MGTA-145 in Combination with Plerixafor Rapidly Mobilizes High Numbers of Hematopoietic Stem Cells and Graft-Versus-Host Disease Inhibiting Myeloid-Derived Suppressor Cells in Non-Human Primates

Abstract Background. The majority of bone marrow transplants (BMTs) are performed with granulocyte-colony stimulating factor (G-CSF) mobilized peripheral blood (mPB) as the source of hematopoietic stem cells (HSCs) for patients. Up to 80% of mPB allogeneic recipients, however, will experience graft-versus-host disease (GvHD). Despite higher levels of CD3+ T cells in mPB grafts compared to BM, the level of acute GvHD observed following transplant of HLA-matched mPB is comparable to HLA-matched BM. One explanation is that G-CSF mobilized grafts contain myeloid-derived suppressor cells (MDSCs) possessing potent immunosuppressive properties capable of inhibiting T cell proliferation in vitro. The percentage of MDSCs is variable in grafts mobilized with G-CSF and clinical data suggest that patients transplanted with mPB grafts that contain higher numbers of MDSCs may have better outcomes including lower rates of acute GvHD (Vendramin et al., BBMT 2014). Identification of a mobilizing regimen that consistently produces high numbers of HSCs and MDSCs may be preferred. We recently reported that MGTA-145 (GroβT), a CXCR2 agonist, when combined with the CXCR4 inhibitor, plerixafor, robustly mobilizes HSCs (Blood 2017 130:1920). In this study, non-human primates (NHPs) were mobilized with a single dose of MGTA-145, plerixafor, or MGTA-145/plerixafor versus a multi-dose regimen of G-CSF, and mPB was harvested to allow detailed immune profiling at 0 through 24 hours. We observed a significant and rapid increase in number of HSCs and CD34dim monocytes with potent in vitro and in vivo immunosuppressive properties. Results. MGTA-145/plerixafor consistently produced a 16-fold increase in number of CD34+CD90+CD45RA- HSCs within four hours of dosing (p=0.0003, n=11). Profiling of graft subsets from these primates also showed a 10-fold increase over baseline in the number of CD34dim monocytes at 4 hours post treatment (p<0.0001, n=11, Figure 1A) that corresponded to 2-3-fold higher frequency and number compared to G-CSF or plerixafor alone (p<0.01, n=2-5) and correlated with degree of HSC mobilization (p<0.0001). To determine if this monocytic cell population had immunosuppressive properties, CD34dim cells were sorted from peripheral blood of NHPs treated with MGTA-145/plerixafor and co-cultured with anti-CD2, anti-CD3 and anti-CD28-stimulated autologous T cells. MGTA-145/plerixafor CD34dim monocytes suppressed T cell proliferation, as measured by CFSE staining after four days. To assess whether these immunosuppressive monocytes may prevent GvHD, we developed a xenograft GvHD model in NSG mice. MGTA-145/plerixafor mPB (6 x 106 PBMCs) containing a high percentage of CD34dim monocytes were injected into sublethally irradiated NSG mice. This was compared to unmobilized primate PBMCs (6 x 106 PBMCs) containing relatively low numbers of CD34dim cells. At day 20, all mice (8/8) transplanted with unmobilized PBMCs had died of acute GvHD compared to none of the mice transplanted with MGTA-145/plerixafor mPB. Mice transplanted with unmobilized PBMCs also demonstrated 3-fold higher numbers of T-cells and increased T-cell activation compared to mice transplanted with MGTA-145/plerixafor mobilized PBMCs (p<0.01, n=6-8). At day 60 post-transplant, 7/8 mice remained alive (Figure 1B, p<0.0001). To assess whether this immunosuppressive effect is due to CD34dim monocytes, we sorted these cells and transplanted PBMCs depleted of CD34dim monocytes into NSG mice. In addition, experiments comparing the number and function of primate HSCs mobilized by MGTA-145/plerixafor or G-CSF alone using the NSG engraftment model and using autologous NHP transplant coupled with ex vivo HSC gene therapy are ongoing. Conclusions. Co-administration of MGTA-145/plerixafor in NHPs results in both rapid and efficacious mobilization of highly enriched HSCs and a CD34dim monocyte population with potent immunosuppressive activity compared to cells mobilized with plerixafor alone or with the current standard of care, G-CSF. The increased number of these immunosuppressive monocytes compared to G-CSF has the potential to reduce GvHD in the allogenic transplant setting. Thus, MGTA-145/plerixafor may offer an advantageous graft in the allogeneic setting where the risk of GvHD remains a significant clinical problem. IND-enabling studies of MGTA-145 are in progress to assess this regimen for mPB collection and transplant. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Falahee:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Li:Magenta Therapeutics: Employment, Equity Ownership. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Morrow:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

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Identification of Small Molecule Modulators to Enhance the Therapeutic Properties of Chimeric Antigen Receptor T Cells

Blood ◽

10.1182/blood.v128.22.4712.4712 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4712-4712

Author(s):

