Superior Immunogenicity of Idiotype Fab Fragments As Compared to Entire Immunoglobulin for Active Lymphoma Immunotherapy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1633-1633
Author(s):  
Marcelo A. Navarrete ◽  
Benjamin Kisser ◽  
Hendrik J. Veelken
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
B Cell Lymphomas ◽  
Cd8 Cells ◽  
Fab Fragments ◽  
The Individual

Abstract Abstract 1633 Introduction: The individual collection of epitopes within the variable regions of the unique immunoglobulin expressed by every mature B-cell lymphoma (idiotype, or Id) represents a tumor-specific antigen and lends itself as a target for therapeutic vaccination strategies. Immunization with tumor Id has the capacity to elicit polyclonal antibody responses as well as CD8+ and CD4+ T cells recognizing Id-derived peptides presented on class I and class II HLA molecules, respectively. Due to a perceived low immunogenicity of lymphoma-derived Id, most Id vaccines tested in clinical trials so far have been formulated as conjugates with the strongly immunogenic carrier keyhole limpet hemocyanin (KLH). In contrast, we have consistently observed high rates of humoral and cellular anti-Id immune responses in consecutive trials of active immunization with unconjugated recombinant Fab fragments of Id in indolent B-cell lymphomas (Bertinetti et al., Cancer Res. 2006; Navarrete et al., BLOOD 2011). We therefore hypothesized that Id Fab fragment might be intrinsically more immunogenic than entire Id Ig and tested this hypothesis by comparative in vitro experiments. Methods: Monocyte-derived dendritic cells (DC) where loaded with human monoclonal IgG, papain-digested Fab fragments, Fc fragments, or recombinant lymphoma-derived Fab fragments. Functional DC phenotypes were assessed by flow cytometry of crucial maturation and activation markers. IL-10 and IL-12 was measured in DC culture supernatants by ELISA. Antigen-loaded DC where subsequently used for priming of CFSE-labeled autologous peripheral blood mononuclear cells. Stimulated T cell populations were analyzed by multicolor flow cytometry. Results: Loading of DC with Fab, Fc, IgG, or mixtures of Fab and Fc fragments did not alter surface expression of CD11c, CD80, CD83, CD86, HLA-DR, PDL-1 and PDL-2 on DC. Likewise, the various antigens did not influence the cytokine release by DC during the loading or maturation process. DC loaded with isolated Fab fragments induced significantly higher proliferation of both CD4+ and CD8+ T cells than undigested IgG. The mean proliferation rate of CD4+ cells stimulated with Fab fragments was 18.5% versus 5.6% for undigested IgG stimulation (p=0.021); proliferation rates of CD8+ cells were 14.2% versus 6.2% (p=0.034). These results were reproduced for 4 different monoclonal IgGs tested on 4 different donors. The addition of Fc fragments to Fab reduced the proliferation rates of CD4+ and CD8+ cells to 10.2% and 8.6% respectively. In addition, DC loaded with undigested IgG induced a relative increase in the number of CD25high/FoxP3+ regulatory T cells compared with Fab stimulation (8.2% versus 1.4%; p<0.01). Conclusions: Isolated Fab fragments, i.e. the Id portions that contain the individual candidate antigenic epitopes of B-cell lymphomas, prime autologous T cells in vitro more efficiently than entire IgG. This finding is consistent with the high immune response rate against recombinant unconjugated Fab fragments observed in vivo in our clinical vaccination trials. Peptide sequences shared between Ig molecules that are predominantly located in the IgG Fc fragment appear to exert an inhibitory effect on T-cell priming. In accordance with our recent in vivo data in a syngeneic mouse model of Id vaccination (Warncke et al., Cancer Immunol. Immunother. 2011), this effect may be mediated by effective activation of Treg. Fab fragments therefore appear to be the more immunogenic and therefore preferable Ig antigenic format for active anti-Id immunotherapy. Furthermore, the inhibitory effects of IgG Fc offers a potential explanation for the recently reported lack of efficacy of Id vaccination in IgG-expressing follicular lymphomas in a randomized phase III trial, in which patients with IgM-expressing lymphomas, in contrast, had a significant benefit from Id vaccination in intention-to-treat analyses (Schuster et al., JCO 2011). Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
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.

scholarly journals CD28 Costimulation Is Required for In Vivo Induction of Peripheral Tolerance in CD8 T Cells

2002 ◽  
Vol 197 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Melanie S. Vacchio ◽  
Richard J. Hodes
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Tolerance Induction ◽  
Peripheral Tolerance ◽  
Cd8 Cells ◽  
Costimulatory Signal ◽  
Cd28 Costimulation

Whereas ligation of CD28 is known to provide a critical costimulatory signal for activation of CD4 T cells, the requirement for CD28 as a costimulatory signal during activation of CD8 cells is less well defined. Even less is known about the involvement of CD28 signals during peripheral tolerance induction in CD8 T cells. In this study, comparison of T cell responses from CD28-deficient and CD28 wild-type H-Y–specific T cell receptor transgenic mice reveals that CD8 cells can proliferate, secrete cytokines, and generate cytotoxic T lymphocytes efficiently in the absence of CD28 costimulation in vitro. Surprisingly, using pregnancy as a model to study the H-Y–specific response of maternal T cells in the presence or absence of CD28 costimulation in vivo, it was found that peripheral tolerance does not occur in CD28KO pregnants in contrast to the partial clonal deletion and hyporesponsiveness of remaining T cells observed in CD28WT pregnants. These data demonstrate for the first time that CD28 is critical for tolerance induction of CD8 T cells, contrasting markedly with CD28 independence of in vitro activation, and suggest that the role of CD28/B7 interactions in peripheral tolerance of CD8 T cells may differ significantly from that of CD4 T cells.

