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T-cell-engaging bispecifics have great clinical potential for the treatment of cancer and infectious diseases. The binding affinity and kinetics of a bispecific molecule for both target and T-cell CD3 have substantial effects on potency and specificity, but the rules governing these relationships are not fully understood. Using immune mobilizing monoclonal TCRs against cancer (ImmTAC) molecules as a model, we explored the impact of altering affinity for target and CD3 on the potency and specificity of the redirected T-cell response. This class of bispecifics binds specific target peptides presented by human leukocyte antigen on the cell surface via an affinity-enhanced T-cell receptor and can redirect T-cell activation with an anti-CD3 effector moiety. The data reveal that combining a strong affinity TCR with an intermediate affinity anti-CD3 results in optimal T-cell activation, while strong affinity of both targeting and effector domains significantly reduces maximum cytokine release. Moreover, by optimizing the affinity of both parts of the molecule, it is possible to improve the selectivity. These results could be effectively modelled based on kinetic proofreading with limited signalling. This model explained the experimental observation that strong binding at both ends of the molecules leads to reduced activity, through very stable target-bispecific-effector complexes leading to CD3 entering a non-signalling dark state. These findings have important implications for the design of anti-CD3-based bispecifics with optimal biophysical parameters for both activity and specificity., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Immunology.)

Purpose: Tumor infiltration by different T lymphocyte subsets is known to be associated with favorable prognosis in colorectal cancer. Still debated is the role of innate immune system. We investigated clinical relevance, phenotypes, and functional features of colorectal cancer-infiltrating CD66b + neutrophils and their crosstalk with CD8 + T cells. Experimental Design: CD66b + and CD8 + cell infiltration was analyzed by IHC on a tissue microarray including >650 evaluable colorectal cancer samples. Phenotypic profiles of tissue-infiltrating and peripheral blood CD66b + cells were evaluated by flow cytometry. CD66b + /CD8 + cells crosstalk was investigated by in vitro experiments. Results: CD66b + cell infiltration in colorectal cancer is significantly associated with increased survival. Interestingly, neutrophils frequently colocalize with CD8 + T cells in colorectal cancer. Functional studies indicate that although neutrophils are devoid of direct antitumor potential, coculture with peripheral blood or tumor-associated neutrophils (TAN) enhances CD8 + T-cell activation, proliferation, and cytokine release induced by suboptimal concentrations of anti-CD3 mAb. Moreover, under optimal activation conditions, CD8 + cell stimulation in the presence of CD66b + cells results in increasing numbers of cells expressing CD45RO/CD62L "central memory" phenotype. Importantly, combined tumor infiltration by CD66b + and CD8 + T lymphocytes is associated with significantly better prognosis, as compared with CD8 + T-cell infiltration alone. Conclusions: Neutrophils enhance the responsiveness of CD8 + T cells to T-cell receptor triggering. Accordingly, infiltration by neutrophils enhances the prognostic significance of colorectal cancer infiltration by CD8 + T cells, suggesting that they might effectively promote antitumor immunity. Clin Cancer Res; 23(14); 3847-58. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Background: OX40 is a TNF receptor family member expressed by activated T cells. Its triggering by OX40 ligand promotes lymphocyte survival and memory generation. Anti-OX40 agonistic monoclonal antibodies (mAb) are currently being tested in cancer immunotherapy. We explored the prognostic significance of tumor infiltration by OX40+ cells in a large colorectal cancer (CRC) collective., Methods: OX40 gene expression was analyzed in 50 freshly excised CRC and corresponding healthy mucosa by qRT-PCR. A tissue microarray including 657 clinically annotated CRC specimens was stained with anti-OX40, -CD8 and -FOXP3 mAbs by standard immunohistochemistry. The CRC cohort was randomly split into training and validation sets. Correlations between CRC infiltration by OX40+ cells alone, or in combination with CD8+ or FOXP3+ cells, and clinical-pathological data and overall survival were comparatively evaluated., Results: OX40 gene expression in CRC significantly correlated with FOXP3 and CD8 gene expression. High CRC infiltration by OX40+ cells was significantly associated with favorable prognosis in training and validation sets in univariate, but not multivariate, Cox regression analysis. CRC with OX40(high)/CD8(high) infiltration were characterized by significantly prolonged overall survival, as compared to tumors with OX40(low)/CD8(high), OX40(high)/CD8(low) or OX40(low)/CD8(low) infiltration in both uni- and multivariate analysis. In contrast, prognostic significance of OX40+ and FOXP3+ cell infiltration was not enhanced by a combined evaluation. Irrespective of TNM stage, CRC with OX40(high)/CD8(high) density infiltrates showed an overall survival similar to that of all stage I CRC included in the study., Conclusions: OX40(high)/CD8(high) density tumor infiltration represents an independent, favorable, prognostic marker in CRC with an overall survival similar to stage I cancers.

