Programmed cell death protein 1/programmed cell death protein ligand1 (PD-1/PD-L1) interaction is an important immune checkpoint targeted by anti-PD-1/PD-L1 immunotherapies. However, the observed ...prognostic significance of PD-1/PD-L1 expression in diffuse large B-cell lymphoma treated with the standard of care has been inconsistent and even contradictory. To clarify the prognostic role of PD-1/PD-L1 expression and interaction in diffuse large B-cell lymphoma, in this study we used 3-marker fluorescent multiplex immunohistochemistry and Automated Quantitative Analysis Technology to assess the CD3
, PD-L1
, and PD-1
CD3
expression in diagnostic samples and PD-1/PD-L1 interaction as indicated by presence of PD-1
CD3
cells in the vicinity of PD-L1
cells, analyzed their prognostic effects in 414 patients with de novo diffuse large B-cell lymphoma, and examined whether PD-1/PD-L1 interaction is required for the prognostic role of PD-1
/PD-L1
expression. We found that low T-cell tissue cellularity, tissue PD-L1
expression (irrespective of cell types), PD-1
CD3
expression, and PD-1/PD-L1 interaction showed hierarchical adverse prognostic effects in the study cohort. PD-1/PD-L1 interaction showed higher sensitivity and specificity than PD-1
and PD-L1
expression in predicting inferior prognosis in patients with high CD3
tissue cellularity ("hot"/inflammatory tumors). However, both PD-1
and PD-L1
expression showed adverse prognostic effects independent of PD-1/PD-L1 interaction, and PD-1/PD-L1 interaction showed favorable prognostic effect in PD-L1
patients without high CD3
tissue cellularity. Macrophage function and tumor-cell MYC expression may contribute to the PD-1-independent adverse prognostic effect of PD-L1
expression. In summary, low T-cell tissue cellularity has unfavorable prognostic impact in diffuse large B-cell lymphoma, and tissue PD-L1
expression and T-cell-derived PD-1
expression have significant adverse impact only in patients with high T-cell infiltration. PD-1/PD-L1 interaction in tissue is essential but not always responsible for the inhibitory effect of PD-L1
/PD-1
expression. These results suggest the benefit of PD-1/PD-L1 blockade therapies only in patients with sufficient T-cell infiltration, and the potential of immunofluorescent assays and Automated Quantitative Analysis in the clinical assessment of PD-1/PD-L1 expression and interaction.
Platinum-based chemotherapy is usually curative for patients with testicular germ cell tumors (TGCT), but a subset of patients experience disease progression and poor clinical outcomes. Here, we ...tested whether immune profiling of TGCT could identify novel prognostic markers and therapeutic targets for this patient cohort. We obtained primary and metastatic TGCT samples from one center. We performed immune profiling using multiplexed fluorescence immunohistochemistry (FIHC) for T-cell subsets and immune checkpoints, and targeted gene expression profiling (Nanostring nCounter Immune panel). Publically available data sets were used to validate primary sample analyses. Nearly all samples had some degree of T-cell infiltration and immune checkpoint expression. Seminomas were associated with increased CD3
+
T-cell infiltration, decreased Regulatory T-cells, increased PD-L1, and increased PD-1/PD-L1 spatial interaction compared with non-seminomas using FIHC. Gene expression profiling confirmed these findings and also demonstrated increased expression of T-cell markers (e.g., IFNγ, and LAG3) and cancer/testis antigens (e.g., PRAME) in seminomas, whereas non-seminomas demonstrated high neutrophil and macrophage gene signatures. Irrespective of histology, advanced TGCT stage was associated with decreased T-cell and NK-cell signatures, while Treg, neutrophil, mast cell and macrophage signatures increased with advanced stage. Importantly, cancer/testis antigen, neutrophil, and CD8
+
/regulatory T-cell signatures correlated with recurrence free survival. Thus, deep immune characterization of TGCT using IHC and gene expression profiling identified activated T-cell infiltration which correlated with seminoma histology and good prognosis. These results may provide a rationale for testing of anti-PD-1/PD-L1 agents and suggest prognostic markers.
