scholarly journals A BAFF ligand-based CAR-T cell targeting three receptors and multiple B cell cancers

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Derek P. Wong ◽  
Nand K. Roy ◽  
Keman Zhang ◽  
Anusha Anukanth ◽  
Abhishek Asthana ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Viral Gene ◽  
Car T Cells ◽  
Car T ◽  
Almost All ◽  
Viral Gene Delivery

AbstractB cell-activating factor (BAFF) binds the three receptors BAFF-R, BCMA, and TACI, predominantly expressed on mature B cells. Almost all B cell cancers are reported to express at least one of these receptors. Here we develop a BAFF ligand-based chimeric antigen receptor (CAR) and generate BAFF CAR-T cells using a non-viral gene delivery method. We show that BAFF CAR-T cells bind specifically to each of the three BAFF receptors and are effective at killing multiple B cell cancers, including mantle cell lymphoma (MCL), multiple myeloma (MM), and acute lymphoblastic leukemia (ALL), in vitro and in vivo using different xenograft models. Co-culture of BAFF CAR-T cells with these tumor cells results in induction of activation marker CD69, degranulation marker CD107a, and multiple proinflammatory cytokines. In summary, we report a ligand-based BAFF CAR-T capable of binding three different receptors, minimizing the potential for antigen escape in the treatment of B cell cancers.

scholarly journals 552 Amphiphile-peptide boosting with FMC63-binding surrogate peptide mimotopes induces activation and potent effector function in CAR-T cells targeting CD19

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A582-A582
Author(s):  
Peter DeMuth ◽  
Amy Tavares ◽  
Ana Castano
Keyword(s):  
T Cells ◽  
B Cell ◽  
Solid Tumors ◽  
Lymphoblastic Leukemia ◽  
Effector Functions ◽  
Car T Cells ◽  
Car T ◽  
Peptide Mimotopes

BackgroundGenetic engineering of T cells to express anti-CD19 Chimeric Antigen Receptors (CAR-T cells) has been FDA approved for the treatment of refractory/relapsing acute lymphocytic leukemia and diffuse large B cell lymphoma. With more patients receiving treatment with CAR-T cells it has been observed that approximately 10–20% of patients fail to enter remission after therapy,1 and 30–50% of patients who achieve remission with anti-CD19 CAR T cells have disease relapse.2 In prior studies, CAR-binding amphiphile (AMP)-peptides were shown to effectively localize in lymph nodes (LN), where they decorate endogenous antigen-presenting cells (APC) and stimulate CAR signaling to promote potent CAR-T responses against solid tumors.3 In this study, we describe how CD19 mimotope peptides specific for FMC63-based CARs can be modified with AMP technology to enhance peptide accumulation in LNs, enable presentation on APCs to CAR-Ts, and promote activation and effector functionality of CAR-T cells.MethodsWe performed phage-screening and enrichment for CD19 surrogate peptides recognized by FMC-63-scFv. Surface Plasmon Resonance (SPR) was utilized to evaluate the affinity of the peptides to immobilized FMC-63. AMP versions of peptides were generated. In vitro, human dendritic cells (DCs) were preconditioned with AMP-CD19 or soluble peptides and cocultured with autologous T cells engineered to express CD19 CARs (FMC63-28z and FMC63-41BBz). Markers for activation, proliferation, cytotoxicity, and effector functions were evaluated. In vivo experiments were performed to evaluate the biodistribution of peptides. Luciferase-expressing murine CAR-T cells were engineered to evaluate the expansion and biodistribution of CAR-T cells in combination with AMP or soluble regimens.ResultsWe found surrogate CD19 peptide mimotopes that bind to FMC-63 with different affinities evaluated by ELISA and SPR. Assessment in human autologous DC/CAR-T cell cocultures demonstrated that AMP-CD19 peptides can decorate DCs effectively and promote potent activation (OX40, 41BB, CD69), proliferation, cytokine production (IFNγ, TNFα, and IL2), cytotoxicity (CD107a), and phenotypic enhancement of CD19-specific CAR-T cells. Assessment in vivo showed that AMPs are effectively delivered to LN where endogenous APCs are decorated to promote the activity of murine CAR-T cells.ConclusionsIn vitro, AMP modification of CAR-binding peptide mimotopes induces activation, cytotoxicity, and effector functions of CAR-T cells. These AMP-peptides effectively accumulate in LN and boost CAR-T activation and expansion in vivo. This platform can potentially be utilized as a mechanism to expand and functionally enhance CAR-T cells in vivo for blood and solid tumors.ReferencesMaude SL et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med 2018;378:439–448.Park JH et al. Long-term follow-up of CD19 CAR therapy in acute lymphoblastic leukemia. N Engl J Med 2018;378:449–459.Ma L et al. Enhanced CAR–T cell activity against solid tumors by vaccine boosting through the chimeric receptor. Science 2019;365(6449):162–168.Ethics ApprovalAll animal experiments in this study were performed in accordance with the approval of IACUC Protocol CR-0039.