Colorectal cancer (CRC) infiltration by cells expressing myeloperoxidase (MPO) or CD8 positive T lymphocytes has been shown to be independently associated with favorable prognosis. We explored the relationship occurring between CD8+ and MPO+ cell CRC infiltration, its impact on clinical-pathological features and its prognostic significance in a tissue microarray (TMA) including 1,162 CRC. We observed that CRC showing high MPO+ cell infiltration are characterized by a prognosis as favorable as that of cancers with high CD8+ T cell infiltration. However, MPO+ and CD8+ CRC infiltrating cells did not synergize in determining a more favorable outcome, as compared with cancers showing MPO high /CD8 low or MPO low /CD8 high infiltrates. Most importantly, we identified a subgroup of CRC with MPO low /CD8 low tumor infiltration characterized by a particularly severe prognosis. Intriguingly, although MPO+ and CD8+ cells did not co-localize in CRC infiltrates, an increased expression of TIA-1 and granzyme-B was detectable in T cells infiltrating CRC with high MPO+ cell density.

Purpose: Colorectal cancer infiltration by CD16(+) myeloid cells correlates with improved prognosis. We addressed mechanistic clues and gene and protein expression of cytokines potentially associated with macrophage polarization., Experimental Design: GM-CSF or M-CSF-stimulated peripheral blood CD14(+) cells from healthy donors were cocultured with colorectal cancer cells. Tumor cell proliferation was assessed by (3)H-thymidine incorporation. Expression of cytokine genes in colorectal cancer and autologous healthy mucosa was tested by quantitative, real-time PCR. A tumor microarray (TMA) including >1,200 colorectal cancer specimens was stained with GM-CSF- and M-CSF-specific antibodies. Clinicopathological features and overall survival were analyzed., Results: GM-CSF induced CD16 expression in 66% ± 8% of monocytes, as compared with 28% ± 1% in cells stimulated by M-CSF (P = 0.011). GM-CSF but not M-CSF-stimulated macrophages significantly (P < 0.02) inhibited colorectal cancer cell proliferation. GM-CSF gene was expressed to significantly (n = 45, P < 0.0001) higher extents in colorectal cancer than in healthy mucosa, whereas M-CSF gene expression was similar in healthy mucosa and colorectal cancer. Accordingly, IL1β and IL23 genes, typically expressed by M1 macrophages, were expressed to significantly (P < 0.001) higher extents in colorectal cancer than in healthy mucosa. TMA staining revealed that GM-CSF production by tumor cells is associated with lower T stage (P = 0.02), "pushing" growth pattern (P = 0.004) and significantly (P = 0.0002) longer survival in mismatch-repair proficient colorectal cancer. Favorable prognostic effect of GM-CSF production by colorectal cancer cells was confirmed by multivariate analysis and was independent from CD16(+) and CD8(+) cell colorectal cancer infiltration. M-CSF expression had no significant prognostic relevance., Conclusions: GM-CSF production by tumor cells is an independent favorable prognostic factor in colorectal cancer., (©2014 American Association for Cancer Research.)

Aims: A trend towards local excision of early rectal cancers has prompted us to investigate if immunoprofiling might help in predicting lymph node involvement in this subgroup., Methods: A tissue microarray of 126 biopsies of early rectal cancer (T1 and T2) was stained for several immunomarkers of the innate and the adaptive immune response. Patients' survival and nodal status were analyzed and correlated with infiltration of the different immune cells., Results: Of all tested markers, only CD8 (P = 0.005) and TIA-1 (P = 0.05) were significantly more frequently detectable in early rectal cancer biopsies of node negative as compared to node positive patients. Although these two immunomarkers did not display prognostic effect "per se," CD8+ and, marginally, TIA-1 T cell infiltration could predict nodal involvement in univariate logistic regression analysis (OR 0.994; 95% CI 0.992-0.996; P = 0.009 and OR 0.988; 95% CI 0.984-0.994; P = 0.05, resp.). An algorithm significantly predicting the nodal status in early rectal cancer based on CD8 together with vascular invasion and tumor border configuration could be calculated (P < 0.00001)., Conclusion: Our data indicate that in early rectal cancers absence of CD8+ T-cell infiltration helps in predicting patients' nodal involvement.

Background: Colorectal cancer (CRC) infiltration by adaptive immune system cells correlates with favorable prognosis. The role of the innate immune system is still debated. Here we addressed the prognostic impact of CRC infiltration by neutrophil granulocytes (NG)., Methods: A TMA including healthy mucosa and clinically annotated CRC specimens (n = 1491) was stained with MPO and CD15 specific antibodies. MPO+ and CD15+ positive immune cells were counted by three independent observers. Phenotypic profiles of CRC infiltrating MPO+ and CD15+ cells were validated by flow cytometry on cell suspensions derived from enzymatically digested surgical specimens. Survival analysis was performed by splitting randomized data in training and validation subsets., Results: MPO+ and CD15+ cell infiltration were significantly correlated (p<0.0001; r = 0.76). However, only high density of MPO+ cell infiltration was associated with significantly improved survival in training (P = 0.038) and validation (P = 0.002) sets. In multivariate analysis including T and N stage, vascular invasion, tumor border configuration and microsatellite instability status, MPO+ cell infiltration proved an independent prognostic marker overall (P = 0.004; HR = 0.65; CI:±0.15) and in both training (P = 0.048) and validation (P = 0.036) sets. Flow-cytometry analysis of CRC cell suspensions derived from clinical specimens showed that while MPO+ cells were largely CD15+/CD66b+, sizeable percentages of CD15+ and CD66b+ cells were MPO-., Conclusions: High density MPO+ cell infiltration is a novel independent favorable prognostic factor in CRC.