•Neoadjuvant ipilimumab and IFNα were evaluated in regionally advanced melanoma.•Immune cellular profiling investigated tumor immune susceptibility and resistance.•Higher levels of peripheral Th1 ...cell subsets predicted favorable clinical outcomes.•Higher levels of peripheral Th2 cells was associated with poor prognosis.•Significant reductions in peripheral T-reg and MDSC were seen in responders.
Neoadjuvant therapy with ipilimumab in combination with high dose IFNα was evaluated in patients with locally/regionally advanced melanoma in a previously reported clinical trial NCT01608594. In this study, peripheral immune cell profiling was performed in order to investigate the underlying mechanisms of tumor immune susceptibility and resistance. Peripheral blood mononuclear cells (PBMCs) from treated patients (N = 28) were collected at baseline and then at 6-weeks, 3-months and 12-months. High complexity (14-color) flow cytometry, designed to detect key immunological biomarkers was used to evaluate the frequencies of immune cell subsets. Statistical significance was determined using R-package employing Kruskal's test. We found that higher levels of Th1 cells at baseline (defined as CD45RA- CCR6- CXCR3+ CCR4-) correlated with the preoperative radiological response (p = 0.007) while higher Th2 cells (defined as CD45RA- CCR6- CXCR3- CCR4+) were associated with progressive disease (p = 0.009). A multimarker score consisting of higher levels of Th1 cells and CD8+ central memory T-cells was associated with pathologic complete response (pCR) (p = 0.041) at surgical resection. On the other hand, high TIM3 expression on T-cells correlated with gross viable tumor (p = 0.047). With regard to immune related toxicity, higher levels of phenotypically naive (defined as CCR7+CD45RA+) and effector memory (defined as CCR7-CD45RO+) CD8+ T-cells (p = 0.014) or lower levels of Th2 cells were associated with lower toxicity (p = 0.024). Furthermore, a multimarker score consisting of higher CD19+ and CD8+ cells was associated with lower toxicity (p = 0.0014). In conclusion, our study yielded mechanistic insights related to the immune impact of CTLA4 blockade and IFNα and potential biomarkers of immune response and toxicity.
Although the immune system is capable of mounting a response against many cancers, that response is insufficient for tumor eradication in most patients due to factors in the tumor microenvironment ...that defeat tumor immunity. We previously identified the immune-suppressive molecule CD200 as up-regulated on primary B cell chronic lymphocytic leukemia (B-CLL) cells and demonstrated negative immune regulation by B-CLL and other tumor cells overexpressing CD200 in vitro. In this study we developed a novel animal model that incorporates human immune cells and human tumor cells to address the effects of CD200 overexpression on tumor cells in vivo and to assess the effect of targeting Abs in the presence of human immune cells. Although human mononuclear cells prevented tumor growth when tumor cells did not express CD200, tumor-expressed CD200 inhibited the ability of lymphocytes to eradicate tumor cells. Anti-CD200 Ab administration to mice bearing CD200-expressing tumors resulted in nearly complete tumor growth inhibition even in the context of established receptor-ligand interactions. Evaluation of an anti-CD200 Ab with abrogated effector function provided evidence that blocking of the receptor-ligand interaction was sufficient for control of CD200-mediated immune modulation and tumor growth inhibition in this model. Our data indicate that CD200 expression by tumor cells suppresses antitumor responses and suggest that anti-CD200 treatment might be therapeutically beneficial for treating CD200-expressing cancers.