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.

scholarly journals Engineering better chimeric antigen receptor T cells

2020 ◽  
Vol 9 (1) ◽  
Author(s):  
Hao Zhang ◽  
Pu Zhao ◽  
He Huang
Keyword(s):  
T Cells ◽  
Intracellular Signaling ◽  
Influence Factors ◽  
Target Antigen ◽  
Car T Cells ◽  
Car T ◽  
Underlying Mechanisms ◽  
Almost All

AbstractCD19-targeted CAR T cells therapy has shown remarkable efficacy in treatment of B cell malignancies. However, relapse of primary disease remains a major obstacle after CAR T cells therapy, and the majority of relapses present a tumor phenotype with retention of target antigen (antigen-positive relapse), which highly correlate with poor CAR T cells persistence. Therefore, study on factors and mechanisms that limit the in vivo persistence of CAR T cells is crucial for developing strategies to overcome these limitations. In this review, we summarize the rapidly developing knowledge regarding the factors that influence CAR T cells in vivo persistence and the underlying mechanisms. The factors involve the CAR constructs (extracellular structures, transmembrane and intracellular signaling domains, as well as the accessory structures), activation signaling (CAR signaling and TCR engagement), methods for in vitro culture (T cells collection, purification, activation, gene transduction and cells expansion), epigenetic regulations, tumor environment, CD4/CD8 subsets, CAR T cells differentiation and exhaustion. Of note, among these influence factors, CAR T cells differentiation and exhaustion are identified as the central part due to the fact that almost all factors eventually alter the state of cells differentiation and exhaustion. Moreover, we review the potential coping strategies aiming at these limitations throughout this study.

scholarly journals Fratricide-resistant CD1a-specific CAR T cells for the treatment of cortical T-cell acute lymphoblastic leukemia

Blood ◽  
2019 ◽  
Vol 133 (21) ◽  
pp. 2291-2304 ◽  
Author(s):  
Diego Sánchez-Martínez ◽  
Matteo L. Baroni ◽  
Francisco Gutierrez-Agüera ◽  
Heleia Roca-Ho ◽  
Oscar Blanch-Lombarte ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Antileukemic Activity ◽  
Car T Cells ◽  
Car T ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Relapsed/refractory T-cell acute lymphoblastic leukemia (T-ALL) has a dismal outcome, and no effective targeted immunotherapies for T-ALL exist. The extension of chimeric antigen receptor (CAR) T cells (CARTs) to T-ALL remains challenging because the shared expression of target antigens between CARTs and T-ALL blasts leads to CART fratricide. CD1a is exclusively expressed in cortical T-ALL (coT-ALL), a major subset of T-ALL, and retained at relapse. This article reports that the expression of CD1a is mainly restricted to developing cortical thymocytes, and neither CD34+ progenitors nor T cells express CD1a during ontogeny, confining the risk of on-target/off-tumor toxicity. We thus developed and preclinically validated a CD1a-specific CAR with robust and specific cytotoxicity in vitro and antileukemic activity in vivo in xenograft models of coT-ALL, using both cell lines and coT-ALL patient–derived primary blasts. CD1a-CARTs are fratricide resistant, persist long term in vivo (retaining antileukemic activity in re-challenge experiments), and respond to viral antigens. Our data support the therapeutic and safe use of fratricide-resistant CD1a-CARTs for relapsed/refractory coT-ALL.

