanistic explanations of the links between RT and anti-tumor immune responses, and describes how optimized combinations and schedules of immunomodulation and radiation may tip the immune balance in favor of the host, sufficiently to lead to tumor shrinkage or rejection.

Abstract Background T-cell checkpoint blockade and MEK inhibitor combinations are under clinical investigation. Despite progress elucidating the immuno-modulatory effects of MEK inhibitors as standalone therapies, the impact of MEK inhibition on the activity of T-cell checkpoint inhibitors remains incompletely understood. Here we sought to characterize the combined effects of MEK inhibition and anti-CTLA-4 mAb (anti-CTLA-4) therapy, examining effects on both T-cells and tumor microenvironment (TME). Methods In mice, the effects of MEK inhibition, via selumetinib, and anti-CTLA-4 on immune responses to keyhole limpet haemocyanin (KLH) immunization were monitored using ex vivo functional assays with splenocytes. In a KRAS-mutant CT26 mouse colorectal cancer model, the impact on the tumor microenvironment (TME) and the spleen were evaluated by flow cytometry. The TME was further examined by gene expression and immunohistochemical analyses. The combination and sequencing of selumetinib and anti-CTLA-4 were also evaluated in efficacy studies using the CT26 mouse syngeneic model. Results Anti-CTLA-4 enhanced the generation of KLH specific immunity following KLH immunization in vivo; selumetinib was found to reduce, but did not prevent, this enhancement of immune response by anti-CTLA-4 in vivo. In the CT26 mouse model, anti-CTLA-4 treatment led to higher expression levels of the immunosuppressive mediators, Cox-2 and Arg1 in the TME. Combination of anti-CTLA-4 with selumetinib negated this up-regulation of Cox-2 and Arg1, reduced the frequency of CD11+ Ly6G+ myeloid cells, and led to the accumulation of differentiating monocytes at the Ly6C+ MHC+ intermediate state in the tumor. We also report that MEK inhibition had limited impact on anti-CTLA-4-mediated increases in T-cell infiltration and T-cell activation in CT26 tumors. Finally, we show that pre-treatment, but not concurrent treatment, with selumetinib enhanced the anti-tumor activity of anti-CTLA-4 in the CT26 model. Conclusion These data provide evidence that MEK inhibition can lead to changes in myeloid cells and immunosuppressive factors in the tumor, thus potentially conditioning the TME to facilitate improved response to anti-CTLA-4 treatment. In summary, the use of MEK inhibitors to alter the TME as an approach to enhance the activities of immune checkpoint inhibitors warrants further investigation in clinical trials.

Peripheral immune regulation is critical for the maintenance of self-tolerance. Here we have investigated signaling processes that distinguish T cells with regulatory capability from effector T cells. The murine Tg4 T cell receptor recognizes a peptide derived from the self-antigen myelin basic protein. T cells from Tg4 T cell receptor transgenic mice can be used to generate effector T cells and three types of T cells with regulatory capability, inducible regulatory T cells, T cells tolerized by repeated in vivo antigenic peptide exposure or T cells treated with the tolerogenic drug UCB9608 (a phosphatidylinositol 4 kinase IIIβ inhibitor). We comparatively studied signaling in all of these T cells by activating them with the same antigen presenting cells presenting the same myelin basic protein peptide. Supramolecular signaling structures, as efficiently detected by large-scale live cell imaging, are critical mediators of T cell activation. The formation of a supramolecular signaling complex anchored by the adaptor protein linker for activation of T cells (LAT) was consistently terminated more rapidly in Tg4 T cells with regulatory capability. Such termination could be partially reversed by blocking the inhibitory receptors CTLA-4 and PD-1. Our work suggests that attenuation of proximal signaling may favor regulatory over effector function in T cells.

ABSTRACTInnovative approaches in the design of T cell-engaging (TCE) molecules are ushering in a new wave of promising immunotherapies for the treatment of cancer. Their mechanism of action, which generates an in trans interaction to create a synthetic immune synapse, leads to complex and interconnected relationships between the exposure, efficacy, and toxicity of these drugs. Challenges thus arise when designing optimal clinical dose regimens for TCEs with narrow therapeutic windows, with a variety of dosing strategies being evaluated to mitigate key side effects such as cytokine release syndrome, neurotoxicity, and on-target off-tumor toxicities. This review evaluates the current approaches to dose optimization throughout the preclinical and clinical development of TCEs, along with perspectives for improvement of these strategies. Quantitative approaches used to aid the understanding of dose-exposure-response relationships are highlighted, along with opportunities to guide the rational design of next-generation TCE molecules, and optimize their dose regimens in patients.