Jonathan Rosen ◽

Betsy Rezner ◽

David Robbins ◽

Ian Hardy ◽

Eigen Peralta ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

T Cell ◽

Small Molecules ◽

Small Molecule ◽

Employment Equity ◽

Car T Cells ◽

Car T ◽

Equity Ownership

Abstract Adoptive cellular therapies using engineered chimeric antigen receptor T cells (CAR-T cells) are rapidly emerging as a highly effective treatment option for a variety of life-threatening hematological malignancies. Small molecule-mediated modulation of T cell differentiation during the in vitro CAR-T manufacturing process has great potential as a method to optimize the therapeutic potential of cellular immunotherapies. In animal models, T cells with a central or stem memory (TCM/SCM) phenotype display enhanced in vivoefficacy and persistence relative to other T cell subpopulations. We sought to identify small molecules that promote skewing towards a TCM/SCM phenotype during the CAR-T manufacturing process, with associated enhanced viability, expansion and metabolic profiles of the engineered cells. To this end, we developed a high-throughput functional screening platform with primary human T cells using a combination of high-content immunophenotyping and gene expression-based readouts to analyze cells following a high-throughput T cell culture platform that represents a scaled-down model of clinical CAR-T cell production. Multicolor flow cytometry was used to measure expansion, cell viability and the expression levels of cell surface proteins that define TCM cells (e.g., CCR7, CD62L and CD27) and markers of T cell exhaustion (e.g., PD1, LAG3, and TIM3). In parallel, a portion of each sample was evaluated using high content RNA-Seq based gene expression analysis of ~100 genes representing key biological pathways of interest. A variety of known positive and negative control compounds were incorporated into the high-throughput screens to validate the functional assays and to assess the robustness of the 384-well-based screening. The ability to simultaneously correlate small molecule-induced changes in protein and gene expression levels with impacts on cell proliferation and viability of various T cell subsets, enabled us to identify multiple classes of small molecules that favorably enhance the therapeutic properties of CAR-T cells. Consistent with results previously presented by Perkins et al. (ASH, 2015), we identified multiple PI3K inhibitors that could modify expansion of T cells while retaining a TCM/SCM phenotype. In addition, we identified small molecules, and small molecule combinations, that have not been described previously in the literature that could improve CAR-T biology. Several of the top hits from the screens have been evaluated across multiple in vitro (e.g., expansion, viability, CAR expression, serial restimulation/killing, metabolic profiling, and evaluation of exhaustion markers) and in vivo (e.g., mouse tumor models for persistence and killing) assays. Results from the initial screening hits have enabled us to further refine the optimal target profile of a pharmacologically-enhanced CAR-T cell. In addition, we are extending this screening approach to identify small molecules that enhance the trafficking and persistence of CAR-T cells for treating solid tumors. In conclusion, the approach described here identifies unique small molecule modulators that can modify CAR-T cells during in vitro expansion, such that improved profiles can be tracked and selected from screening through in vitro and in vivo functional assays. Disclosures Rosen: Fate Therapeutics: Employment, Equity Ownership. Rezner:Fate Therapeutics, Inc: Employment, Equity Ownership. Robbins:Fate Therapeutics: Employment, Equity Ownership. Hardy:Fate Therapeutics: Employment, Equity Ownership. Peralta:Fate Therapeutics: Employment, Equity Ownership. Maine:Fate Therapeutics: Employment, Equity Ownership. Sabouri:Fate Therapeutics: Employment, Equity Ownership. Reynal:Fate Therapeutics: Employment. Truong:Fate Therapeutics: Employment, Equity Ownership. Moreno:Fate Therapeutics, Inc.: Employment, Equity Ownership. Foster:Fate Therapeutics: Employment, Equity Ownership. Borchelt:Fate Therapeutics: Employment, Equity Ownership. Meza:Fate Therapeutics: Employment, Equity Ownership. Thompson:Juno Therapeutics: Employment, Equity Ownership. Fontenot:Juno Therapeutics: Employment, Equity Ownership. Larson:Juno Therapeutics: Employment, Equity Ownership. Mujacic:Juno Therapeutics: Employment, Equity Ownership. Shoemaker:Fate Therapeutics: Employment, Equity Ownership.

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Prophylactic Itacitinib (INCB039110) for the Prevention of Cytokine Release Syndrome Induced By Chimeric Antigen Receptor T-Cells (CAR-T-cells) Therapy

Blood ◽

10.1182/blood-2019-128288 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1934-1934 ◽

Cited By ~ 3

Author(s):

Eduardo Huarte ◽

Roddy S O'Connor ◽

Melissa Parker ◽

Taisheng Huang ◽

Michael C. Milone ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cytokine Release ◽

Employment Equity ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Equity Ownership

Background: T-cells engineered to express a chimeric antigen receptor (CAR-T-cells) are a promising cancer immunotherapy. Such targeted therapies have shown long-term relapse survival in patients with B cell leukemia and lymphoma. However, cytokine release syndrome (CRS) represents a serious, potentially life-threatening, side effect often associated with CAR-T cells therapy. The Janus kinase (JAK) tyrosine kinase family is pivotal for the downstream signaling of inflammatory cytokines, including interleukins (ILs), interferons (IFNs), and multiple growth factors. CRS manifests as a rapid (hyper)immune reaction driven by excessive inflammatory cytokine release, including IFN-g and IL-6. Itacitinib is a potent, selective JAK1 inhibitor which is being clinically evaluated in several inflammatory diseases. Aims: To evaluate in vitro and in vivo the potential of itacitinib to modulate CRS without impairing CAR-T cell anti-tumor activity. Materials and Methods: In vitro proliferation and cytotoxic activity of T cells and CAR-T cells was measured in the presence of increasing concentrations of itacitinib or tocilizumab (anti-IL-6R). To evaluate itacitinib effects in vivo, we conducted experiments involving adoptive transfer of human CD19-CAR-T-cells in immunodeficient animals (NSG) bearing CD19 expressing NAMALWA human lymphoma cells. The effect of itacitinib on cytokine production was studied on CD19-CAR-T-cells expanded in the presence of itacitinib or tocilizumab. Finally, to study whether itacitinib was able to reduce CRS symptoms in an in vivo setting, naïve mice were stimulated with Concanavalin-A (ConA), a potent T-cell mitogen capable of inducing broad inflammatory cytokine releases and proliferation. Results: In vitro, itacitinib at IC50 relevant concentrations did not significantly inhibit proliferation or anti-tumor killing capacity of human CAR-T-cells. Itacitinib and tocilizumab (anti-IL-6R) demonstrated a similar effect on CAR T-cell cytotoxic activity profile. In vivo, CD19-CAR-T-cells adoptively transferred into CD19+ tumor bearing immunodeficient animals were unaffected by oral itacitinib treatment. In an in vitro model, itacitinib was more effective than tocilizumab in reducing CRS-related cytokines produced by CD19-CAR-T-cells. Furthermore, in the in vivo immune hyperactivity (ConA) model, itacitinib reduced serum levels of CRS-related cytokines in a dose-dependent manner. Conclusion: Itacitinib at IC50 and clinically relevant concentrations did not adversely impair the in vitro or in vivo anti-tumor activity of CAR-T cells. Using CAR-T and T cell in vitro and in vivo systems, we demonstrate that itacitinib significantly reduces CRS-associated cytokines in a dose dependent manner. Together, the data suggest that itacitinib may have potential as a prophylactic agent for the prevention of CAR-T cell induced CRS. Disclosures Huarte: Incyte corporation: Employment, Equity Ownership. Parker:Incyte corporation: Employment, Equity Ownership. Huang:Incyte corporation: Employment, Equity Ownership. Milone:Novartis: Patents & Royalties: patents related to tisagenlecleucel (CTL019) and CART-BCMA; Novartis: Research Funding. Smith:Incyte corporation: Employment, Equity Ownership.

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Protein Kinase C-Theta Interacts with mTORC2 and Vimentin to Limit Regulatory T-Cell Function

Blood ◽

10.1182/blood.v126.23.849.849 ◽

2015 ◽

Vol 126 (23) ◽

pp. 849-849

Author(s):

Cameron McDonald-Hyman ◽

Govindarajan Thangavelu ◽

James Muller ◽

Guoan Zhang ◽

Sudha Kumari ◽

...

