scholarly journals Multispecific anti-HIV duoCAR-T cells display broad in vitro antiviral activity and potent in vivo elimination of HIV-infected cells in a humanized mouse model

2019 ◽  
Vol 11 (504) ◽  
pp. eaav5685 ◽  
Author(s):  
Kim Anthony-Gonda ◽  
Ariola Bardhi ◽  
Alex Ray ◽  
Nina Flerin ◽  
Mengyan Li ◽  
...  
Keyword(s):  
T Cells ◽  
Mouse Model ◽  
Hiv Infection ◽  
Lentiviral Vectors ◽  
Car T ◽  
Anti Hiv ◽  
Hiv 1

Adoptive immunotherapy using chimeric antigen receptor–modified T cells (CAR-T) has made substantial contributions to the treatment of certain B cell malignancies. Such treatment modalities could potentially obviate the need for long-term antiretroviral drug therapy in HIV/AIDS. Here, we report the development of HIV-1–based lentiviral vectors that encode CARs targeting multiple highly conserved sites on the HIV-1 envelope glycoprotein using a two-molecule CAR architecture, termed duoCAR. We show that transduction with lentiviral vectors encoding multispecific anti-HIV duoCARs confer primary T cells with the capacity to potently reduce cellular HIV infection by up to 99% in vitro and >97% in vivo. T cells are the targets of HIV infection, but the transduced T cells are protected from genetically diverse HIV-1 strains. The CAR-T cells also potently eliminated PBMCs infected with broadly neutralizing antibody-resistant HIV strains, including VRC01/3BNC117-resistant HIV-1. Furthermore, multispecific anti-HIV duoCAR-T cells demonstrated long-term control of HIV infection in vivo and prevented the loss of CD4+T cells during HIV infection using a humanized NSG mouse model of intrasplenic HIV infection. These data suggest that multispecific anti-HIV duoCAR-T cells could be an effective approach for the treatment of patients with HIV-1 infection.

scholarly journals Preferential infection and depletion of Mycobacterium tuberculosis–specific CD4 T cells after HIV-1 infection

2010 ◽  
Vol 207 (13) ◽  
pp. 2869-2881 ◽  
Author(s):  
Christof Geldmacher ◽  
Njabulo Ngwenyama ◽  
Alexandra Schuetz ◽  
Constantinos Petrovas ◽  
Klaus Reither ◽  
...  
Keyword(s):  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
Hiv Infection ◽  
Cd4 T Cells ◽  
Staphylococcal Enterotoxin B ◽  
Opportunistic Pathogens ◽  
Rapid Loss ◽  
Hiv 1

HIV-1 infection results in the progressive loss of CD4 T cells. In this study, we address how different pathogen-specific CD4 T cells are affected by HIV infection and the cellular parameters involved. We found striking differences in the depletion rates between CD4 T cells to two common opportunistic pathogens, cytomegalovirus (CMV) and Mycobacterium tuberculosis (MTB). CMV-specific CD4 T cells persisted after HIV infection, whereas MTB-specific CD4 T cells were depleted rapidly. CMV-specific CD4 T cells expressed a mature phenotype and produced very little IL-2, but large amounts of MIP-1β. In contrast, MTB-specific CD4 T cells were less mature, and most produced IL-2 but not MIP-1β. Staphylococcal enterotoxin B–stimulated IL-2–producing cells were more susceptible to HIV infection in vitro than MIP-1β–producing cells. Moreover, IL-2 production was associated with expression of CD25, and neutralization of IL-2 completely abrogated productive HIV infection in vitro. HIV DNA was found to be most abundant in IL-2–producing cells, and least abundant in MIP-1β–producing MTB-specific CD4 T cells from HIV-infected subjects with active tuberculosis. These data support the hypothesis that differences in function affect the susceptibility of pathogen-specific CD4 T cells to HIV infection and depletion in vivo, providing a potential mechanism to explain the rapid loss of MTB-specific CD4 T cells after HIV infection.

