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Summary Success in solid tumor chimeric antigen receptor (CAR) T-cell therapy requires overcoming several barriers, including lung sequestration, inefficient accumulation within the tumor, and target-antigen heterogeneity. Understanding CAR T-cell kinetics can assist in the interpretation of therapy response and limitations and thereby facilitate developing successful strategies to treat solid tumors. As T-cell therapy response varies across metastatic sites, the assessment of CAR T-cell kinetics by peripheral blood analysis or a single-site tumor biopsy is inadequate for interpretation of therapy response. The use of tumor imaging alone has also proven to be insufficient to interpret response to therapy. To address these limitations, we conducted dual tumor and T-cell imaging by use of a bioluminescent reporter and positron emission tomography in clinically relevant mouse models of pleural mesothelioma and non-small cell lung cancer. We observed that the mode of delivery of T cells (systemic versus regional), T-cell activation status (presence or absence of antigen-expressing tumor), and tumor-antigen expression heterogeneity influence T-cell kinetics. The observations from our study underscore the need to identify and develop a T-cell reporter—in addition to standard parameters of tumor imaging and antitumor efficacy—that can be used for repeat imaging without compromising the efficacy of CAR T cells in vivo., Graphical abstract, Imaging the kinetics—trafficking, biodistribution, infiltration, and accumulation—of adoptively transferred CAR T cells in heterogenous antigen-expressing solid tumors can reveal critical information to interpret the response to immunotherapy. The authors performed serial quantitative imaging of tumor burden and CAR T cells in clinically relevant models of thoracic cancers.

The aim of adoptive T-cell therapy is to promote tumor-infiltrating immune cells following the transfer of either tumor-harvested or genetically engineered T lymphocytes. A new chapter in adoptive T-cell therapy began with the success of chimeric antigen receptor (CAR) T-cell therapy. T cells harvested from peripheral blood are transduced with genetically engineered CARs that render the ability to recognize cancer cell-surface antigen and lyse cancer cells. The successes in CAR T-cell therapy for B-cell leukemia and lymphoma have led to efforts to expand this therapy to solid tumors. Herein, we discuss the rationale behind the preclinical development and clinical trials of T-cell therapies in patients with malignant pleural mesothelioma. Furthermore, we highlight the ongoing investigation of combination immunotherapy strategies to synergistically potentiate endogenous as well as adoptively transferred immunity.

Plasma-derived polyclonal antibody therapeutics, such as intravenous immunoglobulin, have multiple drawbacks, including low potency, impurities, insufficient supply, and batch-to-batch variation. Here we describe a microfluidics and molecular genomics strategy for capturing diverse mammalian antibody repertoires to create recombinant multivalent hyperimmune globulins. Our method generates thousands-diverse mixtures of recombinant antibodies, enriched for specificity and activity against therapeutic targets. Each hyperimmune globulin product comprised thousands to tens of thousands of antibodies derived from convalescent or vaccinated human donors, or immunized mice. Using this approach, we generated hyperimmune globulins with potent neutralizing activity against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in under three months, Fc-engineered hyperimmune globulins specific for Zika virus that lacked antibody-dependent enhancement of disease, and hyperimmune globulins specific for lung pathogens present in patients with primary immune deficiency. To address the limitations of rabbit-derived anti-thymocyte globulin (ATG), we generated a recombinant human version and demonstrated its efficacy in mice against graft-versus-host disease.

In pursuit of a preventive therapeutic for maternal autoantibody-related (MAR) autism, we assessed the toxicity, biodistribution, and clearance of a MAR specific peptide-functionalized dextran iron oxide nanoparticle system in pregnant murine dams. We previously synthesized ~15 nm citrate-coated dextran iron oxide nanoparticles (DIONPs), surface-modified with polyethylene glycol and MAR peptides to produce systems for nanoparticle-based autoantibody reception and entrapments (SNAREs). First, we investigated their immunogenicity and MAR lactate dehydrogenase B antibody uptake in murine serum in vitro. To assess biodistribution and toxicity, as well as systemic effects, we performed in vivo clinical and post mortem pathological evaluations. We observed minimal production of inflammatory cytokines—interleukin 10 (IL-10) and IL-12 following in vitro exposure of macrophages to SNAREs. We established the maximum tolerated dose of SNAREs to be 150 mg/kg at which deposition of iron was evident in the liver and lungs by histology and magnetic resonance imaging but no concurrent evidence of liver toxicity or lung infarction was detected. Further, SNAREs exhibited slower clearance from the maternal blood in pregnant dams compared to DIONPs based on serum total iron concentration. These findings demonstrated that the SNAREs have a prolonged presence in the blood and are safe for use in pregnant mice as evidenced by no associated organ damage, failure, inflammation, and fetal mortality. Determination of the MTD dose sets the basis for future studies investigating the efficacy of our nanoparticle formulation in a MAR autism mouse model.