109 Dominant-negative TGFβ receptor 2 enhances GPC3-targeting CAR-T cell efficacy against hepatocellular carcinoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A121-A121
Author(s):  
Nina Chu ◽  
Michael Overstreet ◽  
Ryan Gilbreth ◽  
Lori Clarke ◽  
Christina Gesse ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Dominant Negative ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundChimeric antigen receptors (CARs) are engineered synthetic receptors that reprogram T cell specificity and function against a given antigen. Autologous CAR-T cell therapy has demonstrated potent efficacy against various hematological malignancies, but has yielded limited success against solid cancers. MEDI7028 is a CAR that targets oncofetal antigen glypican-3 (GPC3), which is expressed in 70–90% of hepatocellular carcinoma (HCC), but not in normal liver tissue. Transforming growth factor β (TGFβ) secretion is increased in advanced HCC, which creates an immunosuppressive milieu and facilitates cancer progression and poor prognosis. We tested whether the anti-tumor efficacy of a GPC3 CAR-T can be enhanced with the co-expression of dominant-negative TGFβRII (TGFβRIIDN).MethodsPrimary human T cells were lentivirally transduced to express GPC3 CAR both with and without TGFβRIIDN. Western blot and flow cytometry were performed on purified CAR-T cells to assess modulation of pathways and immune phenotypes driven by TGFβ in vitro. A xenograft model of human HCC cell line overexpressing TGFβ in immunodeficient mice was used to investigate the in vivo efficacy of TGFβRIIDN armored and unarmored CAR-T. Tumor infiltrating lymphocyte populations were analyzed by flow cytometry while serum cytokine levels were quantified with ELISA.ResultsArmoring GPC3 CAR-T with TGFβRIIDN nearly abolished phospho-SMAD2/3 expression upon exposure to recombinant human TGFβ in vitro, indicating that the TGFβ signaling axis was successfully blocked by expression of the dominant-negative receptor. Additionally, expression of TGFβRIIDN suppressed TGFβ-driven CD103 upregulation, further demonstrating attenuation of the pathway by this armoring strategy. In vivo, the TGFβRIIDN armored CAR-T achieved superior tumor regression and delayed tumor regrowth compared to the unarmored CAR-T. The armored CAR-T cells infiltrated HCC tumors more abundantly than their unarmored counterparts, and were phenotypically less exhausted and less differentiated. In line with these observations, we detected significantly more interferon gamma (IFNγ) at peak response and decreased alpha-fetoprotein in the serum of mice treated with armored cells compared to mice receiving unarmored CAR-T, demonstrating in vivo functional superiority of TGFβRIIDN armored CAR-T therapy.ConclusionsArmoring GPC3 CAR-T with TGFβRIIDN abrogates the signaling of TGFβ in vitro and enhances the anti-tumor efficacy of GPC3 CAR-T against TGFβ-expressing HCC tumors in vivo, proving TGFβRIIDN to be an effective armoring strategy against TGFβ-expressing solid malignancies in preclinical models.Ethics ApprovalThe study was approved by AstraZeneca’s Ethics Board and Institutional Animal Care and Use Committee (IACUC).

T Cells Engineered with a Chimeric Antigen Receptor (CAR) Targeting CD19 (CTL019) Have Long Term Persistence and Induce Durable Remissions in Children with Relapsed, Refractory ALL

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 380-380 ◽  
Author(s):  
Stephan A. Grupp ◽  
Shannon L Maude ◽  
Pamela Shaw ◽  
Richard Aplenc ◽  
David M. Barrett ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Research Funding ◽  
Dose Range ◽  
Single Chain

