A Phase I trial of Nab-Paclitaxel/Bevacizumab (AB160) Nano-Immunoconjugate Therapy for Gynecologic Malignancies.

PURPOSE: AB160 is a 160 nm nano-immunoconjugate consisting of nab-paclitaxel (ABX) nanoparticles non-covalently coated with bevacizumab (BEV) for targeted delivery into tissues expressing high levels of VEGF. Preclinical data showed that AB160 resulted in greater tumor targeting and tumor inhibition compared to sequential treatment with ABX then BEV. Given individual drug activity, we investigated the safety and toxicity of AB160 in patients with gynecologic cancers. PATIENTS AND METHODS: A 3+3 phase I trial was conducted with 3 potential dose levels in patients with previously treated endometrial (EC), cervical (CC), and platinum-resistant ovarian cancer (OC) patients to ascertain the recommended Phase II dose (RP2D). AB160 was administered intravenously on Days 1, 8 and 15 of a 28-day cycle (ABX 75-175 mg/m2, BEV 30-70 mg/m2). Pharmacokinetic analyses were performed. RESULTS: No dose-limiting toxicities (DLTs) were seen among the 3 DLs tested. Grade 3/4 toxicities included neutropenia, thromboembolic events, and leukopenia. DL2 (ABX 150 mg/m2, BEV 60 mg/m2) was chosen as the RP2D. Seven of the 19 patients with measurable disease (36.8%) had confirmed partial responses (95% CI: 16.3%-61.6%). Pharmacokinetic analyses demonstrated that AB160 allowed 50% higher paclitaxel dosing and that paclitaxel clearance mirrored that of therapeutic antibodies. CONCLUSIONS: The safety profile and clinical activity of AB160 supports further clinical testing in patients with gynecologic cancers; the RP2D is DL2 (ABX 150 mg/m2, BEV 60 mg/m2).

CD19 occupancy with tafasitamab increases therapeutic index of CART19 cell therapy and diminishes severity of CRS.

ABSTRACT: In the development of various strategies of anti-CD19 immunotherapy for the treatment of B-cell malignancies, it remains unclear whether CD19 monoclonal antibody therapy impairs subsequent CD19-targeted chimeric antigen receptor T-cell (CART19) therapy. We evaluated the potential interference between the CD19-targeting monoclonal antibody tafasitamab and CART19 treatment in preclinical models. Concomitant treatment with tafasitamab and CART19 showed major CD19 binding competition, which led to CART19 functional impairment. However, when CD19+ cell lines were pretreated with tafasitamab overnight and the unbound antibody was subsequently removed from the culture, CART19 function was not affected. In preclinical in vivo models, tafasitamab pretreatment demonstrated reduced incidence and severity of cytokine release syndrome and exhibited superior antitumor effects and overall survival compared with CART19 alone. This was associated with transient CD19 occupancy with tafasitamab, which in turn resulted in the inhibition of CART19 overactivation, leading to diminished CAR T apoptosis and pyroptosis of tumor cells.

AXL Inhibition Improves the Antitumor Activity of Chimeric Antigen Receptor T Cells.

The receptor tyrosine kinase AXL is a member of the TYRO3, AXL, and proto-oncogene tyrosine-protein kinase MER family and plays pleiotropic roles in cancer progression. AXL is expressed in immunosuppressive cells, which contributes to decreased efficacy of immunotherapy. Therefore, we hypothesized that AXL inhibition could serve as a strategy to overcome resistance to chimeric antigen receptor T (CAR T)-cell therapy. To test this, we determined the impact of AXL inhibition on CD19-targeted CAR T (CART19)-cell functions. Our results demonstrate that T cells and CAR T cells express high levels of AXL. Specifically, higher levels of AXL on activated Th2 CAR T cells and M2-polarized macrophages were observed. AXL inhibition with small molecules or via genetic disruption in T cells demonstrated selective inhibition of Th2 CAR T cells, reduction of Th2 cytokines, reversal of CAR T-cell inhibition, and promotion of CAR T-cell effector functions. AXL inhibition is a novel strategy to enhance CAR T-cell functions through two independent, but complementary, mechanisms: targeting Th2 cells and reversing myeloid-induced CAR T-cell inhibition through selective targeting of M2-polarized macrophages.