Anti-CTLA-4 antibodies such as ipilimumab were among the first immuno-oncology agents to show significantly improved outcomes for patients. However, existing anti-CTLA-4 therapies fail to induce a response in a majority of patients and can induce severe, immune-related adverse events. It has been assumed that checkpoint inhibition, i.e., blocking the interaction between CTLA-4 and its ligands, is the primary mechanism of action for ipilimumab. Here we present evidence that checkpoint inhibition may not be the primary mechanism of action for efficacy of anti-CTLA-4 antibodies. Instead, the primary mechanism for efficacy may be FcR-mediated Treg depletion in the tumor microenvironment. First, we identified a monoclonal antibody (mAb), GIGA-564, that binds to CTLA-4 at an epitope that differs from ipilimumab’s by only a few amino acids, yet has limited checkpoint inhibitor activity. Surprisingly, GIGA-564 has superior anti-tumor activity compared to ipilimumab in a murine model. GIGA-564 also induces less Treg proliferation and has increased ability to induce in vitro FcR signaling and in vivo depletion of intratumoral Tregs. Further experiments showed that the enhanced FcR activity of GIGA-564 likely contributes to its enhanced anti-tumor activity. Importantly, we also showed that GIGA-564 was associated with lower toxicity in murine models. Our work suggests that new anti-CTLA-4 drugs should be optimized for Treg depletion rather than checkpoint inhibition. Citation Format: Erica L Stone, Kyle P Carter, Ellen K Wagner, Michael A Asensio, Emily Benzie, Yao Yuan Chiang, Garry L Coles, Chelsea Edgar, Bishal K Gautam, Ashley Gras, Jackson Leong, Renee Leong, Vishal A Manickam, Rena A Mizrahi, Ariel R Niedecken, Jasmeen Saini, Savreet K Sandhu, Jan Fredrick Simons, Kacy Stadtmiller, Brendan Tinsley, LaRee Tracy, Nicholas P Wayham, Yoong Wearn Lim, Adam S Adler, David S Johnson. GIGA-564, a third generation anti-CTLA-4 with minimal ability to block CTLA-4 binding to B7 ligands, has enhanced efficacy but reduced toxicity compared to ipilimumab in pre-clinical models [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A32.

Anti-CTLA-4 antibodies such as ipilimumab were among the first immune-oncology agents to show significantly improved outcomes for patients. However, existing anti-CTLA-4 therapies fail to induce a response in a majority of patients and can induce severe, immune-related adverse events. It has been assumed that checkpoint inhibition, i.e., blocking the interaction between CTLA-4 and its ligands, is the primary mechanism of action for ipilimumab. In this study we present evidence that checkpoint inhibition is not a primary mechanism of action for efficacy of anti-CTLA-4 antibodies. Instead, the primary mechanism for efficacy is FcR-mediated Treg depletion in the tumor microenvironment. First, we identified a monoclonal antibody (mAb) that binds to CTLA-4 at an epitope that differs from ipilimumab’s by only a few amino acids, yet has limited checkpoint inhibitor activity. Surprisingly, the weak checkpoint inhibitor has superior anti-tumor activity compared to ipilimumab in a murine model. The weak checkpoint inhibitor also induces less Treg proliferation and has increased ability to inducein vitroFcR signaling andin vivodepletion of intratumoral Tregs. Further experiments showed that the enhanced FcR activity of the weak checkpoint inhibitor likely contributes to its enhanced anti-tumor activity. Importantly, we also showed that weak checkpoint inhibition was associated with lower toxicity in murine models. Our work suggests that new anti-CTLA-4 drugs should be optimized for Treg depletion rather than checkpoint inhibition.