Abstract BACKGROUND CARs combine a single chain variable fragment (scFv) of an antibody with intracellular signaling domains. We have previously reported on CTL019 cells expressing an anti-CD19 CAR. Infusion of these cells results in 100 to 100,000x in vivo proliferation, durable anti-tumor activity, and prolonged persistence in pts with B cell tumors, including sustained CRs in adults and children with ALL (Grupp et al., NEJM 2013, Maude et al., NEJM 2014). We now report on outcomes and longer follow up of the first 30 pts with relapsed, refractory ALL treated on our pilot trial in pediatric ALL. METHODS T cells were lentivirally transduced with a CAR composed of anti-CD19 scFv/4-1BB/CD3ζ, activated/expanded ex-vivo with anti-CD3/anti-CD28 beads, and then infused into children with relapsed or refractory CD19+ ALL. 26/30 pts received lymphodepleting chemotherapy the week prior to CTL019 infusion. The targeted T cell dose range was 107 to 108 cells/kg with a transduction efficiency of 11-45%. T cells for manufacturing were collected from the pt regardless of prior SCT status, not allo donors. RESULTS 30 children median age 10y (5-22y) with CD19+ ALL were treated. 25/30 pts had detectable disease on the day before CTL019 cell infusion, while 5 were MRD(-). A median of 3.6x106 CTL019 cells/kg (1.1-18x106/kg) were infused over 1-3 days. There were no infusional toxicities >grade 2, although 9 pts developed fevers within 24 hrs of infusion and did not receive a planned 2nd infusion of CTL019 cells. 27 pts (90%) achieved a CR, including a patient with T cell ALL aberrantly expressing CD19+. 3 did not respond. MRD measured by clinical flow cytometry was negative in 23 responding pts and positive at 0.1% (negative at 3 mo), 0.09%, 0.22%, and 1.1% in 4 pts. With median follow up 8 mo (1-26 mo), 16 pts have ongoing CR, with only 3 patients in the cohort receiving subsequent treatment such as donor lymphocyte infusion or SCT, 6-month EFS measured from infusion is 63% (95% CI, 47-84%), and OS is 78% (95% CI, 63-95%). CTL019 cells were detected in the CSF of 17/19 pts and 2 pts with CNS2a disease experienced a CR in CSF. 10 pts with a CR at 1 mo have subsequently relapsed, half with CD19(-) blasts. 2/5 pts who relapsed with CD19(-) disease had previously been refractory to CD19-directed blinatumomab and subsequently went into CR with CTL019. Figure 1 Figure 1. All responding pts developed grade 1-4 cytokine release syndrome (CRS) at peak T cell expansion. Detailed cytokine analysis showed marked increases of IL6 and IFNγ (both up to 1000x), and IL2R. Treatment for CRS was required for hemodynamic or respiratory instability in 37% of patients and was rapidly reversed in all cases with the IL6-receptor antagonist tocilizumab, together with corticosteroids in 5 pts. Although T cells collected from the 21 pts who had relapsed after allo SCT were median 100% donor origin, no GVHD has been seen. Grade 4 CRS was strongly associated with high disease burden prior to infusion and with elevations in IL-6, ferritin (suggesting macrophage activation syndrome) and C reactive protein after infusion. Persistence of CTL019 cells detected by flow cytometry and/or QPCR, and accompanied by B cell aplasia, continued for 1-26 months after infusion in pts with ongoing responses. QPCR showed very high levels of CTL019 proliferation, with all patients achieving peak levels >5000 copies/ug gDNA and 26 patients with peak levels >15,000 copies/ug gDNA. B cell aplasia has been treated with IVIg without significant infectious complications. Probability of 6-mo CTL019 persistence by flow was68% (95% CI, 50-92%) andrelapse-free B cell aplasia was 73% (95% CI, 57-94%). CONCLUSIONS: CTL019 cells can undergo robust in-vivo expansion and can persist for 2 years or longer in pts with relapsed ALL, allowing for the possibility of long-term disease response without subsequent therapy such as SCT. This approach also has promise as a salvage therapy for patients who relapse after allo-SCT with a low risk of GVHD. CTL019 therapy is associated with a significant CRS that responds rapidly to IL-6-targeted anti-cytokine treatment. CTL019 cells can induce potent and durable responses for patients with relapsed/refractory ALL; however, recurrence with cells that have lost CD19 is an important mechanism of CLT019 resistance. CTL019 therapy has received Breakthrough Therapy designation from the FDA in both pediatric and adult ALL, and phase II multicenter trials have been initiated. Disclosures Grupp: Novartis: Consultancy, Research Funding. Barrett:Novartis: Research Funding. Chew:Novartis: Research Funding. Lacey:Novartis: Research Funding. Levine:Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Research Funding. Rheingold:Novartis: Consultancy. Shen:Novartis: Employment. Wood:Novartis Pharma: Employment. Porter:Novartis: managed according to U Penn Policy Patents & Royalties, Research Funding. June:Novartis: Research Funding, Royalty income Patents & Royalties.

scholarly journals P025 Identification and validation of predictors for tofacitinib through supervised and unbiased approaches in Inflammatory Bowel Disease

2021 ◽  
Vol 15 (Supplement_1) ◽  
pp. S141-S141
Author(s):  
B Liu ◽  
M Spalinger ◽  
L G Perez ◽  
A Machicote ◽  
N Gagliani ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Signaling Pathway ◽  
Cd4 T Cells ◽  
Cytokine Production ◽  
Stat Signaling ◽  
Deficient Mice