scholarly journals 133 CRISPR/Cas9 gene-edited allogeneic CAR-T cells targeting CD33 show high preclinical efficacy against AML without long-term hematopoietic toxicity

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A143-A143
Author(s):  
Jonathan Terrett ◽  
Brigid Mcewan ◽  
Daniel Hostetter ◽  
Luis Gamboa ◽  
Meghna Kuppuraju ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antibody Drug Conjugate ◽  
T Cell Therapy ◽  
In Vivo Models ◽  
Car T Cells ◽  
Car T

BackgroundCD33 is the most consistently expressed antigen in AML, with high levels and homogeneous expression observed in malignant AML cells from most patients, including those with relapsed disease. Normal myelomonocytic cell lineages and a percentage of hematopoietic progenitors also express CD33, and the extreme myeloablation caused by the CD33-targeted antibody-drug conjugate (ADC) gemtuzumab ozogamicin reinforced concerns about targeting this antigen with more potent agents such as T-cell engaging bispecific antibodies and CAR-T cells. We have shown previously that allogeneic CRISPR/Cas9 gene-edited CAR-T cells targeting CD33 with TRAC disruption to reduce GvHD and B2M disruption to reduce allogeneic host rejection could eliminate tumors in xenograft models of AMLMethodsGiven that off-target activity of the toxin could contribute to the myeloablation seen with CD33-targeted ADCs, we created in vitro and in vivo models to examine reconstitution of the myeloid compartment following treatment of CD33-targeted allogeneic CAR-T cells.ResultsAlthough co-culture of CD34+ stem cells in vitro with our CD33-targeted allogeneic CAR-T cells did significantly deplete the cell population, colonies still formed after removal of the CAR-T cells as the presumably CD33-negative stem/progenitor cells expanded and differentiated. A similar phenomenon was observed in vivo with CD34 humanized mice bearing an AML tumor (THP-1 cells) and treated with the CD33-targeted allogeneic CAR-T cells. The CAR-T cells completely eradicated the THP-1 tumor but did not lead to long-term myelosuppression or B cell aplasia.ConclusionsThus, allogeneic CRISPR/Cas9 multiplex gene-edited CD33-targeted CAR-T cell therapy may be both efficacious and tolerable in AML.

scholarly journals A New Promising Third Generation CAR-CD30 T-Cell Therapy for CD30+ Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2069-2069
Author(s):  
Biagio De Angelis ◽  
Marika Guercio ◽  
Domenico Orlando ◽  
Stefano Di Cecca ◽  
Matilde Sinibaldi ◽  
...  
Keyword(s):  
T Cells ◽  
Board Of Directors ◽  
Cell Lines ◽  
Short Term ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T