CD200 is an immunosuppressive molecule overexpressed in multiple hematologic malignancies such as B cell chronic lymphocytic leukemia, multiple myeloma, and acute myeloid leukemia. We previously ...demonstrated that up-regulation of CD200 on tumor cells suppresses antitumor immune responses and that antagonistic anti-human CD200 mAbs enabled human PBMC-mediated tumor growth inhibition in xenograft NOD/SCID human (hu)-mouse models. Ab variants with effector function (IgG1 constant region (G1)) or without effector function (IgG2/G4 fusion constant region (G2G4)) exhibited high antitumor activity in a human tumor xenograft model in which CD200 was expressed. In this report, we seek to select the best candidate to move forward into the clinic and begin to decipher the mechanisms of tumor cell killing by comparing anti-CD200-G1 vs anti-CD200-G2G4 in two related animal models. In a CD200-expressing xenograft NOD/SCID hu-mouse model where CD200 ligand/receptor interactions are already established before initiating treatment, we find that anti-CD200-G1 is a less effective Ab compared with anti-CD200-G2G4. Separately, in a model that evaluates the effect of the Abs on the immune cell component of the xenograft NOD/SCID hu-mouse model distinctly from the effects of binding to CD200 on tumor cells, we find that the administration of anti-CD200-G1 Abs completely abolished human PBMC-mediated tumor growth inhibition. Along with supporting in vitro studies, our data indicate that anti-CD200-G1 Abs efficiently mediate Ab-dependent cellular cytotoxicity of activated T cells, critical cells involved in immune-mediated killing. These studies suggest important implications regarding the selection of the constant region in anti-CD200 immunotherapy of cancer patients.
Multiple cancer vaccine trials have been carried out using ex vivo generated autologous dendritic cells (DCs) loaded with tumor antigen before readministration into patients. Though promising, ...overall immunologic potency and clinical efficacy might be improved with more efficient DC-based therapies that avoid ex vivo manipulations, but are instead based on in vivo targeting of DCs. For initial in vivo proof of concept studies, we evaluated targeting of proteins or peptides to DCs through DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN). Because the biology of DC-SIGN is different between mice and humans, we assess human DC-SIGN targeting in the setting of elements of a human immune system in a mouse model. Administration of anti-DC-SIGN antibodies carrying either tetanus toxoid peptides or keyhole limpet hemocyanin (KLH) to Rag2gammaC mice reconstituted with human immune cells raised stimulatory human T-cell responses to the respective antigen without additional adjuvant requirements. Furthermore, administration of anti-DC-SIGN antibody-KLH conjugate enhanced the adjuvant properties of KLH resulting in inhibition of RAJI (Human Burkitt's Lymphoma Cell Line) cell tumor growth in Nonobese Diabetic/Severe Combined Immunodeficient mice transplanted with human immune cells. Thus, mouse models reconstituted with human immune cells seem to be suitable for evaluating DC-targeted vaccines, and furthermore, targeting to DCs in situ via DC-SIGN may provide a promising vaccine platform for inducing strong immune responses against cancer and infectious disease agents.
BackgroundBoth proteins (e.g., PD-L1 IHC) and tumor mutation burden (NGS-based) are known to independently predict clinical response to anti-PD-1/PD-L1 therapies. In a meta-analysis of tumor ...specimens from 8135 patients treated with PD-1/PD-L1 blockers, multiplex fluorescence immunohistochemistry (mFIHC) had significantly higher diagnostic accuracy than PD-L1 IHC, tumor mutational burden (TMB), or gene expression profiling alone in predicting clinical response1 or equivalent to a multimodality approach (e.g., PD-L1IHC + TMB). While the benefits of combining mFIHC (tumor-immune interplay) and NGS approaches in selection of patients for next generation immunotherapies is appealing, tumor tissue is a key limiting factor for multimodality analyses in clinical trials. To address this critical limitation, we developed a novel approach for sequential profiling of tumor and immune cell interactions by 7-parameter mFIHC assays, followed by analyses of nucleic acid extracted from same tissue sections.MethodsFormalin-fixed paraffin-embedded (FFPE) tumor tissue and cell line blocks were sectioned, and then stained using mFIHC followed by isolation of nucleic acids, or direct isolation of total nucleic