Keyword(s):

T Cells ◽

Protein Kinase C ◽

T Cell ◽

Protein Kinase ◽

Acute Gvhd ◽

Hematopoietic Stem ◽

Phosphorylated Proteins ◽

Kinase C ◽

Treg Function

Abstract Regulatory T-cells (Tregs) play a critical role in preventing autoimmune and alloimmune reactions, including graft-versus-host disease (GVHD). Two recent clinical trials demonstrated that in patients undergoing hematopoietic stem cell transplantation, adoptive transfer of Tregs significantly reduced the incidence of grades II-IV GVHD. While Tregs significantly reduced GVHD severity, they did not eliminate GVHD. One potential way to augment Treg-mediated inhibition of GVHD is to increase Treg suppressive potency. We showed previously that Treg-specific inhibition of protein kinase C-theta (PKC-θ) enhances Treg function (Science 328:372, 2010). However, it is unclear whether PKC-θ inhibition can boost Treg function in a systemic inflammatory condition like GVHD. Furthermore, the mechanism by which PKC-θ inhibition augments Treg function is unknown. In this study, we address these unanswered questions. Using a mouse MHC class I/II disparate acute GVHD model, we found that freshly isolated Tregs treated for 30 minutes with 10uM of the clinically available PKC-θ inhibitor AEB071 suppressed GVHD mortality (Fig 1A) and severity significantly better than DMSO treated Tregs. As Tregs exert much of their protective effect against GVHD early in the course of the disease, we analyzed proliferation of GVHD-causing conventional T-cells (Tcon) on D4 after transplant. We observed a significant reduction in Tcon proliferation in mice given AEB071 treated Tregs compared to DMSO treated Tregs. We then performed multi-photon microscopy on D4 after transplant using TEα-GFP Tcon, CD11c-eYFP antigen presenting cells (APCs) and wild-type Tregs. Compared to DMSO, AEB071 treated Tregs significantly increased Tcon velocity and displacement from APCs. Increased velocity and displacement are indicative of decreased Tcon-APC interactions, suggesting reduced priming when AEB071 Tregs are present. Mechanistically, AEB071 vs DMSO treatment of Tregs resulted in augmented expression of the suppressive molecules Neuropilin-1 (Nrp1) and Lymphocyte activation gene 3 (Lag3) after in vitro activation (Fig 1B, C) and in Tregs isolated from acute GVHD mice. Antibody blockade of Nrp1 and Lag3 in in vitro transwell suppression assays reduced the effect of AEB071 treatment, suggesting that these molecules may play a role in enhancing Treg function after PKC-θ inhibition. Flow cytometry analysis of phosphorylated proteins in activated Tregs revealed that PKC-θ inhibition resulted in reduced phosphorylation of the mTORC2 target FoxO3a, but not mTORC1 targets S6 and 4E-BP1. In addition, the mTORC2-specific phosphorylation site on Akt, serine 473, was reduced, whereas the mTORC1-specific site, threonine 308, was unaltered. Together, these data suggest reduced mTORC2 activity. Reduced phosphorylation increases Foxo3a nuclear translocation, which may result in increased Nrp1 and Lag3 expression, since Foxo3a has binding sites in both gene promoters. As both mTORC1 and 2 are involved in T-cell metabolism, we investigated the effect of AEB071 treatment on Treg oxygen consumption rate (OCR). Compared to DMSO, AEB071 treatment significantly increased Treg baseline and maximal OCRs after activation (Fig 1D). Increased OCR has been associated with increased Treg function. To identify additional alterations in phosphorylated proteins after PKC-θ inhibition, we performed a phosphoproteomic screen using in vitro expanded human Tregs treated with AEB701 or DMSO. We identified significant alterations in phosphorylation sites on 72 proteins, including reduced phosphorylation of an adaptor molecule that links PKC-θ to the intermediate filament vimentin. We found that vimentin is highly upregulated in Tregs compared to Tcon and that in Tregs, vimentin interacts with PKC-θ after activation. AEB071 treatment reduced the interaction between vimentin and PKC-θ. As with AEB071 treatment, Vimentin siRNA significantly increased Treg suppression in vitro compared to control transfected Tregs (Fig 1E), and augmented expression of Nrp1 and Lag3. AEB071 treatment of vimentin siRNA transfected Tregs did not further augment Treg function, suggesting an overlapping mechanism. In summary, our data demonstrate that PKC-θ interacts with mTORC2 and vimentin to modulate multiple aspects of Treg function, and that a brief incubation of Tregs with a PKC-θ inhibitor may be a viable method to enhance the efficacy of Treg therapeutics. Disclosures No relevant conflicts of interest to declare.

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ALLO-715, an Allogeneic BCMA CAR T Therapy Possessing an Off-Switch for the Treatment of Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-119227 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 591-591 ◽

Cited By ~ 8

Author(s):

Cesar Sommer ◽

Bijan Boldajipour ◽

Julien Valton ◽

Roman Galetto ◽

Trevor Bentley ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Expansion ◽

Employment Equity ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Equity Ownership