Resolving the heterogeneous tumor-centric cellular neighborhood through multiplexed, spatial paracrine interactions in the setting of immune checkpoint blockade.

Direct interactions between tumor and immune cells mediate the antitumor effect of all modern cancer immunotherapeutic agents. Simultaneously, tumor cells have evolved mechanisms of evasion including the downregulation of HLA-I potentially disrupting the mechanism of action employed by many immune checkpoint inhibitors. And yet the in situ interplay between these cells within the tumor immune microenvironment (TIME) remains elusive. Recent advances in histologic multiplex bioimaging platforms have enabled in-depth molecular characterization of single cells within spatially-preserved and clinically archived tumor tissues. Herein, we applied multiplex immunofluorescence (MxIF) to excisional lymph node biopsies from 14 patients with metastatic melanoma who experienced clear objective responses to immunotherapy (7 complete response; 7 progressive disease) to determine distinguishing features of the TIME in the pretreatment setting. Distinct regions of the TIME were evaluated using 35 proteins probing tumor, immune and vasculature components across 323 fields of view. Single cell compositional analysis confirmed established prognostic immune cell types including increased prevalence of cytotoxic T cells within the tumor core FOVs of responders. Integrating single cell quantification with the spatial arrangement of cellular neighborhoods surrounding tumor cells revealed novel, spatial immune signatures capable of stratifying TIME based on clinical response. Our analysis revealed dynamic cellular composition of the TCCN based on anatomical subregion, functional expression of HLA-I by the index tumor cell and ultimately clinical response to immunotherapy. Overall, this study provides an analytical framework to resolve the cellular complexity of the TIME, increasingly relevant to the outcomes of modern cancer immunotherapy.

Targeting cancer-associated fibroblasts in the bone marrow prevents resistance to CART-cell therapy in multiple myeloma.

Pivotal clinical trials of B-cell maturation antigen-targeted chimeric antigen receptor T (CART)-cell therapy in patients with relapsed/refractory multiple myeloma (MM) resulted in remarkable initial responses, which led to a recent US Food and Drug Administration approval. Despite the success of this therapy, durable remissions continue to be low, and the predominant mechanism of resistance is loss of CART cells and inhibition by the tumor microenvironment (TME). MM is characterized by an immunosuppressive TME with an abundance of cancer-associated fibroblasts (CAFs). Using MM models, we studied the impact of CAFs on CART-cell efficacy and developed strategies to overcome CART-cell inhibition. We showed that CAFs inhibit CART-cell antitumor activity and promote MM progression. CAFs express molecules such as fibroblast activation protein and signaling lymphocyte activation molecule family-7, which are attractive immunotherapy targets. To overcome CAF-induced CART-cell inhibition, CART cells were generated targeting both MM cells and CAFs. This dual-targeting CART-cell strategy significantly improved the effector functions of CART cells. We show for the first time that dual targeting of both malignant plasma cells and the CAFs within the TME is a novel strategy to overcome resistance to CART-cell therapy in MM.

Development of a Clinically Relevant Reporter for Chimeric Antigen Receptor T-cell Expansion, Trafficking, and Toxicity.

Although chimeric antigen receptor T (CART)-cell therapy has been successful in treating certain hematologic malignancies, wider adoption of CART-cell therapy is limited because of minimal activity in solid tumors and development of life-threatening toxicities, including cytokine release syndrome (CRS). There is a lack of a robust, clinically relevant imaging platform to monitor in vivo expansion and trafficking to tumor sites. To address this, we utilized the sodium iodide symporter (NIS) as a platform to image and track CART cells. We engineered CD19-directed and B-cell maturation antigen (BCMA)-directed CART cells to express NIS (NIS+CART19 and NIS+BCMA-CART, respectively) and tested the sensitivity of 18F-TFB-PET to detect trafficking and expansion in systemic and localized tumor models and in a CART-cell toxicity model. NIS+CART19 and NIS+BCMA-CART cells were generated through dual transduction with two vectors and demonstrated exclusive 125I uptake in vitro. 18F-TFB-PET detected NIS+CART cells in vivo to a sensitivity level of 40,000 cells. 18F-TFB-PET confirmed NIS+BCMA-CART-cell trafficking to the tumor sites in localized and systemic tumor models. In a xenograft model for CART-cell toxicity, 18F-TFB-PET revealed significant systemic uptake, correlating with CART-cell in vivo expansion, cytokine production, and development of CRS-associated clinical symptoms. NIS provides a sensitive, clinically applicable platform for CART-cell imaging with PET scan. 18F-TFB-PET detected CART-cell trafficking to tumor sites and in vivo expansion, correlating with the development of clinical and laboratory markers of CRS. These studies demonstrate a noninvasive, clinically relevant method to assess CART-cell functions in vivo.

