Bellicum Pharmaceuticals (United States)

Abstract Introduction: GoCAR is a chimeric antigen receptor (CAR) platform that utilizes a rimiducid-dependent activation switch (inducible MyD88/CD40; iMC) to enhance CAR-T cell proliferation and to stimulate the host immune system. To understand how these unique CAR signaling molecules compare to conventional costimulatory domains (e.g., CD28 and 4-1BB), we employed a detection and bioinformatics platform to assess the functional attributes of CD19-specific CAR-T cells by simultaneous detection of 32 proteins secreted from single CAR-T cells upon various stimulation conditions. Methods: T cells were transduced with retrovirus encoding CD19-specific CARs with conventional costimulatory molecules (CD19.ζ, CD19.28.ζ or CD19.BB.ζ) or with GoCAR, a vector encoding iMC and a CD19-specific CAR (iMC-CD19.ζ). Control or modified T cells were enriched by anti-CD4 or anti-CD8 microbeads and stimulated with CD19 or ΔNGFR-expressing K562 cells with or without 10 nM rimiducid. After 24-hour, CD4 and CD8 T cells were collected and loaded into an IsoCode chip containing ~12,000 microchambers. Each chamber (~1.2 nL) was pre-patterned with a complete copy of a 32-plex antibody array. Cells on-chip were imaged, and single-cell cytokine signals were captured with a microarray scanner. The T cell polyfunctional profile (2+ cytokines per cell) of single T cells was evaluated across 5 functional groups: Effector: Granzyme B, TNFα, IFNγ, MIP1α, Perforin, TNFβ; Stimulatory: GM-CSF, IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, IL-15, IL-21; Chemoattractive: CCL11, IP-10, MIP-1β, RANTES; Regulatory: IL-4, IL-10, IL-13, IL-22, sCD137, sCD40L, TGFβ1; Inflammatory: IL-6, IL-17A, IL-17F, MCP-1, MCP-4, IL-1β. Results: Single-cell multiplex proteomic analysis demonstrated the synergistic effects of CD19-specific and rimiducid-induced stimulation on polyfunctional upregulation in both CD4 and CD8 GoCAR-T cells across donors. In the “off” state (i.e., no rimiducid), CD19-specific GoCAR-T showed a reduced polyfunctional strength index (PSI) compared to CD28 and 4-1BB CAR-T cells, which was predominated by antitumor-associated proteins. In contrast, activation of iMC dramatically increased GoCAR-T PSI, which was characterized by elevated “stimulatory” (GM-CSF, IL-2, IL-5, IL-8, IL-9), “chemotactic” (MIP-1b) and “regulatory” (IL-4, sCD137) molecules compared to conventional CAR constructs. Polyfunctional heatmaps revealed that GoCAR-T cells co-secreted combinatorial proteins in response to CD19-expressing target cells in combination with iMC-based costimulation. Further, both CD4+ and CD8+ GoCAR-T cells acquired the ability to produce IL-2 as part of their polyfunctional profile, which was only observed with iMC costimulation. Conclusions: Single-cell multiplexed proteomic profiling reveals that polyfunctionality can be regulated by small molecule activation of an inducible costimulatory switch, producing distinct “on/off” states. iMC signaling also enhances production of important stimulatory and chemoattractive molecules which may improve CAR-T cell activity and provoke a host anti-tumor immune response. Thus, the GoCAR-T platform may potentially be used to optimize CAR-T activity through rimiducid administration to maximize efficacy while mitigating CAR-T-related toxicities. Citation Format: Aaron Foster, Joanne Shaw, Matthew Collinson-Pautz, Aruna Mahendravada, Christine Gagliardi, Mariam Khalil, Patrick Paczkowski, Sean Mackay, Jing Zhou. Single-cell multiplex proteomics reveals synergistic activity of antigen and MyD88/CD40 stimulatory signals on promoting polyfunctional chimeric antigen receptor T cells [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 898.

Traditionally, drug development has evaluated dose, safety, activity, and comparative benefit in a sequence of phases using trial designs and endpoints specifically devised for each phase. Innovations in drug development seek to consolidate the phases and rapidly expand accrual with “seamless” trial designs. Although consolidation and rapid accrual may yield efficiencies, widespread use of seamless first-in-human (FiH) trials without careful consideration of objectives, statistical analysis plans, or trial oversight raises concerns. A working group formed by the National Cancer Institute convened to consider and discuss opportunities and challenges for such trials as well as encourage responsible use of these designs. We reviewed all abstracts presented at American Society of Clinical Oncology annual meetings from 2010 to 2017 for FiH trials enrolling at least 100 patients. We identified 1786 early-phase trials enrolling 57 559 adult patients. Fifty-one of the trials (2.9%) investigated 50 investigational new drugs, were seamless, and accounted for 14.6% of the total patients. The seamless trials included a median of 3 (range = 1–13) expansion cohorts. The overall risk of clinically significant treatment-related adverse events (grade 3–4) was 49.1% (range = 0.0–100%), and seven studies reported at least one toxic death. Rapid expansion of FiH trials may lead to earlier drug approval and corresponding widespread patient access to active therapeutics. Nevertheless, seamless designs must adhere to established ethical, scientific, and statistical standards. Protocols should include prospectively planned analyses of efficacy in disease- or biomarker-defined cohorts of sufficient rigor to support accelerated approval.