Abstract Autologous chimeric antigen receptor (CAR) T cells targeting B-Cell Maturation Antigen (BCMA) have demonstrated promising clinical activity, inducing durable responses in patients with relapsed/refractory multiple myeloma (MM). Development of autologous CAR T therapies is however limited by logistical challenges and the time required for manufacturing, which has to be done for each patient. In addition, manufacturing may not be feasible in some patients. An allogeneic approach that utilizes engineered cells from a healthy donor could potentially expand patient access to these therapies by providing a readily available off-the-shelf product. We have previously described the screening of a library of single chain variable fragments (scFvs) with high affinity to human BCMA and the identification of candidate BCMA CARs with potent antitumor activity. Here we sought to further characterize ALLO-715, our lead allogeneic BCMA CAR T cell product, for its specificity to human BCMA, antitumor efficacy in vitro using a long-term killing assay and in xenograft mouse models with physiologic levels of human IL-7 and IL-15, and suitability for scale-up manufacturing. Allogeneic ALLO-715 CAR T cells were generated by lentiviral transduction with a second generation CAR construct incorporating a novel scFv derived from a fully-human antibody with high affinity to BCMA (KD value ~ 5 nM, determined at 37°C) and featuring a rituximab-driven off-switch. Transduced T cells were then transfected with mRNAs encoding Transcription Activator-Like Effector Nucleases (TALEN®) designed to specifically disrupt the T cell receptor alpha chain and CD52 loci. These modifications result in a cell product with a lower risk of TCR-mediated graft-versus-host disease and resistance to the CD52 antibody alemtuzumab, a lymphodepleting agent. BCMA CAR T cells exhibited robust cell expansion, with low levels of tonic signaling that resulted in minimal differentiation (> 50% Tscm/Tcm phenotype). In in vitro assays, ALLO-715 CAR T cells displayed potent cytotoxic activity when co-cultured with the target cell lines MM.1S, Molp-8, and BCMA-REH but negligible cytotoxicity against BCMA-negative REH cells. The high proliferative potential indicated by the high frequency of memory T cells was validated in long-term killing assays, where ALLO-715 CAR T cells showed substantial expansion in the presence of MM.1S cells with no evidence of exhaustion or diminished cytolytic activity after seven days of continuous exposure to target. The potency of ALLO-715 CAR T cells was unaffected by high concentrations of soluble BCMA (>10 ug/mL), which has been shown previously to interfere with the activity of some BCMA-specific CARs. In MM xenograft mouse models, ALLO-715 CAR T cells were highly efficacious at single dose. High serum IL-15 levels have been associated with CAR T cell expansion in clinical trials. To evaluate the impact of homeostatic cytokines on CAR T cell survival and antitumor activity in our xenograft models, mice were administered adeno-associated viruses (AAV) for the expression of human IL-7 and IL-15. In the presence of physiological concentrations of these cytokines, enhanced BCMA CAR T cell expansion and anti-tumor activity were observed. To assess potential off-target interactions of ALLO-715 CAR, tissue cross-reactivity studies were carried out on standard human tissue panels using a scFv-human IgG fusion protein. Consistent with the limited expression pattern of BCMA, reactivity was seen on scattered cells in lymphoid tissues such as tonsil and abundantly on BCMA-expressing cell lines, but no appreciable staining was detected in other tissues. We examined BCMA CAR T cells manufactured following a proprietary GMP-like clinical scale process and found that cell expansion and viability, T cell phenotype and in vivo antitumor efficacy were preserved. These results demonstrate the potential of ALLO-715 as a novel allogeneic BCMA CAR T therapy for the treatment of relapsed/refractory MM and other BCMA-positive malignancies. Disclosures Sommer: Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties. Boldajipour:Pfizer Inc.: Employment, Patents & Royalties. Valton:Cellectis.Inc: Employment, Equity Ownership, Patents & Royalties. Galetto:Cellectis SA: Employment, Equity Ownership, Patents & Royalties. Bentley:Allogene Therapeutics: Employment, Equity Ownership. Sutton:Allogene Therapeutics: Employment, Equity Ownership. Ni:Allogene Therapeutics: Employment, Equity Ownership. Leonard:Allogene Therapeutics: Employment, Equity Ownership. Van Blarcom:Allogene Therapeutics: Employment, Equity Ownership. Smith:Cellectis. Inc: Employment, Patents & Royalties. Chaparro-Riggers:Pfizer Inc.: Employment, Patents & Royalties. Sasu:Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties.

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Anti-CD19 ARTEMISTM Therapy Drastically Reduces Cytokine Release without Compromising Efficacy Against Preclinical Lymphoma Models

Blood ◽

10.1182/blood.v128.22.3354.3354 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3354-3354

Author(s):

Hong Liu ◽

Li Long ◽

Shon Green ◽

Lucas H Horan ◽

Bryan Zimdahl ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Growth ◽

Cytokine Release ◽

Raji Cells ◽

Car T Cells ◽

Nsg Mice ◽

Car T ◽

Equity Ownership

Abstract Anti-CD19 chimeric antigen receptor (CAR) T cell therapies for B cell malignancies have demonstrated the remarkable curative potential of T cell immunotherapies. However, in clinical trials anti-CD19-CAR T cells continue to trigger life threatening adverse events that are often associated with excessive cytokine release and excessive T-cell proliferation. We reasoned that the activation pathway of current CAR T cells could be altered to better regulate proliferation and cytokine secretion, and thus disentangle the correlation between cytokine release syndrome (CRS) and efficacy of T cell-based therapies. Through protein engineering, we developed the ARTEMISTM (1) signaling platform which when expressed on primary T-cells results in a dramatic reduction of cytokine release during tumor cell lysis, without sacrificing efficacy. Using a human phage display library, we also identified several human CD19 antibodies with improved specificity and affinity that will be less immunogenic as compared to the murine-derived anti-CD19 antibodies that are currently used in most trials. Our lead antibody clone CD19(7) was then engineered into both CD28z-CAR and ARTEMISTM platforms for comparison. When tested in vitro, both CD19(7)-ARTEMISTM T cells and CD19(7)-CD28z-CAR T cells specifically lysed multiple CD19+ leukemia and lymphoma cell lines with similar potencies. However, during the 16 hour killing assays, ARTEMIS™ T cells secreted over 1000-fold less IL-2 and dramatically lower levels of IFN-γ, GM-CSF, IL-10 and IL-6. ARTEMISTM T cells also accumulated less PD-1, LAG3, and TIM3 on their surface during culturing and following in vitro killing, indicating a diminished propensity for exhaustion. Furthermore, during in vitro T cell expansion, ARTEMISTM cells were enriched for naïve/central memory subpopulations, had lower expression of granzyme B, a marker of terminal differentiation, and had reduced rates of receptor internalization upon antigen engagement. These characteristics suggest that T-cells activated through the ARTEMISTM receptor will have improved persistence and long-term proliferation potential, as well as a safer, more controlled cytokine release when used for T-cell therapies. When tested in vivo against CD19+ Raji systematic lymphoma xenografts, intravenous administration of CD19(7)-ARTEMISTM T cells caused rapid, complete, and lasting tumor regression that was better than that achieved with an equal dose of CD19(7)-CD28z-CAR T cells (Figure 1). In agreement with our in vitro data, mice treated with ARTEMISTM T cells had nearly undetectable levels of cytokines in their blood at 24 hours post dosing, a time in which CD19(7)-CAR-treated mice had markedly elevated levels of human IFN-γ, IL-2, TNFα, and IL-10. While flow cytometry analysis of the peripheral blood showed that CD19(7)-CAR T cells expanded more rapidly in mice, CD19(7)-ARTEMISTM T cells better controlled Raji tumor growth and were negative for PD-1 expression which was high on circulating CAR T cells. At 7 weeks post dosing, a time when all ARTEMISTM T cell-treated mice had no detectable tumors, they were re-challenged with Raji lymphoma. While tumors grew rapidly in control mice, ARTEMISTM T cell-treated mice resisted the Raji lymphoma re-challenge, indicating that ARTEMISTM T cells persisted in these mice despite the absence of tumors and remained antigen-responsive (Figure 2). Our data demonstrates that CD19(7)-ARTEMISTM T cells are highly potent against lymphoma preclinical models while releasing drastically lower levels of cytokines. Thus we have developed and pre-clinically validated a novel fully human anti-CD19 T cell therapy that has the potential to persist longer in patients and, importantly, presents a lower risk of cytokine-related toxicities without compromising efficacy. A clinical trial testing CD19(7)-ARTEMISTM T cell therapy in humans is expected to begin in 2017. Figure 1 Raji lymphoma tumor growth in NSG mice treated with either donor-matched untransduced T cells (Mock), CD19(7)-CAR, or CD19(7)-ARTEMISTM T cells (5x106 receptor-positive cells per mouse) Figure 1. Raji lymphoma tumor growth in NSG mice treated with either donor-matched untransduced T cells (Mock), CD19(7)-CAR, or CD19(7)-ARTEMISTM T cells (5x106 receptor-positive cells per mouse) Figure 2 Raji lymphoma tumor growth in NSG mice previously treated with CD19(7)-ARTEMISTM T cells who had complete regression (0.5x106 Raji cells/mouse). As controls, Raji-naïve mice were implanted with Raji cells following an injection of Mock T cells. (1)ARTEMISTM is trademarked by Eureka Therapeutics, Inc. Figure 2. Raji lymphoma tumor growth in NSG mice previously treated with CD19(7)-ARTEMISTM T cells who had complete regression (0.5x106 Raji cells/mouse). As controls, Raji-naïve mice were implanted with Raji cells following an injection of Mock T cells. / (1)ARTEMISTM is trademarked by Eureka Therapeutics, Inc. Disclosures Liu: Eureka Therapeutics: Employment, Equity Ownership, Patents & Royalties. Long:Eureka Therapeutics: Employment, Equity Ownership. Green:Eureka Therapeutics: Employment. Horan:Eureka Therapeutics: Employment. Zimdahl:Eureka Therapeutics: Employment. Liu:Eureka Therapeutics: Employment, Equity Ownership, Patents & Royalties.