Abstract Background Inflammatory Bowel Disease (IBD) is characterized by an overwhelming gut inflammation, where CD4+ effector T cells are main mediators of the inflammatory response. Tofacitinib, a small molecular drug recently used in IBD patients, blocks the JAK/STAT signaling pathway necessary for CD4+ effector T-cell activation. However, clinical data show that a percentage of patients do not respond to the treatment. Our main goal is to identify biomarkers predicting the response of patients to tofacitinib. Methods Tofacitinib efficacy was studied in vivo in wild type (WT) and T-cell-specific PTPN2 deficient mice (CD4-Cre;Ptpn2 floxed) in which the JAK/STAT signaling pathway is over activated. WT and PTPN2 deficient mice were gavaged with tofacitinib (50mg/kg, twice daily) or vehicle. Acute DSS-colitis was induced. Colitis development was evaluated by weight loss, colonoscopy and histology. CD4+ T cells were isolated from the colon and analyzed by flow cytometry. To study the effect of tofacitinib on T-cell differentiation, we isolated naïve T cells from mouse spleen and polarized them in vitro to different T-cell subsets with or without tofacitinib. CD4+ T cells differentiation and cytokine production were analyzed by flow cytometry. To evaluate the influence of tofacitinib on human CD4+ T cells, human peripheral blood mononuclear cells (PBMCs) from healthy donors and IBD patients were stimulated in presence of tofacitinib, and analyzed by flow cytometry. Results While no protective effect was found after tofacitinib treatment in WT mice, PTPN2 deficient mice were protected from colitis based on less weight loss, lower endoscopic and histological scores. The expression of pro-inflammatory cytokines such as IL-17 and IFN-γ by colonic CD4+ T cells was also decreased by tofacitinib. Consistent with the in vivo observations, in vitro experiments revealed a strong impact of tofacitinib on CD4+ T-cells cytokine production. In PBMCs from IBD patients, IFN-γ and TNF-α expression was strongly impacted. In contrast, in healthy donors, IL-10 was the most impacted cytokine. Finally, tofacitinib decreased the in vitro differentiation of Th1, Th2, Th17, Th22, Treg and Tr1. Conclusion In the T-cell-specific PTPN2 deficient mice, tofacitinib exerts a protective effect after DSS-induced colitis. In line with the in vivo findings, in vitro experiments show that tofacitinib has a strong impact on pro-inflammatory cytokine production, especially in the IBD patients. Taken together, these data suggest that tofacitinib might be suitable primarily for IBD patients where the JAK/STAT signaling pathway is over activated.

scholarly journals In Vivo Delivery of a CD20 CAR Using a CD8-Targeted Fusosome in Southern Pig-Tail Macaques (M. nemestrina) Results in B Cell Depletion

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2769-2769
Author(s):  
Justine Cunningham ◽  
Sundeep Chandra ◽  
Akinola Emmanuel ◽  
Allyse Mazzarelli ◽  
Carmela Passaro ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Systemic Administration ◽  
Cell Depletion ◽  
B Cell Depletion ◽  
Car T ◽  
Integrating Vector