Abstract Tissue damage contributes to initiation and modulation of an immune response. Tumor progression generally causes distress to the surrounding tissue. However, how tumor-induced parenchymal damage regulates anti-tumor immune response remains to be understood. We found that tumors that invaded brain parenchyma compressed the surrounding neurons causing increased expression of the neuronal chemokine CX3CL1/fractalkine in the peritumoral margin. Intravital two-photon microscopy revealed perivascular recruitment of monocyte-derived CD11c+ dendritic cells and T cells that interacted and killed individual cancer cells in tumor margins. Immune surveillance of brain tumors became inefficient in mice lacking the receptor for fractalkine, CX3CR1, resulting in more aggressive tumor progression. Our results identify tissue stress and associated chemokine signaling as a potential target to orchestrate anti-tumor immune surveillance in the brain.

Abstract T cells engineered to express the α and β chains of antigen-specific T cell receptors (TCRs) have shown promise as a cancer immunotherapy treatment; however, durable responses have been limited by poor persistence of gene-modified T cells. Additionally, severe toxicities, including patient deaths, have occurred upon infusion of large numbers of TCR-modified T cells. To enhance T cell persistence while providing a safeguard against life-threatening toxicity, we developed a dual-switch αβ TCR platform that uses a rapamycin (Rap)-induced caspase-9 (iRC9) together with a rimiducid (Rim)-controlled activation switch, inducible MyD88/CD40 (iMC). The αβ TCR sequence derived from an HLA-A2-restricted, PRAME-specific T cell clone was synthesized and placed in-frame with iMC, comprising signaling domains from MyD88 and CD40 fused to tandem Rim-binding mutant FKBP12v36 domains to generate the iMC-PRAME TCR. Caspase-9 was fused to FRB and wild-type FKBP12 domains and cloned in-frame with a selectable marker, truncated CD19 (ΔCD19), to generate iRC9-ΔCD19 retrovirus. All modules were separated by 2A polypeptide, bond-skipping sequences. Activated human T cells were dual-transduced with iMC-PRAME TCR and iRC9-ΔCD19 viruses and subsequently enriched for CD19 expression using magnetic selection. iMC and iRC9 were activated by exposing transduced T cells to 10 nM Rim or Rap, respectively. Proliferation, cytokine production and cytotoxicity of TCR-modified T cells were assessed in co-culture assays with U266 (myeloma) and THP-1 (AML) cells in the presence or absence of inducible ligands. T cells transduced with iMC-PRAME TCR and iRC9-ΔCD19 showed efficient and stable expression for TCR and ΔCD19 post-CD19 selection (82±9% CD3+Vβ1+, 96±2% CD3+CD19+). In coculture assays, dual-switch PRAME TCR-modified T cells demonstrated specific lysis of HLA-A2+PRAME+ THP-1 and U266 tumor cells compared to an irrelevant TCR (CMVpp65-specific)-modified T cells with or without iMC activation. However, Rim exposure induced a 42-fold induction of IL-2 (9±0.3 versus 385±180 pg/ml IL-2) and resulted in 13-fold greater expansion of TCR-modified T cells. The expression of iRC9 did not interfere with TCR function, nor with the synergy between TCR and iMC activation. Furthermore, exposure to Rap triggered rapid apoptosis of dual-switch TCR-modified T cells (72±5% Annexin-V+ with Rap versus 14±4% without drug), indicating that the suicide switch is also functional. iMC is a potent “Go” switch that utilizes rimiducid to provide costimulation to TCR-engineered T cells. In addition, iRC9 is a novel rapamycin-inducible suicide switch that can eliminate T cells in case of severe toxicity. This iMC-enhanced iRC9-incorporating TCR is the first reported prototype of a dual switch TCR-engineered T cell therapy to increase efficacy, durability and safety of adoptive T cell therapies. Citation Format: Tsvetelina Pentcheva-Hoang, Tania Rodriguez, David Torres, Ana Korngold, Jeannette Crisostomo, MyLinh T. Duong, Matthew Collinson-Pautz, J. Henri Bayle, Mirjam HM Heemskerk, J.H. Frederik Falkenburg, Annemarie Moseley, Kevin M. Slawin, David Spencer, Aaron E. Foster. Dual-switch TCR: A two-ligand system to control PRAME TCR-modified T cell proliferation and death using inducible MyD88/CD40 and caspase-9 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3745. doi:10.1158/1538-7445.AM2017-3745