scholarly journals Automated Generation and Phenotypic Characterization of Clinical Grade CD19 CAR-T Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1675-1675
Author(s):  
Ashish Sharma ◽  
Anne Roe ◽  
Filipa Blasco Lopes ◽  
Ruifu Liu ◽  
Jane Reese ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Cd8 T Cells ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Central Memory ◽  
Car T Cells ◽  
Car T

Abstract BACKGROUND: Chimeric antigen receptor (CAR) T cells have shown enormous promise in the treatment of certain B cell malignancies. Access to treatment is still limited due to a variety of issues, including pricing and centralized manufacturing models. Generation of CAR-T cells using an automated platform, followed by rigorous functional phenotyping, may contribute to the development of a robust long-lasting therapy. METHODS: Here, we used the Miltenyi Prodigy (Miltenyi Biotech, Bergisch Gladbach, Germany) to automate the process of manufacturing genetically manipulated T cells in a closed system. The system obviates the need for clean room infrastructure. We tested the feasibility of utilizing the Miltenyi Prodigy to manufacture CAR-T cells using a CD19 scFV vector with the 4-1BB co-stimulatory domain. (Lentigen Technology, Inc, Gaithersburg, MD). The purity, differentiation capacity and effector function of the enriched CAR-T cells was studied using high-dimensional flow cytometry. Finally, the functional potential of these cells was tested in vitro and by treating NOD-SCID-gamma (NSG) mice infused with B cell lymphoma cells (Raji B cell), with the CAR-T cells. RESULTS: Starting with 1 x 108 CD4 and CD8 cells from donor apheresis products, CAR-T cells were expanded for 12 days in culture media containing with TransAct (Miltenyi Biotech), IL7 and IL15. The mean fold-expansion at day 12 was 44 ± 5.6, range 39-50 (n=3). The mean transduction efficiency of CAR-T vector was 20%, range 10-25% (n=3), which is similar to other reported methods. The CD19 CAR-T product was enriched in both the CD4 and CD8 T cells subsets, and showed high-level of cytotoxicity against CD19+ cell lines in vitro and in vivo (Figure 1: Mice treated with the CD19-CAR T demonstrated a marked reduction in disease burden as compared to T cell control as measured by bioluminescence imaging and flow cytometric analysis). The CAR-T product was enriched in cell subsets with both effector (CD27-CCR7-; ~20% of total cells) and central memory phenotypes (CD27+CCR7+; ~30% of total T cells). The effector CD4 and CD8 T cells showed increased expression of major functional T cell differentiation transcription factors (i.e. T-bet and GATA3) critical for the development of anti-tumor responses. Whereas, the central CD4 and CD8 T cells were enriched for the expression of TCF7 (a stemness related member of the WNT signaling known to increase longevity of these cells). The frequencies and phenotypes of these cells were maintained in peripheral blood of NSG mice infused with B cell lymphoma cells (Raji B cells), 1 week after treatment. A significant expansion of CD8+ effector T cells and a dramatic reduction in tumor burden was observed over the next 4 weeks in all major organs. Interestingly, we observed that smaller proportion of central-memory like cells (with higher TCF7 levels) continued to persist 6 weeks post-treatment, potentially contributing to a long-lived recallable response. Based on these data we have initiated a phase 1 clinical trial to test the therapeutic potential of the CAR-T product in patients with advanced/refractory B cell lymphoma. The first clinical grade manufacturing run resulted in a CD19 + cell yield of 1.4 x109. CONCLUSION: Our data highlight that the automated CAR-T generation platform (i.e. Miltenyi Prodigy) is effective at the generating purified functionally competent CAR-T cells. Further, findings from our phenotyping analyses show that the CAR-T product is enriched in both effector and central memory subsets and is effective at tumor clearance in vivo. Thus far, we have treated one patient with CD19 CAR-T manufactured on this platform and 2 more have been enrolled. Though this initial study is based on CD19 CAR-T cells, the approach described here could easily be utilized to genetically engineer T cells with gene constructs that are more relevant for specific cancers and infectious diseases. Disclosures No relevant conflicts of interest to declare.