Prognosis of a significant proportion of patients with chemotherapy-refractory or multiply-relapsed CD30+ Non-Hodgkin's Lymphoma (NHL) or Hodgkin lymphoma (HL) still remain poor. Targeting CD30 with monoclonal antibodies in HL and anaplastic large cell lymphoma was shown to induce remarkable clinical activity; however, occurrence of adverse events (mainly neuropathy) may result into treatment discontinuation in many patients. Immunotherapeutic approaches targeting CD30 by chimeric antigen receptor (CAR) has been demonstrated to be of value in two independent clinical trials, although clinical benefit was sub-optimal. We designed a new CAR construct characterized by an anti-CD30 single-chain variable-fragment cassette (AC10), linked to CD3ζ by the signaling domains of two costimulatory molecules, namely either CD28.4-1BB or CD28.OX40. The inducible Caspase-9 (iCasp9) safety switch was included in both constructs with the goal of promptly controlling undue toxicity. As a selectable marker, we added in frame the CD34 antigen. The in vitro anti-tumor efficacy was evaluated by using either the NHL cell line: Karpas299, or the HL cell lines: L428, in both short-term cytotoxic assay (51Cr release assays) and long-term co-cultures for 6 days. Supernatant from co-culture experiments was analyzed by Elisa. We assessed the antitumor effect of CAR.CD30 T cells in a in vivo NSG mouse model engrafted i.v. with lymphoma FF-luciferase cell lines Karpas299 or L428, and monitored tumor growth by IVIS Imaging system. For tumor re-challenging, mice of the NHL model surviving until day +140, were i.v. infused with 0.2x106 Karpas299 cells, and subsequently followed for additional 110 days. Persistence of CAR.CD30 T cells was evaluated, together with a deep characterization of memory profile of T cells. Independently from the costimulatory domains CD28.OX40 or CD28.4-1BB, the generated retroviral vectors showed similar transduction efficiency of T cells (86.5±5.1% and 79.3±5.3%, respectively). Nevertheless, CD28.OX40 costimulatory domains was associated with more stable expression of the CAR over time, during extensive in vitro culture (84.72±5.30% vs 63.98±11.51% CD28.4-1BB CAR T cells at 30 days after transduction; p=0.002). For both CAR constructs, we did not observe any significant difference in the suicide gene iCasp9 activity, both in vitro and in vivo. In short-term cytotoxic assay, both CAR.CD30 T cells significantly and specifically lysed CD30+ NHL and HL tumor cell lines. In long-term co-culture, CD28.OX40 showed a superior anti-lymphoma in vitro activity as compared to CD28.41BB T cells, when challenged at very high tumor/effector ratio (8:1) (for Karpas 299; p=0.03). Moreover, the antigen stimulation was associated to higher levels of Th1 cytokine production, with CD28.OX40 T cells secreting a significantly higher amount of IFNγ, IL2 and TNFα as compared to CD28.41BB T cells (p= 0.040; p=0.008; p=0.02; respectively). Bioluminescence in HL (L428) tumor-bearing mice, treated with NT T cells, rapidly increased up to 5 log in less than 50 days and mice either died or were sacrificed due to morbidity. The best outcome was observed in mice treated with CD28.OX40, as three out of five mice were still alive at the experimental end-point of day+165, as compared with mice treated with CD28.4-1BB (60% vs 0%, p=0.0021). In NHL (Karpas 299) mouse models, CD28.OX40 had an extensive anti-tumor control superior to that of CD28.41BB T cells, leading to a significant reduction of tumor bioluminescence at day 45 (3.32x10 vs 2.29x10, p=0.04). The median survival of mice treated with NT and CD28.4-1BB CAR T cells was 45.5 and 58 days respectively, but undetermined for mice treated with CD28.OX40 CAR T cells (p=0.0002). After 140 days, cured mice were re-challenged with Karpas 299; mice were followed for other 100 days. Bioluminescence analysis showed rapid progression of the tumor in the control mice cohort, as well as in CD28.4-1BB treated mice. In contrast, in CD28.OX40 treated mice, at day+240 days, 4 out of 6 mice were tumor-free, resulting into a statistically significant survival benefit (p=0.0014). Only in mice treated with 28.OX40 T cells, we observed a long-lasting persistence of circulating CAR-T cells up to day +221. In summary, we have developed a novel CAR.CD30 construct displaying features that make it a particularly suitable candidate for a clinical trial in patients suffering from CD30+ tumors. Disclosures Merli: Novartis: Honoraria; Sobi: Consultancy; Amgen: Honoraria; Bellicum: Consultancy. Locatelli:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BluebirdBio: Consultancy; Miltenyi: Honoraria; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Evaluating the Safety and Efficacy of Anti-HIV Transgenic Cells in a Humanized Mouse Model (NOD/SCIDγc−/−) for HIV Stem Cell Gene Therapy.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2352-2352
Author(s):  
Joseph S. Anderson ◽  
Jon Walker ◽  
Louisa Wirthlin ◽  
John Javien ◽  
Jan A. Nolta ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
B Cells ◽  
Mouse Model ◽  
Lentiviral Vector ◽  
Human Cd34 ◽  
Cell Engraftment ◽  
Anti Hiv ◽  
Hiv 1