Abstract Introduction: Ex vivo manufactured chimeric antigen receptor (CAR) T cell therapies are highly effective for treating B cell malignancies. However, the complexity, cost and time required to manufacture CAR T cells limits access. To overcome conventional ex vivo CAR T limitations, a novel gene therapy platform has been developed that can deliver CAR transgenes directly to T cells through systemic administration of a fusosome, an engineered, target-directed novel paramyxovirus-based integrating vector that binds specific cell surface receptors for gene delivery through membrane fusion. Here, we demonstrate that systemic administration of a CD8a-targeted, integrating vector envelope (i.e., fusogen) encoding an anti-CD20 CAR into Southern pig-tail macaques (M. nemestrina), which is a species permissive to the integrating vector-mediated transduction, results in T cell transduction and B cell depletion with no treatment-related toxicities. Methods: CD8a-specific single chain variable fragments (scFvs) were generated and measured for target specificity versus non-CD8-expressing cells in vitro. Cross-reactivity of the CD8a-specific fusogen for human and nemestrina T cells was confirmed in vitro. Targeted fusogens were then used to pseudotype integrating vector expressing an anti-CD20 CAR containing the 4-1BB and CD3zeta signaling domains (CD8a-anti-CD20CAR). Transduction and B cell killing was confirmed on human and nemestrina PBMCs. To evaluate in vivo activity, normal, healthy nemestrina macaques were treated with a single dose of CD8a-targeted anti-CD20 CAR fusosome (n=6) or saline (n=2) via intravenous infusion at 10mL/kg/hr for 1-hour and evaluated for up to 52 days for evidence of adverse effects, B cell depletion, CAR-mediated cytokine production, CAR T cell persistence and vector biodistribution using ddPCR and anti-CD20CAR transgene by RT-ddPCR to detect transgene levels. Histopathology of several organs and immunohistochemistry for CD3 and CD20 on lymph nodes, spleen, and bone marrow were performed at termination (days 35 and 52). Tolerability of the treatment was assessed by body weight, body temperature, neurological exams, serum chemistry panel, and complete blood counts pre-dose and post-dose up to 52 days. Results: The CD8a-targeted fusogen demonstrated CD8a-specificity versus human CD8 negative cell lines, and cross-reactivity and transduction efficiency in nemestrina PBMCs in vitro. Compared to a control vector (GFP), anti-CD20CAR-modified T cells showed a dose-dependent depletion of B cells using in vitro assays. Following infusion of CD8a-anti-CD20CAR fusosomes into macaques, pharmacological activity in peripheral blood was detected by a reduction of B cells in 4 of 6 animals after 7 to 10 days. Two animals showed persistent B cell depletion until study termination, with two others showing a temporary response. The presence of vector copy could be detected in the peripheral blood of all treated animals between days 3 and 10, and in isolated spleen cells in 5 of 6 animals. All control animals (saline) were negative for vector. RT-ddPCR mRNA expression similarly revealed the presence of anti-CD20CAR transcripts in isolated spleen cells from treated animals; no expression was detected in tissues from control animals. Elevations in inflammatory cytokines could be detected in the serum of treated animals between days 3 and 14. Fusosome treatment was well-tolerated in all animals with no evidence of adverse effects. Moreover, T cell transduction and B cell depletion was not associated with cytokine-related toxicities, and blood chemistry and histopathology were within normal limits. Conclusion: These data obtained in an immunologically competent animal demonstrate the proof-of-concept that systemic administration of a CD8a-anti-CD20CAR fusosome can specifically transduce T cells in vivo without pre-conditioning or T cell activation, resulting in B cell depletion in the absence of vector- or CAR T-related toxicities. Therefore, fusosome technology represents a novel therapeutic opportunity to treat patients with B cell malignancies and potentially overcome some of the treatment barriers that exist with conventional CAR T therapies. Disclosures Cunningham: Sana Biotechnology: Current Employment. Chandra: Sana Biotechnology: Current Employment. Emmanuel: Sana Biotechnology: Current Employment. Mazzarelli: Sana Biotechnology: Current Employment. Passaro: Sana Biotechnology: Current Employment. Baldwin: Sana Biotechnology: Current Employment. Nguyen-McCarty: Sana Biotechnology: Current Employment. Rocca: Sana Biotechnology: Current Employment. Joyce: Sana Biotechnology: Current Employment. Kim: Sana Biotechnology: Current Employment. Vagin: Sana Biotechnology: Current Employment. Kaczmarek: Sana Biotechnology: Current Employment. Chavan: Sana Biotechnology: Current Employment. Jewell: Sana Biotechnology: Current Employment. Lipsitz: Sana Biotechnology: Current Employment. Shamashkin: Sana Biotechnology: Current Employment. Hlavaty: Sana Biotechnology: Current Employment. Rodriguez: Sana Biotechnology: Current Employment. Co: Sana Biotechnology: Current Employment. Cruite: Sana Biotechnology: Current Employment. Ennajdaoui: Sana Biotechnology: Current Employment. Duback: Sana Biotechnology: Current Employment. Elman: Sana Biotechnology: Current Employment. Amatya: Sana Biotechnology: Current Employment. Harding: Sana Biotechnology: Current Employment. Lyubinetsky: Sana Biotechnology: Current Employment. Patel: Sana Biotechnology: Current Employment. Pepper: Sana Biotechnology: Current Employment. Ruzo: Sana Biotechnology: Current Employment. Iovino: Sana Biotechnology: Current Employment. Varghese: Sana Biotechnology: Current Employment. Foster: Sana Biotechnology: Current Employment. Gorovits: Sana Biotechnology: Current Employment. Elpek: Sana Biotechnology: Current Employment. Laska: Sana Biotechnology: Current Employment. McGill: Sana Biotechnology: Current Employment. Shah: Sana Biotechnology: Current Employment. Fry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Dambach: Sana Biotechnology: Current Employment.

scholarly journals A Novel Fully Human Bispecific CD19 x CD3 Antibody That Kills Lymphoma Cells with Minimal Cytokine Secretion

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1671-1671
Author(s):  
Harbani Malik ◽  
Ben Buelow ◽  
Brian Avanzino ◽  
Aarti Balasubramani ◽  
Andrew Boudreau ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cytokine Secretion ◽  
Lymphoma Cells ◽  
Nog Mice