scholarly journals 5-Aza-2'-Deoxycytidine Promotes Cytotoxic Effect of CART-Cells on Leukaemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5812-5812
Author(s):  
Alla Dolnikov ◽  
Swapna Rossi ◽  
Ning Xu ◽  
Guy Klamer ◽  
Sylvie Shen ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Target Cells ◽  
Car T Cells ◽  
Anti Tumour Activity ◽  
Car T ◽  
Pre Treatment ◽  
Tumour Activity

Abstract T cells modified to express CD19-specific chimeric antigen receptors (CAR) have shown anti-tumour efficacy in early phase clinical trials in patients with relapsed and refractory B-cell malignancies. In addition to direct cytotoxicity, chemotherapeutic drugs can have an immunomodulatory effect both through enhancing the tumour-specific immune response and increasing the tumour’s susceptibility to immune mediated destruction. Hence, combining immunomodulatory chemotherapy and CAR T-cells is an attractive approach for eliminating tumours, particularly in advanced stages. 5-aza-2'-deoxycytidine (5-AZA) is a hypomethylating agent that induces terminal differentiation, senescence or apoptosis in haematological malignancies. Here, we have explored a CAR-based immunotherapy combined with 5-AZA to maximise the effect of the CAR T-cells against CD19+ B-cell leukaemia. A second generation CAR including CD3zeta and the CD28 co-stimulatory domain was cloned into the PiggyBac-transposon vector and was used to generate CAR19 -T cells. Cord blood -derived mononuclear cells (MNC) were transfected with CAR19-transposon/transposase plasmids and expanded with CD3/28 beads for 2 weeks in the presence of 20ng/ml IL2 and 10ng/ml IL7. CAR19 T-cells efficiently induced cytolysis of CD19+ leukaemia cells in vitro and exhibited anti-tumour activity in vivo in a xenograft mouse model of leukaemia. Pre-treatment with 5-AZA produced greater leukaemia cell cytolysis in vitro and maximised anti-tumour activity of CAR19 T-cells in vivo against xenograft primary leukaemia compared to 5-AZA or CAR19 T-cells alone. In vitro analysis revealed that pre-treatment with 5-AZA up-regulates CD19 expression in leukaemia cells and improves CAR19 T-cell recognition of target cells increasing the formation of effector/ target cell conjugates and target cell cytolysis. Therefore using 5-AZA pre-treatment can be particularly useful for B-cell leukaemias with reduced expression of CD19. We have also demonstrated that pre-treatment of target cells with 5-AZA potentiates the effect of CAR19 T-cells used at low dose or low effector:target (E:T) suggesting that even small numbers of CAR19 T-cells can mediate a potent antitumor effect when combined with 5-AZA pre-treatment of target cells. This is particularly important for patients receiving limited numbers of CAR T-cells or for patients with large leukaemic burden. In addition, we speculate that the enhanced cellular cytotoxicity produced by 5-AZA-conditioning may allow the infusion of decreased numbers of CAR19 T-cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Novel full-human CD22-CAR therapy overcomes resistance to previous CD19/22-CAR regimens in ALL