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TNB-486, a Novel Fully Human Bispecific CD19 x CD3 Antibody That Kills CD19-Positive Tumor Cells with Minimal Cytokine Secretion

Blood ◽

10.1182/blood-2019-123226 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4070-4070

Author(s):

Harbani Malik ◽

Ben Buelow ◽

Udaya Rangaswamy ◽

Aarti Balasubramani ◽

Andrew Boudreau ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

B Cell ◽

Tumor Cells ◽

Cytokine Secretion ◽

Employment Equity ◽

Equity Ownership

Introduction The restricted expression of CD19 in the B-cell lineage makes it an attractive target for the therapeutic treatment of B-cell malignancies. Many monoclonal antibodies and antibody drug conjugates targeting CD19 have been developed, including bispecific T-cell redirecting antibodies (T-BsAbs). In addition, anti-CD19 chimeric antigen receptor T-cells (CAR-T) have been approved to treat leukemia and lymphoma. However, despite the impressive depth of responses achieved by T-cell redirecting approaches such as T-BsAbs and CAR-T cells, toxicity from over-activation of T-cells remains a substantial limitation for this type of therapy, in particular neurotoxicity. In designing TNB-486, a novel CD19 x CD3 T-BsAb, we endeavored to retain activity against CD19-positive tumor cells while limiting the cytokine secretion thought to underlie toxicity from T-cell redirecting therapies. Utilizing TeneoSeek, a next generation sequencing (NGS)-based discovery pipeline that leverages in silico analysis of heavy chain only/fixed light chain antibody (HCA/Flic, respectively) sequences to enrich for antigen specific antibodies, we made a high affinity αCD19 HCA and a library of αCD3 Flic antibodies that showed a >2 log range of EC50s for T cell activation in vitro. Of note, the library contained a low-activating αCD3 that induced minimal cytokine secretion even at concentrations that mediated saturating T-cell dependent lysis of lymphoma cells (when paired with an αCD19 HCA). We characterized the relative efficacy and potential therapeutic window of this unique molecule, TNB-486, in vitro and in vivo and compared it to two strongly activating bispecific CD19 x CD3 antibodies similar to those currently available and in clinical development. Methods Affinity measurements of the αCD19 moiety were made via Biacore (protein) and flow cytometry (cell surface). Stability measurements were made by subjecting the molecule to thermal stress and the %aggregation was measured by Size Exclusion Chromatography. T-cell activation was measured via flow cytometry (CD69 and CD25 expression) and cytokine was measured by ELISA (IL-2, IL-6, IL-10, INF-ɣ, and TNFα) in vitro. Lysis of B-cell tumor cell lines (Raji, RI-1, and Nalm6) was measured via flow cytometry in vitro. In vivo, NOG mice were engrafted subcutaneously with NALM-6 or SUDHL-10 cells and intravenously with human peripheral blood mononuclear cells (huPBMC), and the mice treated with multiple doses of TNB-486 or negative or positive control antibody. Tumor burden was evaluated via caliper measurement. Pharmacodynamic/Pharmacokinetic (PK/PD) studies were performed in NOG mice. A pharmacokinetic (PK) study was performed in BALB/c mice, and a tolerability and PK study are ongoing in cynomolgus monkeys. Results TNB-486 bound to cell surface CD19 with single digit nanomolar affinity (~3nM). EC50s for cytotoxicity were in the single-digit nanomolar range for TNB-486, and sub-nanomolar for the strongly activating controls; TNB-486 maximum achievable lysis was identical to the positive controls. TNB-486 induced significantly less cytokine release for all cytokines tested compared to the positive controls even at doses saturating for tumor lysis. No off-target activation was observed in the absence of CD19 expressing target cells. In vivo, TNB-486 eradicated all CD19-positive tumors tested (NALM-6 and SUDHL10) at doses as little as 1µg administered every four days after tumors had reached ~200mm3. TNB-486 showed a PK profile consistent with other IgG molecules in mice (T1/2 ~6 days in mice). Conclusions TNB-486 induced comparable lysis of CD19-positive tumor cells as the strongly activating control bispecific antibodies while inducing significantly reduced cytokine secretion, even at doses saturating for tumor lysis in vitro. In vivo TNB-486 eradicated all tested CD19 positive tumor cell lines in established tumor models. No off-target binding was observed. In summary, TNB-486 shows promise as a lymphoma therapeutic differentiated from T-cell targeted therapies currently in the clinic and in clinical trials. Disclosures Malik: Teneobio, Inc.: Employment, Equity Ownership. Buelow:Teneobio, Inc.: Employment, Equity Ownership. Rangaswamy:Teneobio, Inc.: Employment, Equity Ownership. Balasubramani:Teneobio, Inc.: Employment, Equity Ownership. Boudreau:Teneobio, Inc.: Employment, Equity Ownership. Dang:Teneobio, Inc.: Employment, Equity Ownership. Davison:Teneobio, Inc.: Employment, Equity Ownership. Force Aldred:Teneobio, Inc.: Equity Ownership. Iyer:Teneobio, Inc.: Employment, Equity Ownership. Jorgensen:Teneobio, Inc.: Employment, Equity Ownership. Pham:Teneobio, Inc.: Employment, Equity Ownership. Prabhakar:Teneobio, Inc.: Employment, Equity Ownership. Schellenberger:Teneobio, Inc.: Employment, Equity Ownership. Ugamraj:Teneobio, Inc.: Employment, Equity Ownership. Trinklein:Teneobio, Inc.: Employment, Equity Ownership. Van Schooten:Teneobio, Inc.: Employment, Equity Ownership.