Plasma-derived polyclonal antibodies are polyvalent drugs used for many important clinical indications that require modulation of multiple drug targets simultaneously, including emerging infectious disease and transplantation. However, plasma-derived drugs suffer many problems, including low potency, impurities, constraints on supply, and batch-to-batch variation. In this study, we demonstrated proofs-of-concept for a technology that uses microfluidics and molecular genomics to capture diverse mammalian antibody repertoires as multivalent recombinant drugs. These “recombinant hyperimmune” drugs comprised thousands to tens of thousands of antibodies and were derived from convalescent human donors, or vaccinated human donors or immunized mice. Here we used our technology to build a highly potent recombinant hyperimmune for Severe Acute Respiratory Syndrome Coronavirus-2 (SARS CoV-2) in less than three months. We also validated a recombinant hyperimmune for Zika virus disease that abrogates antibody-dependent enhancement (ADE) through Fc engineering. For patients with primary immune deficiency (PID), we built high potency polyvalent recombinant hyperimmunes against pathogens that commonly cause serious lung infections. Finally, to address the limitations of rabbit-derived anti-thymocyte globulin (ATG), we generated a recombinant human version and demonstrated in vivo function against graft-versus-host disease (GVHD). Recombinant hyperimmunes are a novel class of drugs that could be used to target a wide variety of other clinical applications, including cancer and autoimmunity.

Zirconium substituted zinc–nickel ferrite nanoparticles with chemical composition of Zr x Zn 0.5− x Ni 0.5 Fe 2 O 4 (0≤ x ≤0.25) have been synthesized successfully by solution combustion method using high purity nitrates and fueling agent urea. Powder XRD study confirms the single phase formation of composite ferrite which belongs to cubic spinel structure. TEM further reveals the morphology of well dispersed Zn–Ni ferrite nanoparticles to be spherical. Effect of change of doping concentration of Zirconium is observed using FTIR. The saturation magnetization gradually decreases with the increase in Zr substitution and reaches minimum when x =0.25 whereas the coercivity value reaches minimum when x =0.15. DC electrical resistivity has been found to vary with increasing Zr content. The good electrical resistivity (>10 7 Ωcm) qualify the ceramic for high frequency transformer applications.