Abstract Introduction Along with CD20 and CD22, the restricted expression of CD19 to the B-cell lineage makes it an attractive target for the therapeutic treatment of B-cell malignancies. Many monoclonal antibodies and antibody drug conjugates specific to CD19 have been described, including bispecific T-cell redirecting antibodies (T-BsAbs). In addition, anti-CD19 chimeric antigen receptor T-cells (CAR-Ts) have been approved to treat leukemia. To date, toxicity from over-activation of T-cells and large-scale production of CAR-Ts still hinder this approach. Bispecific T-cell engaging antibodies redirecting T cells to CD19 circumvent the latter problem but to date have shown similar T-cell over-activation, as well as significant neurotoxicity. Utilizing TeneoSeek, a next generation sequencing (NGS)-based discovery pipeline that uses 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 selectively-activating αCD3 that induced potent T-cell dependent lysis of lymphoma cells (when paired with an αCD19 HCA) with minimal cytokine secretion. To characterize the relative efficacy and potential therapeutic window of this unique molecule, we compared the low-activating (and Fc-containing) CD19 x CD3 to two pan T-cell activating bispecific CD19 x CD3 antibodies (blinatumomab and another developed in-house) in vitro and in vivo for T-cell activation, efficacy in killing lymphoma cells, and toxicity. Methods T-cell activation was measured via flow cytometry (CD69 and CD25 expression) and cytokine ELISA (IL-2, IL-6, IL-10, INF-ɣ, and TNFα) in vitro. Lysis of B-cell tumor cell lines (Raji, Ramos, and Nalm6) was measured via calcein release in vitro. In vivo, NOG mice were engrafted with human peripheral blood mononuclear cells (huPBMC) and human lymphoma cell lines, and the mice treated with weekly injections of T-BsAbs. Tumor burden was evaluated via caliper measurement. Pharmacokinetic (PK) studies were performed in NOG mice using ELISA. Results EC50s for cytotoxicity were in the single-digit nanomolar range for the selective T cell activating T-BsAb and sub-nanomolar for the pan T-cell activating controls. The selective T cell activator showed markedly reduced cytokine release for all cytokines tested compared to the pan T-cell controls even at saturating concentrations. In vivo, established CD19 positive B-cell tumors were cleared in NOG mice in the presence of huPBMC. PK profiles of both molecules generated in-house (selective and pan T-cell activators) were consistent with those of an IgG in mice. No activation of T-cells was observed in vitro or in vivo in the absence of CD19 expressing target cells. Conclusions Both the selectively-activating and the pan T-cell activating control bispecific antibodies killed lymphoma cells in vitro and in vivo in a CD19-dependent manner. While the pan T-cell activating controls showed T-cell activation comparable to other CD3-engaging bispecifics, the selective activator induced significantly reduced cytokine secretion by T-cells and demonstrated a half-life consistent with other IgG antibodies. In summary, our selectively activating CD19 x CD3 T-BsAb shows promise as a lymphoma therapeutic differentiated from current T-cell targeted therapies currently in the clinic and in clinical trials. Disclosures Malik: Teneobio, Inc.: Employment. Buelow:Teneobio Inc.: Employment. Avanzino:Teneobio, Inc.: Employment. Balasubramani:Teneobio, Inc.: Employment. Boudreau:Teneobio, Inc.: Employment. Clarke:Teneobio, Inc.: Employment. Dang:Teneobio, Inc.: Employment. Davison:Teneobio, Inc.: Employment. Force Aldred:Teneobio Inc.: Employment. Harris:Teneobio, Inc.: Employment. Jorgensen:Teneobio, Inc.: Employment. Li:Teneobio, Inc.: Employment. Medlari:Teneobio, Inc.: Employment. Narayan:Teneobio, Inc.: Employment. Ogana:Teneobio, Inc.: Employment. Pham:Teneobio Inc.: Employment. Prabhakar:Teneobio, Inc.: Employment. Rangaswamy:Teneobio, Inc.: Employment. Sankaran:Teneobio, Inc.: Employment. Schellenberger:Teneobio, Inc.: Employment. Ugamraj:Teneobio, Inc.: Employment. Trinklein:Teneobio, Inc.: Employment. Van Schooten:Teneobio, Inc.: Employment.

ALL Progression Induces PD1 on T Cells and Blockade of PD1 Enhances Adoptive Immunotherapy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2587-2587 ◽  
Author(s):  
Chad R Burk ◽  
William Fix ◽  
Haiying Qin ◽  
Terry J Fry
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Poor Response ◽  
Cell Phenotype ◽  
Early Progression ◽  
Pd1 Blockade