2021 ◽  
Author(s):  
Yue Tan ◽  
Haodong Cai ◽  
Chuo Li ◽  
Biping Deng ◽  
Weiliang Song ◽  
...  
Keyword(s):  
T Cells ◽  
Lymphoblastic Leukemia ◽  
Considerable Proportion ◽  
Target Antigen ◽  
Single Chain ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T

Abstract BackgroundCD19- and/or CD22-targeted chimeric antigen receptor (CAR) T cells efficiently induced remission in patients with B acute lymphoblastic leukemia (B-ALL), but a considerable proportion of patients relapsed after both CD19- and CD22-CAR therapies associated with the loss or downregulation of target antigen. Re-infusions of the prior used CAR T cells were usually ineffective. In contrast to the frequent loss of CD19, low level of CD22 is usually present on leukemia cells post CAR therapy, suggesting that newly designed CD22-CAR therapies may be effective in these patients.MethodsA yeast full-human single-chain variable fragment (scFv) library and a high-throughput NFAT reporter assay were utilized to screen several full-human CD22-CAR candidates; CD107 assay and in vitro cytotoxicity assay was used to evaluate the effector function of CAR T cells; membrane proteome assay was conducted to determine the specificity of the CAR toward the target antigen; a leukemia animal models was used to test the in vivo efficacy of CAR T cells. A phase I trial (ChiCTR2000028793) was conducted to assess the safety and effectiveness of CD22-CARFH80 therapy in 8 children with B-ALL resistant to or relapsed after prior CD19- and CD22-CAR treatment.ResultsWe identified a full-human CD22-CAR construct termed CD22CARFH80 which could mediate superior anti-leukemia activity in vitro and in a leukemia animal model and had good specificity to the target antigen. Data from the trial showed that with CD22-CARFH80 T-cell therapy, 6/8 (75%) patients including 2 who had CD22low blasts achieved complete remission; 1 patient had a partial response. CAR T cells efficiently expanded in vivo, while the toxic effect is low in most patients. At a median follow-up of 5 months, 4/6 (57%) patients remained in remission.ConclusionsTherapy with a newly invented CD22-CARFH80 overcomes the resistance to prior versions of CD19- and CD22-CAR formats and elicits potent anti-leukemia responses with an acceptable safety profile, representing a promising salvage regimen for B-ALL that fails in prior CD19- and CD22-CAR treatments.Trial registrationClinicalTrials.gov: ChiCTR2000028793; registered 4 January, 2020. http://www.chictr.org.cn/showproj.aspx?proj=47857

scholarly journals Application of Novel T Cell Immunotherapeutics to Drive Antigen-Specific Activation, Expansion, and Differentiation of CD19 Chimeric Antigen Receptor T Cells (CAR T-cells)

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 34-35
Author(s):  
Moriah Rabin ◽  
Mengyan Li ◽  
Scott Garforth ◽  
Jacqueline Marino ◽  
Jian Hua Zheng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cd8 T Cells ◽  
Effector Memory ◽  
Mhc Molecules ◽  
Car T Cells ◽  
Car T