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Preclinical Evaluation of Human Placental-Derived Allogeneic CD19 CAR-T Cells Against B Cell Malignancies

Blood ◽

10.1182/blood-2019-130782 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3222-3222

Author(s):

Kathy Karasiewicz ◽

Shuyang He ◽

Mary Ng ◽

Kristina Tess ◽

Weifang Ling ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Receptor ◽

Employment Equity ◽

Car T Cells ◽

Car T ◽

Equity Ownership ◽

Robust Process

Celularity, Inc. is developing a CD19 CAR-T Cell therapy using an allogeneic platform derived from postpartum human placental cells. T cells isolated from placenta/ umbilical cord blood and genetically modified to express CD19 chimeric antigen receptor (CAR), termed Placental-derived (P-) CD19 CAR T cells, are in development for the treatment of B cell malignancies. Unlike adult peripheral blood mononuclear cell (PBMC)-derived T cells, P-T cells are mostly naïve (CD45RA+) and can be readily expanded while maintaining an earlier differentiation phenotype such as greater expression of naïve/ memory markers, lower expression of effector/ exhaustion markers, allowing for greater proliferative potential of these cells ex vivo. These cells are also known to have greater immune tolerance to HLA mismatch and display impaired allogeneic activation, contributing to lower incidences of severe graft-verse-host disease (GvHD) (Barker, et. al. Blood, 2001; Chen, et al. Biology of Blood and Marrow Transplantation, 2006), making them an attractive cell population for use as an allogeneic, adoptive cell therapy. A robust process for the isolation, transduction, and expansion of placental-derived T cells to generate "off-the-shelf" allogeneic P-CD19 CAR T cells was developed. Twenty-One day expanded, non-modified P-T cells (N=3) were compared to adult PBMCs for their allo-reactivity in a Xenogeneic GvHD model in NCG mice. P-T cells did not induce xeno-GvHD whereas PBMCs did, as evidenced by significant weight loss and death of all mice (N=5) by Day 28 post infusion. Despite expanded P-T cells demonstrating lack of in vivo GvHD, current manufacture of P-CD19 CAR T cells does include a CRISPR-mediated T-cell receptor a constant (TRAC) knockout (KO) step as an additional risk-mitigation strategy to circumvent any potential GvHD stemming from expression of endogenous T cell receptor. CD19 CAR transduction using a retrovirus provided by Sorrento Therapeutics, Inc., followed by TRAC knockout with CRISPR results in both high efficiency of CD19 CAR expression (~30% CD19 Fc+) and TCR KO (>96% CD3-/ TCR a/b-). In vitro, the functional activity of P-CD19 CAR-TRAC KO T cells against CD19+ Burkitt's Lymphoma (Daudi) and Acute lymphoblastic Leukemia (NALM6) cell lines was assessed in cytotoxicity and cytokine release assays. P-CD19 CAR T cells specifically lyse CD19+ Daudi/ Nalm6 targets in both 4-hour endpoint FACS and ACEA kinetic cytotoxicity assays, and in most cases at levels equivalent to or greater than PBMC-derived CD19 CAR T cells. When P-CD19 CAR T cells were co-cultured with CD19+ Daudi/ Nalm6 target cells for 24-hours, they secreted pro-inflammatory cytokines and effector proteins in an antigen-specific manner. In vivo, the anti-tumor activity of P-CD19 CAR T cells was assessed using a disseminated lymphoma xenograft model in NSG mice. Luciferase expressing Daudi cells (3×106) were intravenously (IV) injected on Day 0, followed by IV injection of P-CD19 CAR T cells (14×106) on Day 7. Bioluminescence Imaging (BLI) and survival were used as primary study endpoints. P- CD19 CAR T cells were well tolerated and safe. P-CD19 CAR T cells significantly reduced tumor burden, and improved survival. Four weeks after treatment, the vehicle group had a 100% mortality rate, while all animals from P-CD19 CAR T-treated group (N=5) remained alive without clinical symptoms including weight loss or changes in their fur. In summary, Celularity has defined a robust process for the generation and expansion of CD19 CAR T cells from human placenta. These cells exhibit potent anti-tumor activity both in vitro and in vivo with little evidence of acute GvHD induction, highlighting their potential as an allogeneic, adoptive cell therapeutic agent. Future in vivo GvHD studies will include assessment of both CD19 CAR and TRAC KO genetically modified P-T cells. Disclosures Karasiewicz: Celgene: Equity Ownership; Celularity, Inc.: Employment, Equity Ownership, Patents & Royalties: Patent Inventor. He:Celularity Inc: Employment. Ng:Celularity, Inc.: Employment. Tess:Celularity, Inc.: Employment. Ling:Celularity Inc: Employment. Kaufmann:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Zeldis:Sorrento Therapeutics Inc: Employment, Equity Ownership. Ji:Celularity, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Sorrento Therapeutics Inc: Employment, Equity Ownership, Patents & Royalties. Hariri:Celularity Inc: Employment. Zhang:Celularity Inc: Employment.

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ALLO-819, an Allogeneic Flt3 CAR T Therapy Possessing an Off-Switch for the Treatment of Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-117605 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3335-3335

Author(s):

Cesar Sommer ◽

Ivana Djuretic ◽

Julien Valton ◽

Duy Nguyen ◽

Janette Sutton ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Antitumor Efficacy ◽