Abstract Abstract 2587 Background: Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and, despite tremendous success in therapy over the past 3 decades, remains a primary cause of cancer-related mortality in children. Enthusiasm for the use cellular immunotherapy for ALL has been tempered by the poor response to donor lymphocyte infusions following allogeneic hematopoietic stem cell transplantation. However, ALL blasts are susceptible to T cell and NK cell mediated lysis in vitro suggesting that poor response to in vivo immune interventions may be due to events occurring during the priming of the immune response. Using a murine model of precursor B cell ALL we examined the impact of leukemia progression on T cells in vivo. Methods: We developed a transplantable syngeneic model of pediatric ALL derived from transgeneic mice expressing human E2aPBX1, a recurring translocation present in 5% of pediatric leukemia (Bijl et al, Genes and Development, 2005). This murine line displays a precursor B cell phenotype and results in 100% lethality following injection of 100,000 cells (Qin et al, ASH, 2010). Using congenic (CD45.1) B6 recipients, we tracked the early progression of ALL in vivo and examined the T cells in the leukemia-containing compartments by flow cytometry and PCR. Results: Using congenic markers, ALL cells can be detected in bone marrow as early as 3 days following intravenous injection of 1,000,000 cells with a sensitivity of 0.01%. Spleen and lymph node involvement was seen later (10 days) followed by the detection of circulating blasts by 2 weeks. E2aPBX1 cells express variable levels of costimulatory molecules in vitro with no change in expression during in vivo progression. Notably, PDL1 and PDL2 are expressed both in vitro and in vivo at higher levels than on non-malignant precursor B cells in leukemia-bearing mice. Remarkably, although PD1+ T cells are not seen in the bone marrow of non-leukemia-bearing mice, PD1 expression on bone marrow T cells was markedly increased during progression such that 60–80% of all bone marrow CD4 and CD8 T cells were positive by 2 weeks following leukemia injection (figure). In addition to expression of PD1, these T cells also co-expressed Tim3, a phenotype associated with T cell exhaustion. Blockade of PD1 or PDL1 starting 3 days following leukemia injection had no impact on leukemia progression. However, combining PD1 blockade with the adoptive transfer of T cells from leukemia-primed donors resulted in improved survival compared to primed T cells alone (p=0.0004). Conclusions: Early progression of ALL results in the induction of PD1 and Tim3 on T cells in vivo. Combination of PD1 blockade plus adoptive T cell therapy results in therapeutic benefit suggesting that this axis may be an attractive target in ALL. Disclosures: No relevant conflicts of interest to declare.

Activation of T and NK Cells Following Lenalidomide Therapy in Patients with Follicular Lymphoma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2766-2766
Author(s):  
Seema Rawal ◽  
Nathan Fowler ◽  
Min Zhang ◽  
Zhiqiang Wang ◽  
Tariq Muzzafar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Nk Cells ◽  
Research Funding ◽  
Memory T Cells ◽  
T Cell Subsets ◽  
Central Memory T Cells

Abstract Abstract 2766 Background: Lenalidomide plus rituximab therapy is a highly effective and well-tolerated therapy in patients (pts) with follicular lymphoma (FL). In a Phase II trial, this combination induced a complete remission rate of 87% in pts with advanced stage untreated FL (Fowler et al, Ann Oncol, 2011; 22; suppl 4:137). A randomized Phase III trial was recently initiated to compare this combination with current standard of care therapies in pts with FL. Although lenalidomide is known to be an immunomodulatory drug with effects on a variety of immune cells in vitro, its effects have not been well studied in vivo in humans. Understanding the in vivo effects of lenalidomide could lead to novel combination strategies to enhance the efficacy and improve clinical outcome in FL and other malignancies. Methods: Pts received lenalidomide 20 mg/day on days 1–21 of each 28-day cycle and rituximab was given at 375 mg/m2on day 1 of each cycle. Peripheral blood mononuclear cells (PBMC) were phenotyped by multiparametric flow cytometry at baseline, on cycle 2 day 15 (C2D15), and at the end of cycle 6. In addition, peripheral blood (PB) samples were collected in PAXgene Blood RNA tubes at baseline and on C2D15 for whole genome gene expression profiling (GEP). Results: Immunophenotyping of baseline and end of cycle 6 PBMC (n=17) showed that the percentages and absolute numbers of CD3+, CD4+, CD8+, TCRgd, and Foxp3+ regulatory T cells; and NK, NKT, and myeloid dendritic cells were not significantly different between the two time points. However, a significant increase in CD4+CD45RO+ (p<0.01) and CD8+CD45RO+ (p=0.04) memory T cells was observed post-therapy. Further characterization of CD4+ T cells showed a significant increase in central memory T cells (p<0.001) and a decrease in naïve (p<0.01) and terminally differentiated (p<0.01) T cells, but no change in effector memory T cells. The increase in CD8+ central memory T cells was marginally significant (p=0.06). Plasmacytoid dendritic cells (PDC) were also significantly increased (p=0.02). In contrast, no such changes in T cell subsets or PDC were observed in FL pts (n=9) treated with 6 cycles of R-CHOP chemotherapy that received equal number of rituximab doses and analyzed at similar time points (baseline and end of cycle 6). To understand lenalidomide-induced changes on a molecular level, we compared GEP data at C2D15 vs. baseline for 7 pairs of PB samples. The paired significance analysis of microarrays method, based on Student's t test, identified 1,748 differentially expressed genes (DEG; 713 up, 1035 down), without a fold-change threshold, in C2D15 samples vs. baseline. Results were influenced by rituximab-induced depletion of B cells in C2D15 samples, but there were many changes that suggested altered PBMC physiology. Noteworthy up-regulated genes (>1.5 fold) included genes associated with T and NK cell activation including BATF, CCR2, CD1B, CD2, CD160, CTLA4, CXCR3, ICOS, and LAG3; and CD163 and CD209, phagocytic receptors expressed on monocytes/macrophages. Down-regulated genes (>1.5 fold) included CXCR5, which mediates B cell migration into follicles; and IL1B and TNFSF13B (BAFF), which are produced by activated macrophages and induce B cell proliferation. Gene set enrichment analysis of all GEP results, and Ingenuity Pathway Analysis of DEGs, indicated up regulation of multiple pathways and processes including ribosomal and mitochondrial components involved in translation and oxidative phosphorylation, CTLA4 signaling in cytotoxic T cells, and differentiation and signaling by ICOS and CD28 in T helper cells. We confirmed up regulation of CTLA4, ICOS, and LAG3 at the protein level in C2D15 PBMC by flow cytometry. Furthermore, treatment of PBMC derived from untreated FL pts with lenalidomide in vitro resulted in up regulation of these molecules in T and/or NK cells consistent with our in vivo results. Conclusions: In FL pts, lenalidomide induced multiple changes in the immune system including increases in PDC and memory T cell subsets, activation of T and NK cells, and down-regulation of certain genes mediating B cell migration and proliferation. These results provide insights into the mechanism of action of lenalidomide and suggest that it can be combined with other immunostimulatory agents such as therapeutic vaccines, adoptive T cell therapy strategies, and immune checkpoint inhibitors to further enhance its efficacy in FL and other malignancies. Disclosures: Fowler: Celgene: Research Funding. Heise:Celgene Corporation: Employment, Equity Ownership. Lacerte:Celgene: Honoraria. Samaniego:Celgene: Research Funding. Neelapu:Celgene Corporation: Research Funding.