Cancer immune control dynamics: a clinical data driven model of systemic immunity in patients with metastatic melanoma.

BACKGROUND: Recent clinical advances in cancer immuno-therapeutics underscore the need for improved understanding of the complex relationship between cancer and the multiple, multi-functional, inter-dependent, cellular and humoral mediators/regulators of the human immune system. This interdisciplinary effort exploits engineering analysis methods utilized to investigate anomalous physical system behaviors to explore immune system behaviors. Cancer Immune Control Dynamics (CICD), a systems analysis approach, attempts to identify differences between systemic immune homeostasis of 27 healthy volunteers versus 14 patients with metastatic malignant melanoma based on daily serial measurements of conventional peripheral blood biomarkers (15 cell subsets, 35 cytokines). The modeling strategy applies engineering control theory to analyze an individual's immune system based on the biomarkers' dynamic non-linear oscillatory behaviors. The reverse engineering analysis uses a Singular Value Decomposition (SVD) algorithm to solve the inverse problem and identify a solution profile of the active biomarker relationships. Herein, 28,605 biologically possible biomarker interactions are modeled by a set of matrix equations creating a system interaction model. CICD quantifies the model with a participant's biomarker data then computationally solves it to measure each relationship's activity allowing a visualization of the individual's current state of immunity. RESULTS: CICD results provide initial evidence that this model-based analysis is consistent with identified roles of biomarkers in systemic immunity of cancer patients versus that of healthy volunteers. The mathematical computations alone identified a plausible network of immune cells, including T cells, natural killer (NK) cells, monocytes, and dendritic cells (DC) with cytokines MCP-1 [CXCL2], IP-10 [CXCL10], and IL-8 that play a role in sustaining the state of immunity in advanced cancer. CONCLUSIONS: With CICD modeling capabilities, the complexity of the immune system is mathematically quantified through thousands of possible interactions between multiple biomarkers. Therefore, the overall state of an individual's immune system regardless of clinical status, is modeled as reflected in their blood samples. It is anticipated that CICD-based capabilities will provide tools to specifically address cancer and treatment modulated (immune checkpoint inhibitors) parameters of human immunity, revealing clinically relevant biological interactions.

Outcomes on anti-VEGFR-2/paclitaxel treatment after progression on immune checkpoint inhibition in patients with metastatic gastroesophageal adenocarcinoma.

Through our involvement in KEYNOTE-059, we unexpectedly observed durable responses in two patients with metastatic gastroesophageal adenocarcinoma (mGEA) who received ramucirumab (anti-VEGFR-2)/paclitaxel after immune checkpoint inhibition (ICI). To assess the reproducibility of this observation, we piloted an approach to administer ramucirumab/paclitaxel after ICI in more patients, and explored changes in the immune microenvironment. Nineteen consecutive patients with mGEA received ICI followed by ramucirumab/paclitaxel. Most (95%) did not respond to ICI, yet after irRECIST-defined progression on ICI, all patients experienced tumor size reduction on ramucirumab/paclitaxel. The objective response rate (ORR) and progression-free survival (PFS) on ramucirumab/paclitaxel after ICI were higher than on the last chemotherapy before ICI in the same group of patients (ORR, 58.8% vs 11.8%; PFS 12.2 vs 3.0 months; respectively). Paired tumor biopsies examined by imaging mass cytometry showed a median 5.5-fold (range 4-121) lower frequency of immunosuppressive forkhead box P3+ regulatory T cells with relatively preserved CD8+ T cells, post-treatment versus pre-treatment (n = 5 pairs). We then compared the outcomes of these 19 patients with a separate group who received ramucirumab/paclitaxel without preceding ICI (n = 68). Median overall survival on ramucirumab/paclitaxel was longer with (vs without) immediately preceding ICI (14.8 vs 7.4 months) including after multivariate analysis, as was PFS. In our small clinical series, outcomes appeared improved on anti-VEGFR-2/paclitaxel treatment when preceded by ICI, in association with alterations in the immune microenvironment. However, further investigation is needed to determine the generalizability of these data. Prospective clinical trials to evaluate sequential treatment with ICI followed by anti-VEGF(R)/taxane are underway.