acids. NanoString, qPCR, and NGS were performed on isolated nucleic acids. Nucleic acid quality, transcript abundance, and TMB scores were compared before and after mFIHC staining.ResultsmFIHC revealed a broad range of immune cell phenotypes and spatial interactions, including T cells, B cells, NK cells, monocytes, neutrophils, and their functional status. Isolation of testable quantities of DNA from mFIHC treated slides was achieved when using a DNA-only isolation method, and TMB scores were robust across tested conditions. Cell phenotypes identified by mFIHC were compared to TMB scores across the tested samples. Following mFIHC treatment, RNA yields were reduced relative to the non-mFIHC treated replicates, but still sufficient for optimal input into a 770-target NanoString gene expression panel. However, for mFIHC treated samples, transcript levels were not distinguishable from background for the assessed targets.ConclusionsIn summary, integrating mFIHC testing and TMB analysis on the same samples allows for comprehensive biomarker evaluation. The real world benefits of the combined approach will be described in upcoming clinical trials.ReferenceLu, et al., Comparison of biomarker modalities for predicting response to PD-1/PD-L1 checkpoint blockade, a systematic review and meta-analysis. JAMA Oncology 2019; 5(8):1195–1204
Immunotherapeutic approaches to cancer should focus on novel undertakings that modulate immune responses by synergistic enhancement of antitumor immunological parameters. Cancer vaccines should ...preferably be composed of multiple defined tumor Ag-specific B and T cell epitopes. To develop a multiepitope vaccine, 12 high ranking B cell epitopes were identified from the extracellular domain of the human epidermal growth factor receptor-2 (HER-2) oncoprotein by computer-aided analysis. Four novel HER-2 B cell epitopes were synthesized as chimeras with a promiscuous T cell epitope (aa 288-302) from the measles virus fusion protein (MVF). Two chimeric peptide vaccines, MVF HER-2(316-339) and MVF HER-2(485-503) induced high levels of Abs in outbred rabbits, which inhibited tumor cell growth. In addition, Abs induced by a combination of two vaccines, MVF HER-2(316-339) and MVF HER-2(628-647) down-modulated receptor expression and activated IFN-gamma release better than the individual vaccines. Furthermore, this multiepitope vaccine in combination with IL-12 caused a significant reduction (p = 0.004) in the number of pulmonary metastases induced by challenge with syngeneic tumor cells overexpressing HER-2. Peptide Abs targeting specific sites in the extracellular domain may be used for exploring the oncoprotein's functions. The multiepitope vaccine may have potential application in the treatment of HER-2-associated cancers.
Cancer vaccines designed to elicit an antibody response that target antigenic sites on a tumor antigen must closely mimic the three-dimensional structure of the corresponding region on the antigen. ...We have designed a complex immunogen derived from the extracellular domain of human HER-2/neu-(626–649) that represents a three-dimensional epitope. We have successfully introduced two disulfide bonds into this sequence, thereby recapitulating the natural disulfide pairings observed in the native protein. To evaluate the immunogenicity of the doubly cyclized disulfide-linked peptide versus the free uncyclized peptide we examined the induction of antibody responses in both inbred and outbred mice strains, with both constructs eliciting high titered antibodies. The disulfide-paired specific antibodies exhibited enhanced cross-reactivity to HER-2/neu expressed on BT-474 cell line as determined by flow cytometry. The antitumor activities of the disulfidepaired specific antibodies did not improve the in vitro growth inhibition of human breast cancer cells overexpressing HER-2, but showed superior antitumor responses in the context of ADCC and interferon-γ induction. Inbred mice (FVB/n) vaccinated with the disulfide-paired epitope exhibited a statistically significant reduction in the development of exogenously administered tumors in vivo compared with mice receiving either the free uncyclized or the promiscuous T-cell epitope (MVF) control peptide (p = 0.001). This study demonstrates the feasibility and importance of designing conformational epitopes that mimic the tertiary structure of the native protein for eliciting biologically relevant anti-tumor antibodies. Such approaches are a prerequisite to the design of effective peptide vaccines.
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