A T Cell-Engaging CD3 Recruit-Tandab Potently Kills CD19+ Tumor B Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3721-3721
Author(s):  
Eugene Zhukovsky ◽  
Uwe Reusch ◽  
Carmen Burkhardt ◽  
Stefan Knackmuss ◽  
Ivica Fucek ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Target Cells ◽  
Cytotoxicity Assays

Abstract Abstract 3721 Background: CD19 is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for the development of therapeutic antibodies to treat B cell malignancies. T cells are potent tumor-killing effector cells that cannot be recruited by native antibodies. The CD3 RECRUIT-TandAb AFM11, a humanized bispecific tetravalent antibody with two binding sites for both CD3 and CD19, is a novel therapeutic for the treatment of NHL that harnesses the cytotoxic nature of T cells. Methods: We engineered a bispecific anti-CD19/anti-CD3e tetravalent TandAb with humanized and affinity-matured variable domains. The TandAb's binding properties, T cell-mediated cytotoxic activity, and target-mediated T cell activation were characterized in a panel of in vitro assays. In vivo efficacy was evaluated in a murine NOD/scid xenograft model reconstituted with human PBMC. Results: AFM11 mediates highly potent CD19+ tumor cell lysis in cytotoxicity assays performed on a panel of cell lines (JOK-1, Raji, Nalm-6, MEC-1, VAL, Daudi) and primary B-CLL tumors: EC50 values are in the low- to sub-picomolar range and do not correlate with the expression density of CD19 on the target cell lines. The cytotoxic activity of tetravalent AFM11 is superior to that of alternative bivalent antibody formats possessing only a single binding site for both CD19 and CD3. High affinity binding of AFM11 to CD19 and to CD3 is essential for efficacious T cell recruitment. Both CD8+ and CD4+ T cells mediate cytotoxicity however the former exhibit much faster killing. We observe that AFM11 displays similar cytotoxic efficacy at different effector to target ratios (from 5:1 to 1:5) in cytotoxicity assays; this suggests that T cells are engaged in the serial killing of CD19+ target cells. In the absence of CD19+ target cells in vitro, AFM11 does not elicit T cell activation as manifested by cytokine release (from a panel of ten cytokines associated with T cell activation), their proliferation, or their expression of activation markers. AFM11 activates T cells exclusively in the presence of its targets and mediates lysis of CD19+ cells while sparing antigen-negative bystanders. In the absence of CD19+ target cells, AFM11 concentrations in excess of 500-fold over EC50 induce down-modulation of the CD3/TCR complex. Yet, AFM11-treated T cells can be re-engaged for target cell lysis. All of these features of AFM11-induced T cell activation may contribute additional safety without compromising its efficacy. In vivo AFM11 demonstrates a robust dose-dependent inhibition of subcutaneous Raji tumors in mice. At 5 mg/kg AFM11 demonstrates a complete suppression of tumor growth, and even at 5 ug/kg tumor growth is reduced by 60%. Moreover, we observe that a single administration of AFM11 produces inhibition of tumor growth similar to that of 5 consecutive administrations. Conclusions: In summary, our in vitro and in vivo experiments with AFM11 demonstrate the high potency and efficacy of its anti-tumor cytotoxicity. Thus, AFM11 is a novel highly efficacious drug candidate for the treatment of B cell malignancies with an advantageous safety profile. Disclosures: Zhukovsky: Affimed Therapeutics AG: Employment, Equity Ownership. Reusch:Affimed Therapeutics AG: Employment. Burkhardt:Affimed Therapeutics AG: Employment. Knackmuss:Affimed Therapeutics AG: Employment. Fucek:Affimed Therapeutics AG: Employment. Eser:Affimed Therapeutics AG: Employment. McAleese:Affimed Therapeutics AG: Employment. Ellwanger:Affimed Therapeutics AG: Employment.

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