Abstract In vivo mouse models for safety and efficacy studies of anti-HIV gene therapeutics have been lacking in their ability to evaluate normal systemic hematopoiesis with the development of a functional immune system. These deficiencies in modeling these normal and essential physiologies can now be generated in the recently developed NOD/SCIDγc−/− mouse model. Human CD34+ progenitor cells after intrahepatic injection will undergo multi-organ engraftment generating a functional thymus along with the production of T cells, B cells, and macrophages. Upon injection with various strains of HIV-1, engrafted mice display a disseminated infection due to viral replication in human T cells and macrophages. Infected mice acquire characteristics of increased viremia over time with human CD4+ T cell depletion. Anti-HIV immune responses can also be generated in mice with high viremia. Here we demonstrate the successful engraftment of NOD/SCIDγc−/− mice after injection with human CD34+ progenitor cells isolated from umbilical cord blood. Human cell engraftment was observed in both primary and secondary lymphoid organs including the bone marrow (B cells and macrophages), thymus (T cells), spleen (T cells and B cells), lymph nodes (T cells and B cells), and peripheral blood (T cells and B cells). Human CD34+ cells were found in the liver concluding either organ retention from the initial injection or circulation in the peripheral blood. After displaying a high level of engraftment (>50%), we are now able to proceed with evaluating a number of anti- HIV gene therapeutic constructs for pre-clinical testing. These lentiviral vector constructs include a CCR5 shRNA which has been previously shown to knock down complete expression of CCR5, a human/rhesus macaque chimeric isoform of TRIM5α capable of potently inhibiting HIV-1 infection at the pre-integration step, and combination constructs containing multiple anti-HIV genes. The NOD/SCIDγc−/− mouse model will allow us to evaluate both the safety of these various anti-HIV lentiviral constructs in generating normal functioning transgenic immune system cells and also the efficacy of these anti- HIV genes in inhibiting HIV-1 infection in vivo. With the ability to facilitate multilineage human cell engraftment with the susceptibility for HIV infection, this in vivo model allows for the evaluation of anti-HIV gene therapeutic constructs in a stem cell-based setting. Lentiviral vector transgenic cells can now be tested for their capacity for normal immune system reconstitution.

scholarly journals Anti-Human CD117 CAR T-Cells Efficiently Eliminate Hematopoietic Stem and CD117-Positive AML Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4063-4063 ◽  
Author(s):  
Renier Myburgh ◽  
Jonathan Kiefer ◽  
Norman F Russkamp ◽  
Alexander Simonis ◽  
Surema Pfister ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Cell Lines ◽  
Surface Expression ◽  
Hematopoietic Stem ◽  
Car T Cells ◽  
Car T