Leukemic extracellular vesicles induce chimeric antigen receptor T cell dysfunction in chronic lymphocytic leukemia.

Chimeric antigen receptor (CAR) T cell therapy has yielded unprecedented outcomes in some patients with hematological malignancies; however, inhibition by the tumor microenvironment has prevented the broader success of CART cell therapy. We used chronic lymphocytic leukemia (CLL) as a model to investigate the interactions between the tumor microenvironment and CART cells. CLL is characterized by an immunosuppressive microenvironment, an abundance of systemic extracellular vesicles (EVs), and a relatively lower durable response rate to CART cell therapy. In this study, we characterized plasma EVs from untreated CLL patients and identified their leukemic cell origin. CLL-derived EVs were able to induce a state of CART cell dysfunction characterized by phenotypical, functional, and transcriptional changes of exhaustion. We demonstrate that, specifically, PD-L1+ CLL-derived EVs induce CART cell exhaustion. In conclusion, we identify an important mechanism of CART cell exhaustion induced by EVs from CLL patients.

Public and private human T-cell clones respond differentially to HCMV antigen when boosted by CD3 copotentiation.

Human cytomegalovirus (HCMV) induces long-lasting T-cell immune responses that control but do not clear infection. Typical responses involve private T-cell clones, expressing T-cell antigen receptors (TCRs) unique to a person, and public T-cell clones with identical TCRs active in different people. Here, we report the development of a pretherapeutic immunostimulation modality against HCMV for human T cells, CD3 copotentiation, and the clonal analysis of its effects in recall assays at single-cell resolution. CD3 copotentiation of human T cells required identification of an intrinsically inert anti-CD3 Fab fragment that conditionally augmented signaling only when TCR was coengaged with antigen. When applied in recall assays, CD3 copotentiation enhanced the expansion of both public and private T-cell clones responding to autologous HLA-A2(+) antigen-presenting cells and immunodominant NLVPMVATV (NLV) peptide from HCMV pp65 protein. Interestingly, public vs private TCR expression was associated with distinct clonal expansion signatures in response to recall stimulus. This implied that besides possible differences in their generation and selection in an immune response, public and private T cells may respond differently to pharmacoimmunomodulation. Furthermore, a third clonal expansion profile was observed upon CD3 copotentiation of T-cell clones from HLA-A2(-) donors and 1 HLA-A2(+) presumed-uninfected donor, where NLV was of low intrinsic potency. We conclude that human T-cell copotentiation can increase the expansion of different classes of T-cell clones responding to recall antigens of different strengths, and this may be exploitable for therapeutic development against chronic, persistent infections such as HCMV.

Peptide vaccine with glucopyranosyl lipid A-stable oil-in-water emulsion for patients with resected melanoma.

Aim: We tested the safety and immunogenicity of a novel vaccine in patients with resected high-risk melanoma. Patients & methods: HLA-A2-positive patients with resected Stage II-IV melanoma were randomized to receive up to three vaccinations of melanoma-associated peptide (MART-1a) combined with a stable oil-in-water emulsion (SE) either with the Toll-like receptor 4 agonist glucopyranosyl lipid A (GLA-SE-Schedule 1) or alone (SE-Schedule 2). Safety and immunogenicity of the vaccines were monitored. Results: A total of 23 patients were registered. No treatment-related grade 3 or higher adverse events were observed. Increases in MART-1a-specific T cells were seen in 70 and 63% of Schedule 1 and Schedule 2 patients, respectively. Conclusion: Both vaccine schedules were well-tolerated and resulted in an increase in MART-1a-specific T cells. Clinical Trial registration: NCT02320305 (ClinicalTrials.gov).