Cell Phenotype ◽

Employment Equity ◽

Car T Cells ◽

Car T Cell ◽

Nsg Mice ◽

Car T ◽

Equity Ownership

Abstract Patients with relapsed acute myeloid leukemia (AML) have poor prognosis and limited treatment options. Chimeric antigen receptor (CAR) T cells have demonstrated unprecedented clinical efficacy in hematological malignancies, leading to durable responses in heavily pretreated patients. Adoptive immunotherapies using T cells redirected against AML cells are being pursued as one option with potential curative intent. However, the development of autologous CAR T therapies presents a significant logistical and clinical challenge in a rapidly progressing disease setting such as AML due to the lag time of cell manufacturing. Additionally, harvesting sufficient numbers of healthy T cells from patients with AML may not always be possible. For these reasons the development of an off-the-shelf CAR T cell product may be of benefit. This work details the preclinical evaluation of ALLO-819, an allogeneic CAR T therapy targeting the receptor tyrosine kinase Flt3 (CD135), an AML target with high prevalence in all AML subtypes and limited expression outside of the hematopoietic tissue. To construct a Flt3 CAR, a panel of high affinity (KD values of 0.19 to 233 nM, determined at 37°C) fully-human antibodies was generated using phage display technology. Single-chain variable fragments (scFvs) recognizing different immunoglobulin domains of the extracellular region of Flt3 were inserted into second-generation CAR constructs and tested for their ability to redirect T cell specificity and effector function towards AML cells. A lead CAR exhibiting minimal tonic signaling and potent antitumor activity in orthotopic mouse models of AML (2.5x106 and 1x107 CAR T cells for Eol-1 and Molm-13, respectively) was selected for further engineering to incorporate a safety off-switch in cis. To accomplish this, short amino acid stretches mimicking epitopes for the FDA-approved antibody rituximab were inserted between the hinge and target-binding regions of the CAR. The CAR T cell phenotype and antitumor efficacy were not affected by the presence of the off-switch. In the presence of rituximab, Flt3 CAR T cells were efficiently lysed via complement-dependent cytotoxicity (~ 80 % CAR T cell depletion in 3 hours) in vitro and eliminated in peripheral blood and bone marrow of NSG mice (>100-fold and >300-fold, respectively). Allogeneic ALLO-819 Flt3 CAR T cells with a lower risk of TCR-mediated graft-versus-host disease and resistant to anti-CD52 antibody (alemtuzumab)-mediated lysis were generated by disruption of the T-cell receptor alpha chain (TRAC) and the CD52 loci using TALEN® gene-editing technology. Transient expression of TALEN® in Flt3 CAR T cells resulted in high-efficiency inactivation of both loci and had no impact on T cell phenotype or antitumor efficacy. ALLO-819 Flt3 CAR T cells co-cultured with primary AML blasts ex vivo displayed target-dependent activation, cytokine secretion and cytotoxic activity. Consistent with previous reports, we detected Flt3 expression on a subset of normal hematopoietic stem and progenitor cells (HSPCs) which also showed susceptibility to CAR T cell cytotoxicity. To evaluate off-tumor effects of Flt3 CAR T cells in vivo, NSG mice were administered T cells expressing a CAR with similar affinity to both mouse and human Flt3. Mouse-cross-reactive Flt3 CAR T cells exhibited off-tumor activity that was limited to a subset of bone marrow multipotent progenitors and correlated with antitumor efficacy. Administration of rituximab led to effective depletion of CAR T cells in peripheral blood that was followed by a rapid repopulation of HSPCs to levels observed in naïve mice. In summary, these results support the development of ALLO-819 Flt3 CAR T as a novel immunotherapy for the treatment of AML. Disclosures Sommer: Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties. Djuretic:Pfizer Inc.: Employment. Valton:Cellectis.Inc: Employment, Equity Ownership, Patents & Royalties. Nguyen:Allogene Therapeutics: Employment, Equity Ownership. Sutton:Allogene Therapeutics: Employment, Equity Ownership. Poulsen:Allogene Therapeutics: Employment, Equity Ownership. Smith:Cellectis. Inc: Employment, Patents & Royalties. Djuretic:Pfizer Inc.: Employment. Chaparro-Riggers:Pfizer Inc.: Employment, Patents & Royalties. Sasu:Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties.

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Ex Vivo Expansion with E478 Increases the Rate of CRISPR-Mediated Homology Directed Repair of the Beta-Globin Gene in Human Hematopoietic Stem Cells By >10-Fold and Improves In Vivo Engraftment By >120-Fold

Blood ◽

10.1182/blood-2019-131157 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4634-4634

Author(s):

Kevin A. Goncalves ◽

Megan D. Hoban ◽

Sharon L. Hyzy ◽

Katia S. George ◽

Anthony E. Boitano ◽

...

Keyword(s):

Ex Vivo ◽

Globin Gene ◽

Fold Increase ◽

Ex Vivo Expansion ◽

Gene Correction ◽

Employment Equity ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Equity Ownership

Background . Site-specific gene correction of hematopoietic stem cells (HSCs) via homology directed repair (HDR) has the potential to precisely repair defective genes and provide life-long cures for a variety of blood-based diseases. It is possible to obtain high levels of HDR during in vitro HSC culture, but these cells fail to robustly engraft in vivo, suggesting that the procedure of HDR compromises HSC function or that true HSCs are not undergoing HDR. Cells need to be actively cycling in order to undergo HDR, but conditions that allow HSC replication in vitro without compromising HSC number and function remain elusive. Thus, most HDR protocols minimize time in culture, potentially limiting HDR rates and cell yield. We recently reported that ex vivo expansion of HSCs with an aryl hydrocarbon receptor (AHR) antagonist is a clinically validated method to expand HSCs. The AHR antagonist-expanded CD34+ cell therapy, MGTA-456, results in rapid and durable recovery in patients with hematologic malignancies and inherited metabolic diseases (Wagner et al Cell Stem Cell 2016; Orchard et al AAN 2019). To apply this technology to gene-modified HSCs, we developed a novel AHR antagonist, E478, which expands NSG-engrafting cells 10-fold compared to uncultured primary human mobilized peripheral blood (mPB) CD34+cells in limit dilution studies. We previously showed that expansion with E478 results in up to 10-fold higher engraftment of lentiviral vector (LVV)-transduced cells and CRISPR/Cas9 knockout cells (Hoban et al ASGCT 2019). Here, we demonstrate that ex vivo expansion of mPB CD34+ cells with E478 results in >10-fold increase in rate of HDR and >120-fold increase in NSG engraftment of HDR+ cells compared to conventional approaches. Results . To determine whether more active cycling would lead to higher rates of HDR, we cultured cells for 1, 2, 3, and 4 days prior to electroporation with CRISPR gRNA targeting the beta-globin gene and transduction with a GFP-containing adeno-associated virus (AAV) donor template. Cell cycle analysis revealed that 33±1.8% of cells enriched for HSCs (CD34+CD90+ cells) remain quiescent after 2 days in culture, whereas 0.92±0.06% of CD34+CD90+ cells were quiescent after 3 and 4 days in culture (n=2 mPB donors). We then assessed HDR rates and HSC number after 1, 2, 3, and 4 days of additional culture. Compared to a conventional HDR protocol utilizing a 2-day pre-stimulation period followed by 1 day of culture after electroporation (herein called a 2+1 culture), we observed up to 8-fold increase in HDR with longer pre-stimulation periods, but this was accompanied with differentiation of CD34+CD90+ cells and loss of engraftment in NSG mice (79% decrease, p<0.001). We next evaluated whether E478 could increase the dose of HSCs and maintain high HDR rates. We cultured mPB CD34+ cells with E478 for a 4 day pre-stimulation, performed HDR, and continued the expansion for 4 days with E478 (herein called 4+4 culture). With the 4+4 protocol, we observed a 6-fold increase in the rate of HDR in vitro and a 134-fold increase in the number of CD34+CD90+ cells with E478 relative to 2+1 conditions with DMSO vehicle (n=2, p<0.01). Transplant of these cells into sublethally-irradiated NSG mice resulted in a 4-fold higher rate of engraftment (Figure A, p<0.01, n=8 mice), 12-fold higher rates of HDR (Figure B, p<0.001) and >120-fold increase in the number of HDR+ NSG-engrafting cells relative to 2+1 cultures (Figure C, p<0.001). Further, a 2+1 culture with E478 led to an 8-fold increase in number of HDR+ NSG-engrafting cells (p<0.001) relative to standard 2+1 approaches without a small molecule. Multi-lineage engraftment was observed in all groups. Studies using E478 with bone marrow from patients with sickle cell disease are in progress and will be presented. Conclusions. We demonstrate that ex vivo HSC expansion with E478 enables higher rates of HDR and a high dose of HDR+ HSCs, leading to >120-fold increase in the engraftment of HDR+ HSCs compared to conventional 2+1 approaches. Culture with E478 is a promising approach to realize the full potential of targeted gene correction in HSCs for a variety of genetic diseases. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hoban:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. George:Magenta Therapeutics: Employment, Equity Ownership. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