Abstract Introduction: Acute Myeloid Leukemia (AML) is a clonal disease of the hematopoietic system that originates from immature hematopoietic stem and progenitor cells (HSPC). Because some AML-initiating cells are comparatively resistant to conventional cytotoxic agents, disease relapses are common with current treatment approaches. As an alternative, immunological eradication of leukemic cells by adoptively transferred chimeric-antigen receptor T-cells (CAR T-cells) might be considerably more efficient. To date, however, the search for AML-specific surface antigens has remained largely elusive. To circumvent this problem, we propose to target the stem cell antigen c-Kit (CD117) that is expressed by physiological HSPC as wells as by leukemic blasts in >90% of AML patients. For translation into a clinical setting, CAR T cell treatment must then be followed by depletion of CAR T-cells as well subsequent healthy/allogeneic HSC transplantation. Methods: A lentiviral vector was generated which incorporates the CAR (scFv linked to intracellular CD3ζ and 4-1BB signaling domains via stalk and transmembrane regions derived from CD8), followed by a T2A ribosomal skip sequence and RQR8 as selection marker and depletion gene (surface expression of CD34 and CD20 epitopes). The scFv was extracted from a previously published bivalent anti-CD117 antibody (clone 79D) that was derived from an artificial human phage library (Reshetnyak et al., PNAS, 2013). 79D exhibits high binding affinity to an epitope in the membrane-proximal domain of human CD117. Human CD117 was cloned in human CD117 negative HL-60 AML cells and cell lines with stable expression of CD117 at various levels were derived from these. Results: T-cells were isolated from healthy donors or AML patients in complete remission and both healthy donor and AML pateint derived T-cells exhibited sustained growth after activation with recombinant human IL-2 and CD3/CD28 beads. Lentiviral transduction yielded consistently high transduction rates, ranging from 55 - 75% as determined by staining for RQR8 and the scFv. In co-culture assays, CAR T-cells eliminated more than 90% of CD117high leukemia cell lines within 24 hours at effector-to target ratios (E:T) of 4:1 and 1:1 and more than 50% at E:T of 1:4. CAR-mediated cytotoxicity correlated with levels of CD117 surface expression as the elimination of CD117low target cells was less efficient compared to CD117high and CD117intermediate cells. In long-term cytotoxicity assays (45d), only CD117low cells were able to escape CAR-mediated killing. In the setting of primary cells, anti-CD117 CAR T-cells effectively depleted >90% of lin-CD117+CD34+CD38+ and >70% of lin-CD117+CD34+CD38- cells from healthy bone marrow in vitro within 48 hours. Similarly, >70% of patient derived leukemic blasts were eliminated by autologous anti-CD117 CAR T-cells within 48 hours (1:1 ratio of CAR T cells:blasts). In a long-term assay, no outgrowth of leukemic blasts was observed in the presence of autologous CAR T-cells over 3 weeks. To determine effectivity of CAR T-cells in vivo, humanized mice (NSG & MTRG-SKI) were engrafted with umbilical cord blood derived CD34+ cells. A single injection of 2x106 anti-CD117 CAR T-cells resulted in >90% depletion of CD117+ cells in the bone marrow within 6 days. Finally, humanized mice transplanted with bone marrow from AML patients expressing CD117 were treated with patient-derived autologous CAR T-cells. At 6 weeks after injection of CAR T-cells, >98% of hu-CD45 CD117+ cells were depleted in the bone marrow while control human T-cell treated mice showed full-blown CD117 positive AML. Conclusions: We provide proof of concept for the generation of highly-potent CAR T-cells re-directed against CD117 from healthy human donors and AML patients. Anti-CD117 CAR T-cells exhibit high cytotoxic activity against CD117+ cell lines as well as primary healthy HSPC and patient AML cells in vitro and in vivo in murine xenograft models. Strategies for the complete elimination of CAR T-cells (immunologic or small molecule based) are required before translation of this approach to the clinical setting. Disclosures Neri: Philochem AG: Equity Ownership.

scholarly journals Lentiviral Vectors Interfering with Virus-Induced CD4 Down-Modulation Potently Block Human Immunodeficiency Virus Type 1 Replication in Primary Lymphocytes

2004 ◽  
Vol 78 (23) ◽  
pp. 13072-13081 ◽  
Author(s):  
Hang M. Pham ◽  
Enrique R. Argañaraz ◽  
Bettina Groschel ◽  
Didier Trono ◽  
Juan Lama
Keyword(s):  
Human Immunodeficiency Virus ◽  
Lentiviral Vectors ◽  
Viral Pathogenesis ◽  
Viral Particles ◽  
Immunodeficiency Virus ◽  
Anti Hiv ◽  
Hiv 1

ABSTRACT CD4 down-modulation is essential for the production of human immunodeficiency virus (HIV) infectious particles. Disease progression correlates with enhanced viral induced CD4 down-modulation, and a subset of long-term nonprogressors carry viruses defective in this function. Despite multiple pieces of evidence highlighting the importance of this function in viral pathogenesis in vivo, to date, HIV-induced CD4 down-modulation has not been used as a target for intervention. We describe here HIV-based vectors that deliver truncated CD4 molecules resistant to down-modulation by the viral products Nef and Vpu. Infection of cells previously transduced with these vectors proceeded normally, and viral particles were released in normal amounts. However, the infectivity of the released virions was reduced 1,000-fold. Lentiviral vectors expressing truncated CD4 molecules were efficient at blocking HIV-1 infectivity and replication in several cell lines and in CD4-positive primary lymphocytes. The findings presented here provide proof-of-principle that approaches targeting the virus-induced CD4 down-modulation may constitute the basis for novel anti-HIV therapies.