Identification of novel, immune-mediating extracellular vesicles in human lymphatic effluent draining primary cutaneous melanoma.

Epithelial tumors including melanoma often first metastasize to regional, sentinel lymph nodes (SLN). Thus, the presence of SLN metastases is a critical prognostic factor of survival. Prior to metastasis, accumulating evidence suggests the SLN is immunologically compromised; however, the process by which pre-metastatic niche formation occurs remains unknown. In this prospective study, freshly dissected, afferent lymphatic fluid was obtained during SLN biopsy in three patients with primary cutaneous melanoma. Lymphatic extracellular vesicles (L-EV) were visualized by transmission electron microscopy and proteomic cargo profiled by mass spectrometry. Flow cytometry assessed L-EV effects on autologous dendritic cell maturation in vitro. Immunogold electron microscopy and immunohistochemistry visualized expression of EV cargo within the primary tumor and SLN. Lymphatic extracellular vesicles from each afferent lymphatic channel demonstrated inhibition of autologous dendritic cell maturation. Proteomic profiling identified 81 peptides shared among the L-EV preparations including a signature of 18 immune-modulating proteins including previously established inhibitor of dendritic cell maturation, S100A9. Immunohistochemistry and immunogold electron microscopy confirmed S100A9 tracking along the lymphatic path, from keratinocytes in the primary tumor to sub-capsular macrophages in the SLN. Our findings suggest L-EV cargo may serve as early mediators of tumor-induced immune subversion in regional lymph nodes, by preceding malignant cells and trafficking within the lymphatic vasculature to harbor the first pre-metastatic niche.

Understanding heterogeneous tumor microenvironment in metastatic melanoma.

A systemic analysis of the tumor-immune interactions within the heterogeneous tumor microenvironment is of particular importance for understanding the antitumor immune response. We used multiplexed immunofluorescence to elucidate cellular spatial interactions and T-cell infiltrations in metastatic melanoma tumor microenvironment. We developed two novel computational approaches that enable infiltration clustering and single cell analysis-cell aggregate algorithm and cell neighborhood analysis algorithm-to reveal and to compare the spatial distribution of various immune cells relative to tumor cell in sub-anatomic tumor microenvironment areas. We showed that the heterogeneous tumor human leukocyte antigen-1 expressions differently affect the magnitude of cytotoxic T-cell infiltration and the distributions of CD20+ B cells and CD4+FOXP3+ regulatory T cells within and outside of T-cell infiltrated tumor areas. In a cohort of 166 stage III melanoma samples, high tumor human leukocyte antigen-1 expression is required but not sufficient for high T-cell infiltration, with significantly improved overall survival. Our results demonstrate that tumor cells with heterogeneous properties are associated with differential but predictable distributions of immune cells within heterogeneous tumor microenvironment with various biological features and impacts on clinical outcomes. It establishes tools necessary for systematic analysis of the tumor microenvironment, allowing the elucidation of the "homogeneous patterns" within the heterogeneous tumor microenvironment.

A pilot clinical trial testing topical resiquimod and a xenopeptide as immune adjuvants for a melanoma vaccine targeting MART-1.