Background The prognosis for patients with pancreatic ductal adenocarcinoma (PDAC) remains extremely poor. It has been suggested that the adenosine pathway contributes to the ability of PDAC to evade the immune system and hence, its resistance to immuno-oncology therapies (IOT), by generating extracellular adenosine (eAdo).Methods Using genetically engineered allograft models of PDAC in syngeneic mice with defined and different immune infiltration and response to IOT and autochthonous tumors in KPC mice we investigated the impact of the adenosine pathway on the PDAC tumor microenvironment (TME). Flow cytometry and imaging mass cytometry (IMC) were used to characterize the subpopulation frequency and spatial distribution of tumor-infiltrating immune cells. Mass spectrometry imaging (MSI) was used to visualize adenosine compartmentalization in the PDAC tumors. RNA sequencing was used to evaluate the influence of the adenosine pathway on the shaping of the immune milieu and correlate our findings to published data sets in human PDAC.Results We demonstrated high expression of adenosine pathway components in tumor-infiltrating immune cells (particularly myeloid populations) in the murine models. MSI demonstrated that extracellular adenosine distribution is heterogeneous in tumors, with high concentrations in peri-necrotic, hypoxic regions, associated with rich myeloid infiltration, demonstrated using IMC. Protumorigenic M2 macrophages express high levels of the Adora2a receptor; particularly in the IOT resistant model. Blocking the in vivo formation and function of eAdo (Adoi), using a combination of anti-CD73 antibody and an Adora2a inhibitor slowed tumor growth and reduced metastatic burden. Additionally, blocking the adenosine pathway improved the efficacy of combinations of cytotoxic agents or immunotherapy. Adoi remodeled the TME, by reducing the infiltration of M2 macrophages and regulatory T cells. RNA sequencing analysis showed that genes related to immune modulation, hypoxia and tumor stroma were downregulated following Adoi and a specific adenosine signature derived from this is associated with a poorer prognosis in patients with PDAC.Conclusions The formation of eAdo promotes the development of the immunosuppressive TME in PDAC, contributing to its resistance to conventional and novel therapies. Therefore, inhibition of the adenosine pathway may represent a strategy to modulate the PDAC immune milieu and improve therapy response in patients with PDAC.

The field of Immuno-Oncology (IO) is evolving to utilise novel antibody backbones that can co-target multiple cell-surface stimulatory and inhibitory co-receptors (SICR). This approach necessitates a better understanding of SICR co-expression at the single-cell level on IO-relevant tumor-infiltrating leukocyte (TIL) cell types such as T and natural killer (NK) cells. Using high-dimensional flow cytometry we established a comprehensive SICR profile for tumor-resident T and NK cells across a range of human solid tumors where there is a clear need for improved immunotherapeutic intervention. Leveraging the power of our large flow panel, we performed deep-phenotyping of the critical CD8+CD39+ Cytotoxic T Lymphocyte (CTL) population that is enriched for tumor-reactive cytotoxic cells, revealing subsets that are differentiated by their SICR profile, including three that are uniquely defined by NKG2A expression. This study establishes a comprehensive SICR phenotype for human TIL T and NK cells, providing insights to guide the design and application of the next generation of IO molecules.

Background Immuno-oncology therapies are now part of the standard of care for cancer in many indications. However, durable objective responses remain limited to a subset of patients. As such, there is a critical need to identify biomarkers that can predict or enrich for treatment response. So far, the majority of putative biomarkers consist of features of the tumor microenvironment (TME). However, in preclinical mouse models, the collection of tumor tissue for this type of analysis is a terminal procedure, obviating the ability to directly link potential biomarkers to long-term treatment outcomes.Methods To address this, we developed and validated a novel non-terminal tumor sampling method to enable biopsy of the TME in mouse models based on fine needle aspiration.Results We show that this technique enables repeated in-life sampling of subcutaneous flank tumors and yields sufficient material to support downstream analyses of tumor-infiltrating immune cells using methods such as flow cytometry and single-cell transcriptomics. Moreover, using this technique we demonstrate that we can link TME biomarkers to treatment response outcomes, which is not possible using the current method of terminal tumor sampling.Conclusion Thus, this minimally invasive technique is an important refinement for the pharmacodynamic analysis of the TME facilitating paired evaluation of treatment response biomarkers with outcomes and reducing the number of animals used in preclinical research.