Background: We previously established the safety and antitumor efficacy of regionally delivered mesothelin-targeted M28z chimeric antigen receptor (CAR) T cells combined with programmed death-1 (PD-1) antibody (NCT02414269). As a next step, we developed next-generation CAR T cells equipped with a modified CD3z signaling domain with loss-of-function mutations within 2 of 3 ITAM motifs (1XX), and a PD-1 dominant negative receptor (PD1DNR) that provides T-cell intrinsic checkpoint blockade (M28z1XXPD1DNR CAR T cells). Herein, we provide evidence of the preclinical safety and enhanced antitumor efficacy of clinical-grade M28z1XXPD1DNR CAR T cells. Methods: Comparative cytotoxicity, proliferation, and cytokine secretion of human T cells engineered to express M28z or M28z1XXPD1DNR CAR were assessed by chromium-release, accumulation, and Luminex assays, respectively. The antitumor efficacy of a single dose (1x105 CAR T cells; E:T 1:1000) of intrapleurally administered M28z or M28z1XXPD1DNR CAR T cells was investigated in NSG mice with orthotopic pleural mesothelioma by serial bioluminescence imaging and by comparing survival. Following tumor eradication, functional persistence of CAR T cells was tested by repeated tumor challenge (increasing doses of 2x106 to 10x106 tumor cells). Results: In vitro, both M28z and M28z1XXPD1DNR CAR T cells exhibited antigen-specific cytotoxicity, accumulation, and effector cytokine secretion (table). In vivo, a single dose of M28z1XXPD1DNR CAR T cells led to tumor eradication, mice exhibited enhanced survival with weight gain, and resistance to tumor reestablishment upon 10 tumor rechallenges (table) versus a single dose of M28z CAR T cells. Table.In vitro and in vivo characteristics of M28z and M28z1XXPD1DNR CAR T-cell constructsM28zM28z1XXPD1DNRTargetMesothelinMesothelinCostimulatory domainCD28CD28CD3zNo mutations2 ITAM mutations (1XX)T-cell intrinsic checkpoint blockade (PD1DNR)NoYesIn vitro resultsHuman T-cell transduction, range25%-82%30%-89%PD-1 extracellular domain mRNA expression compared to untransduced, fold4158Cytotoxicity, rangeE:T 10:135%-45%25%-51%E:T 5:128%-44%20%-38%E:T 2:117%-32%14%-24%Accumulation, range, fold110-39053-622Effector cytokines (E:T 1:1, 24 h), rangeIL-214-23 ng/mL9-19 ng/mLTNF-α545-977 pg/mL380-852 pg/mLIFN-γ8-11 ng/mL6-15 ng/mLIn vivo resultsTumor eradication26 days19 daysMedian survival56 daysNot reachedTumor progression as measured by bioluminescence imaging following rechallengeRechallenged 3 times over 15 days+1 log+0.2 logsRechallenged 10 times over 52 days+3-4 logs+0.5 logsCurrent statusIn clinical trialIND submission pending Conclusion: Supported by the safety, tumor eradication, and functional persistence, M28z1XXPD1DNR CAR T cells will advance to IND submission and initiation of a phase I clinical trial in patients with pleural mesothelioma, and further extend our investigation to other mesothelin-expressing solid tumors. Citation Format: Stefan Kiesgen, Camille Linot, Hue T. Quach, Jasmeen Saini, Rebecca Bellis, Srijita Banerjee, Zhaohua Hou, Navin K. Chintala, Michel Sadelain, Prasad S. Adusumilli. Regional delivery of clinical-grade mesothelin-targeted CAR T cells with cell-intrinsic PD-1 checkpoint blockade: Translation to a phase I trial [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-378.

16 Background: We and others have published that antigen-stress induced functional exhaustion of chimeric antigen receptor (CAR) T cells can be rescued by addition of anti-PD1 agents. To avoid the need for multiple administrations of anti-PD1 agent, we developed CAR T-cell intrinsic PD1 dominant negative receptor (PD1 DNR). Herein, we investigated the anti-tumor efficacy of cell-extrinsic versus intrinsic anti-PD1 strategies. Methods: Human T cells transduced with CD28 costimulated mesothelin-targeted CAR T cells (M28z) with or without anti-PD1 agent, and M28zPD1DNR CAR T cells were investigated against mesothelin-expressing cancer cells with inducible or constitutive PDL1 over expression. In vitro, cytotoxicity upon single and multiple antigen stimulation and proliferation, inhibitory receptor expression (PD1, TIM3 LAG3) were tested. In vivo, tumor burden regression kinetics and median survival were determined in an orthotopic model of pleural mesothelioma. Results: Following single antigen stimulation, both CAR T cells exhibited equivalent cytotoxicity. Following multiple antigen stimulations (antigen stress test), M28z PD1DNR CARs showed sustained and relatively higher cytotoxicity and proliferation compared to M28z CAR T cells, even when combined with anti-PD1 agent. PD1, TIM3 and LAG3 expression upregulation was noticed in both CAR T cells. CAR T cell therapy combined with extrinsic PD1 blockade or intrinsic PD1 DNR demonstrated enhanced and sustained tumor regression and prolonged survival (median survival 66 and 60 days) compared to CAR T cell therapy alone (27.5 days). In mouse model with constitutive PDL1 overexpressing cancer, similar trend is observed (74, 69 versus 28 days). Conclusions: A single dose of M28z CAR T cells with cell-intrinsic PD1DNR demonstrated equal anti-tumor efficacy compared to multiple doses of extrinsic anti-PD1 agent administration in models with inducible and constitutive high-PDL1 expressing cancer with no on-target, off-tumor toxicity with either strategy. This data provides the rationale for our ongoing phase II trial of combination therapy with M28z CAR T cells and anti-PD1 agent, and upcoming M28zPD1DNR CAR T cell trial in 2020.