A vaccine that could expand melanoma-specific T cells might reduce the risk of recurrence of resected melanoma and could provide an alternative or adjunct to standard immunotherapy options. We tested the safety and immunogenicity of a vaccine coupling a melanoma-associated peptide with a xenogenic peptide (to promote epitope spreading) and/or resiquimod (to activate antigen-presenting cells). HLA-A2-positive patients with resected stage II, III, and IV melanoma were assigned to treatment on one of three schedules. All patients received three subcutaneous doses of the peptide MART-1a mixed with Montanide. In addition, patients on schedule 1 received the xenoantigen peptide Gag267-274, patients on schedule 2 received topical resiquimod, and patients on schedule 3 received both Gag267-274 and resiquimod. Blood samples were tested for the frequency of antigen-specific T cells by tetramer assay, as well as immune cell subtypes and plasma cytokine levels. Patients enrolled from October 2012 to December 2014, with 10 patients enrolling to each schedule. The most common adverse events were injection site reaction (26 patients) and fatigue (15 patients). Tetramer analysis revealed antigen-specific responses (defined as doubling of MART-1a-specific T cells from pretreatment to post-treatment) in 20, 60, and 40% of patients treated on schedules 1, 2, and 3, respectively. Vaccine treatment consisting of MART-1a peptide, Gag267-274, Montanide, and topical resiquimod was well-tolerated. The addition of the Gag267-274 xenoantigen was not associated with an increase in the response to MART-1a, whereas use of topical resiquimod was associated with a higher frequency of MART-1a-specific T-cell responses that did not meet statistical significance.

GM-CSF inhibition reduces cytokine release syndrome and neuroinflammation but enhances CAR-T cell function in xenografts.

Chimeric antigen receptor T (CAR-T) cell therapy is a new pillar in cancer therapeutics; however, its application is limited by the associated toxicities. These include cytokine release syndrome (CRS) and neurotoxicity. Although the IL-6R antagonist tocilizumab is approved for treatment of CRS, there is no approved treatment of neurotoxicity associated with CD19-targeted CAR-T (CART19) cell therapy. Recent data suggest that monocytes and macrophages contribute to the development of CRS and neurotoxicity after CAR-T cell therapy. Therefore, we investigated neutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) as a potential strategy to manage CART19 cell-associated toxicities. In this study, we show that GM-CSF neutralization with lenzilumab does not inhibit CART19 cell function in vitro or in vivo. Moreover, CART19 cell proliferation was enhanced and durable control of leukemic disease was maintained better in patient-derived xenografts after GM-CSF neutralization with lenzilumab. In a patient acute lymphoblastic leukemia xenograft model of CRS and neuroinflammation (NI), GM-CSF neutralization resulted in a reduction of myeloid and T cell infiltration in the central nervous system and a significant reduction in NI and prevention of CRS. Finally, we generated GM-CSF-deficient CART19 cells through CRISPR/Cas9 disruption of GM-CSF during CAR-T cell manufacturing. These GM-CSFk/o CAR-T cells maintained normal functions and had enhanced antitumor activity in vivo, as well as improved overall survival, compared with CART19 cells. Together, these studies illuminate a novel approach to abrogate NI and CRS through GM-CSF neutralization, which may potentially enhance CAR-T cell function. Phase 2 studies with lenzilumab in combination with CART19 cell therapy are planned.

Non-invasive visualization of tumor infiltrating lymphocytes in patients with metastatic melanoma undergoing immune checkpoint inhibitor therapy: a pilot study.

Early in the course of immunotherapy there is frequently a transient enlargement of tumor masses (pseudo-progression) due to tumor infiltration by TILs. Current clinical imaging modalities are not able to distinguished pseudo-progression from true tumor progression. Thus, patients often remain on treatment 4-8 weeks longer to confirm disease progression. Nuclear medicine offers the possibility to image immune cells and potentially discriminate pseudo-progression and progression. We conducted a pilot study in patients with metastatic melanoma receiving ipilimumab (IPI) or pembrolizumab (PEMBRO) to assess safety and feasibility of SPECT/CT imaging with 99mTc- interleukin-2 (99mTc-HYNIC-IL2) to detect TILs and distinguish between true progression from pseudo- progression. Scans were performed prior to and after 12w treatment. After labelling,99mTc-HYNIC-IL2 was purified and diluted in 10 mL of 5% glucose with 0.1% human serum albumin. Of the 5 patients (2 treated with IPI and 3 with PEMBRO) enrolled, two failed to complete the second scan as they discontinued IPI due grade 3 colitis (1 patient) or patient refusal after developing multiple toxicities attributed to IPI (1 patient). Following the first scan, one patient reported to have a grade 1 pruritus with grade 1 pain. No other toxicities attributed to the radiopharmaceutical infusion were reported. Metastatic lesions could be visualized by 99mTc-IL2 imaging and there was positive correlation between size and 99mTc-HYNIC-IL2 uptake, both before and after 12 weeks of therapy. The results of this pilot study demonstrate the safety and feasibility of 99mTc-IL2 imaging and has led to a number of hypotheses to be tested in future studies.