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Superior Efficacy of CAR-T Cells Using Marrow-Infiltrating Lymphocytes (MILsTM) As Compared to Peripheral Blood Lymphocytes (PBLs)

Blood ◽

10.1182/blood-2019-130629 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4437-4437 ◽

Cited By ~ 2

Author(s):

Eric R. Lutz ◽

Srikanta Jana ◽

Lakshmi Rudraraju ◽

Elizabeth DeOliveira ◽

Jing Zhou ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Employment Equity ◽

Car T Cells ◽

Car T ◽

Equity Ownership

Background The type of T cell used in generating chimeric antigen receptor (CAR) T cells is an important choice. Evidence suggests that T cells that are early in the effector/memory differentiation pathway with more stemness and greater potential to persist are better than more differentiated T cells with less stemness that are more readily exhausted and have less potential to persist. Marrow-infiltrating Lymphocytes (MILsTM) is a novel form of adoptive T cell therapy composed of patient-autologous, polyclonal CD4 and CD8 T cells that are activated and expanded from the bone marrow. Genetically unmodified MILsTM have demonstrated antitumor activity in patients with multiple myeloma and are being developed for several other tumor types, including non-small cell lung cancer and other solid tumors. Distinguishing features of bone marrow T cells used to produce MILsTM include their memory phenotype, inherent tumor antigen-specificity, higher CD8:CD4 ratio and ability to persist long-term when compared to peripheral blood lymphocytes (PBLs) which is the T cell source used to produce currently approved CAR-T therapies. Based on these differences, we hypothesize that MILsTM provide a more robust and better fit platform for CAR-T therapy compared to PBLs. Using a CD38-specific, 4-1BB/CD3z-signaling CAR as an initial model, we have demonstrated the feasibility of producing CAR-modified MILsTM (CAR-MILsTM) and showed that CAR-MILsTM demonstrate superior killing in vitro compared to CAR-T cells generated from patient-matched PBLs (CAR-PBLs). Herein, we build on our previous data and add a second BCMA-specific CAR model. We use the two multiple myeloma model systems to compare cytolytic potential, functionality, and expression of phenotypic markers of memory, stemness and exhaustion between patient-matched CAR-MILsTM and CAR-PBLs. Methods Matched pairs of CAR-MILsTM and CAR-PBLs were produced from the bone marrow and blood of multiple myeloma patients. Two different in vitro cytotoxicity assays, the RTCA xCelligence real-time impedance and FACS assays, were used to evaluate antigen-specific killing of target tumor cells. Functionality of CD4 and CD8 CAR-T cells, at the single-cell level, was evaluated by measuring the secretion of 32 cytokines and chemokines following in vitro antigen-specific stimulation using IsoPlexis IsoCode chips and analyzed using IsoPeak. Expression of markers of T cell memory (CD45RO & CCR7/CD62L), stemness (CD27) and exhaustion (PD1 & TIM3) on CAR-MILsTM and CAR-PBLs prior to and following antigen-specific stimulation was evaluated by flow-cytometry (FACS). Results CAR-MILsTM demonstrated superior killing of tumor target cells in vitro, regardless of the antigen specificity of the CAR, when compared to matched CAR-PBLs and this superiority persisted even upon repeated antigen encounter - a factor that may be critical in guaranteeing better anti-tumor efficacy and persistence. CAR-MILsTM demonstrated increased polyfunctionality (secretion of 2+ cytokines per cell) and an increased polyfunctional strength index (PSI) following antigen-stimulation compared to CAR-PBL in both CD4 and CD8 T cells. The enhanced PSI in CAR-MILsTM was predominately mediated by effector, stimulatory and chemoattractive proteins associated with antitumor activity including Granzyme B, IFNg, IL-8, MIP1a and MIP1b. Coincidentally, increased PSI and enhanced secretion of these same proteins was reported to be associated with improved clinical responses in patients with Non-Hodgkin lymphoma treated with CD19-specific CAR-T therapy. Expression of memory markers on CD4 and CD8 T cells were similar in CAR-MILsTM and CAR-PBLs both prior to and following antigen-stimulation. Although expression of CD27, PD1 and TIM3 were similar at baseline, CAR-MILs maintained higher levels of CD27 and lower levels of PD1 and TIM3 compared to CAR-PBLs following antigen-stimulation in both CD4 and CD8 T cells. Conclusions Collectively, our data suggest that CAR-MILsTM have several advantages over CAR-PBLs, including increased cytolytic potential, enhanced polyfunctionality, increased stemness and less exhaustion. Based on these differences and the inherent antitumor properties of MILsTM, we speculate that CAR-MILsTM would be more potent and effective than currently approved CAR-T products derived from PBLs. Disclosures Lutz: WindMIL Therapeutics: Employment, Equity Ownership. Jana:WindMIL Therapeutics: Employment, Equity Ownership. Rudraraju:WindMIL Therapeutics: Employment, Equity Ownership. DeOliveira:WindMIL Therapeutics: Employment, Equity Ownership. Zhou:Isoplexis: Employment, Equity Ownership. Mackay:Isoplexis: Employment, Equity Ownership. Borrello:Aduro: Patents & Royalties: intellectual property on allogeneic MM GVAX; BMS: Consultancy; WindMIL Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Celgene: Honoraria, Research Funding, Speakers Bureau. Noonan:WindMIL Therapeutics: Employment, Equity Ownership, Patents & Royalties; Aduro: Patents & Royalties: intellectual property on allogeneic MM GVAX.

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