CD22-Targeted Chimeric Antigen Receptor (CAR) T Cells Containing The 4-1BB Costimulatory Domain Demonstrate Enhanced Persistence and Superior Efficacy Against B-Cell Precursor Acute Lymphoblastic Leukemia (ALL) Compared To Those Containing CD28

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1431-1431 ◽  
Author(s):  
Waleed Haso ◽  
Haiying Qin ◽  
Ling Zhang ◽  
Rimas J Orentas ◽  
Terry J Fry
Keyword(s):  
T Cells ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Cell Precursor ◽  
Car T Cells ◽  
Car T ◽  
Anti Tumor Activity

Abstract B cell precursor acute lymphoblastic leukemia (BCP-ALL) remains a leading cause of death from childhood cancers despite survival rates exceeding 80%. Antibody-based CAR-engineered T cells can recognize and eliminate tumors by binding directly to a surface antigen independent from MHC restriction. CAR immunotherapy against BCP-ALL has demonstrated impressive responses and sustained remission in clinical trials targeting CD19. However, some patients receiving the CD19 CAR T cells relapse with a CD19 negative leukemia. Thus, additional CAR targets are needed. CD22 is a Siglec family lectin consisting of 7 extracellular Ig domains that is expressed on the cell surface from the pre-B cell stage of development through mature B cells and is expressed on most B cell hematologic malignancies. We previously generated a second-generation (CD3-Zeta + CD28 costimulatory domain) anti-CD22 CAR derived from a membrane proximal epitope binding scFv (m971-28z) with potent activity in vivo (Haso W et al, Blood 2013). In clinical trials T cells expressing CD19-targeted CAR with 4-1BB costimulatory domains on CD19 CARs show prolonged persistence. To improve long-term persistence of the CD22 CAR, we re-engineered our CAR vector to include a 4-1BB signaling domain (m971-BBz). In vitro data using m971-BBz improved proliferation and expansion compared to m971-28z especially when lower concentrations of IL2 were included in the culture media. When no IL2 was added to the media only the 4-1BB containing CAR expanded. No difference in killing was detected in in vitro cytotoxicity assays. We next evaluated anti-tumor activity and persistence in the NSG mouse model engrafted with the NALM6-GL cell line on day 0 and treated with CAR T cells on day 3 to directly compare the efficacy of m971-28z and m971-BBz modified T cells activated with either OKT3 or anti-CD3/CD28 beads. m971-BBz outperformed m971-28z in terms of in vivo anti-tumor activity and long-term persistence. This effect was only detected when anti-CD3/CD28 beads were used for T cell expansion. OKT3-activated cells failed to persist and demonstrated inferior antitumor activity compared to bead-expanded T cells irrespective of the costimulatory domain and despite a higher percentage of CD8 T cells with significantly better cytotoxicity in vitro. Interestingly, early peripheral blood numbers of CAR T cells in recipients of bead-expanded products demonstrated a predominance of CD4+CAR T cells consistent with preinfusion CD4/CD8 ratios. At later time points this ratio decreased with a predominance of CD8+CAR T cells. In mice receiving m971-28z CAR the CD8+CAR T cells failed to persist resulting in leukemic relapse. Furthermore, direct comparison to the CD19 CAR (FMC63-BBz) in vivo showed that the anti-CD22 CAR (m971-BBz) has equivalent activity. We conclude that anti-CD3/CD28 bead-activated T cells modified to express an anti-CD22 CAR with a 4-1BB costimulatory domain demonstrates potent antitumor activity with long-term leukemic control and offers a promising therapeutic option for pediatric ALL. Disclosures: No relevant conflicts of interest to declare.

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