CD206-positive myeloid cells bind galectin-9 and promote a tumor-supportive microenvironment.

In patients with metastatic melanoma, high blood levels of galectin-9 are correlated with worse overall survival and a bias towards a Th2 inflammatory state supportive of tumor growth. Although galectin-9 signaling through TIM3 on T cells has been described, less is known about the interaction of galectin-9 with macrophages. We aimed to determine whether galectin-9 is a binding partner of CD206 on macrophages and whether the result of this interaction is tumor-supportive. It was determined that incubation of CD68+ macrophages with galectin-9 or anti-CD206 blocked target binding and that both CD206 and galectin-9 were detected by immunoprecipitation of cell lysates. CD206 and galectin-9 had a binding affinity of 2.8 × 10-7  m. Galectin-9 causes CD206+ macrophages to make significantly more FGF2 and monocyte chemoattractant protein (MCP-1), but less macrophage-derived chemokine (MDC). Galectin-9 had no effect on classical monocyte subsets, but caused expansion of the non-classical populations. Lastly, there was a positive correlation between increasing numbers of CD206 macrophages and galectin-9 expression in tumors, and high levels of CD206 macrophages correlated negatively with melanoma survival. These results indicate that galectin-9 binds to CD206 on M2 macrophages, which appear to drive angiogenesis and the production of chemokines that support tumor growth and poor patient prognoses. Targeting this interaction systemically through circulating monocytes may therefore be a novel way to improve local anti-tumor effects by macrophages. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Contraction of T cell richness in lung cancer brain metastases.

Very little is known about how the adaptive immune system responds to clonal evolution and tumor heterogeneity in non-small cell lung cancer. We profiled the T-cell receptor ß complementarity determining region 3 in 20 patients with fully resected non-small cell lung cancer primary lesions and paired brain metastases. We characterized the richness, abundance and overlap of T cell clones between pairs, in addition to the tumor mutation burden and predicted neoantigens. We found a significant contraction in the number of unique T cell clones in brain metastases compared to paired primary cancers. The vast majority of T cell clones were specific to a single lesion, and there was minimal overlap in T cell clones between paired lesions. Despite the contraction in the number of T cell clones, brain metastases had higher non-synonymous mutation burdens than primary lesions. Our results suggest that there is greater richness of T cell clones in primary lung cancers than their paired metastases despite the higher mutation burden observed in metastatic lesions. These results may have implications for immunotherapy.

Identification of a peptide-peptide binding motif in the coating of nab-paclitaxel nanoparticles with clinical antibodies: bevacizumab, rituximab, and trastuzumab.

Antibody directed chemotherapy (ADC) takes advantage of the selectivity of the monoclonal antibody to increase the efficacy of the chemotherapeutic agent, while reducing toxicity. Previously we described three nab-paclitaxel (Abraxane) nanoparticles coated with commercial monoclonal antibodies. Identifying the binding sites responsible for these particles could allow reverse engineering of nab-paclitaxel binding antibodies, creating a modular platform for antibody directed chemotherapeutic nanoparticles. Herein, Biacore surface plasmon resonance is used to identify an antibody binding site, HSA Peptide 40, on human serum albumin with nanomolar affinity for all three monoclonal antibodies. This 18-mer peptide, which lies in Subdomain IIIA of human serum albumin, blocks binding of all three antibodies to nab-paclitaxel when added in excess. We furthermore show the complementary binding region on all three monoclonal antibodies to be the CDR H3 loop of the Fab region, and show that they all have nano to micromolar affinity for HSA Peptide 40 and nab-paclitaxel nanoparticles. The presented data identify the nature of the critical protein-protein interaction that enables antibody coating of nab-paclitaxel.