Background: While chimeric antigen receptor T cells (CAR T-cells) induce dramatic remissions of refractory or recurrent B cell malignancies, the durability of these remissions is frequently limited by subsequent reduction in circulating CAR T-cells and/or by diminution of their effector function. We hypothesized that we could overcome this therapeutic limitation and increase the functional activity and longevity of CAR T-cells by selectively deriving them from virus-specific effector memory T cells. We have developed biologics we termed synTacs (artificial immunological synapse for T-cell activation), which selectively activate and expand antigen-specific CD8+ T cells in vitro and in vivo by recapitulating signals delivered at the immunological synapse. The synTacs consist of dimeric Fc domain scaffolds linking CD28- or 4-1BB-specific ligands to HLA-A2 MHC molecules covalently tethered to virus-derived peptides. Treatment of PBMCs from CMV-exposed donors with synTacs presenting a CMV-derived peptide (pp65-NLVPMVATV) induce vigorous and selective ex vivo and in vivo expansion of highly functional CMV-specific CD8+ T cells, with potent antiviral activity. We used these synTacs to selectively generate CAR T-cells from CMV-specific effector memory CD8+ T cells, which could be further expanded by restimulation with the CMV-specific synTacs. Methods: We treated PBMCs from CMV-exposed donors in media supplemented with either IL-2 or IL-7/12/15 with a synTac containing the CMV-derived pp65 peptide presented by HLA-A2 MHC molecules linked to ligands capable of stimulating CD28- or 4-1BB-dependent costimulatory pathways. PBMCs activated either with anti-CD3/CD28 or the CMV-specific synTacs were transduced with lentivirus expressing an anti-CD19 CAR and a GFP reporter gene. CMV-specific CD8+ T cells were quantified by tetramer staining and CAR T-cells were detected by GFP expression determined by flow cytometric analysis. The functional activity of the CD19 CAR T-cells was determined by a B cell-specific cytotoxic assay. Results: After 7 days, treatment of PBMCs with CMV-specific synTacs rapidly induced robust activation and >50-fold expansion of CMV-specific CD8+ T cells expressing effector memory markers. Treatment of the PBMCs with CMV-specific synTacs selectively activated CMV-specific T cells and enabled them to be specifically transduced with a CD19-specific CAR lentivirus and converted into CD19 CAR T-cells. These CMV-specific CD19 CAR T-cells displayed potent dose-responsive cytotoxic activity targeting purified primary B cells. Furthermore, these CMV-specific CD19 CAR T-cells could be selectively expanded by in vitro treatment with CMV-specific synTacs. Conclusions: SynTacs are versatile immunotherapeutics capable of selective in vitro and in vivo activation and expansion of virus-specific CD8+ T cells with potent antiviral cytotoxic activity. After selective lentiviral transduction and conversion into CD19 CAR T-cells, their co-expression of the CMV-specific T cell receptor enabled them to be potently stimulated and activated by in vitro treatment with CMV synTacs. The modular design of synTacs facilitates efficient coupling of other costimulatory ligands - such as OX40 or GITRL - or cytokines, such as IL-2, IL-7, or IL-15, to enable the selective in vivo delivery of defined costimulatory signals or cytokines to the CAR T-cells expressing CMV-specific TCR. This strategy has the potential to boost the in vivo activity of tumor-specific CAR T-cells after infusion and enable more durable and potent treatment of refractory/recurrent B cell malignancies. Disclosures Almo: Cue Biopharma: Current equity holder in publicly-traded company, Patents & Royalties: Patent number: 62/013,715, Research Funding. Goldstein:Cue Biopharma: Research Funding.

scholarly journals CD72 Nanobody-Based CAR-T Cells Have Potent Anti-Tumor Efficacy in B Cell Malignancies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1717-1717
Author(s):  
Matthew A Nix ◽  
William C Temple ◽  
William Karlon ◽  
Donghui Wang ◽  
Paul Phojanakong ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
B Cell ◽  
In Vitro Cytotoxicity ◽  
B Cell Malignancies ◽  
Cytotoxicity Assays ◽  
Car T Cells ◽  
Car T

Abstract Background: Approximately 50% of pediatric B-ALL patients treated with clinically approved CD19-targeting CAR-T cells do not remain in remission one year after therapy. CD22-targeting CAR-T cells appear to be curative in only a small fraction of CD19-refractory patients and this therapeutic strategy is primarily used as a bridge to stem cell transplant. Additional immunotherapeutic targets thus remain urgently needed. Our laboratory recently used cell surface proteomics to identify CD72 as a B-cell specific marker especially upregulated on poor prognosis, KMT2A/MLL-rearranged B-ALL (Nix et al., Cancer Discovery (2021)). In this published work, we used a best-in-class nanobody library displayed on yeast to develop binders to CD72. We demonstrated for the first time that fully synthetic nanobodies can generate CAR-T cells that are highly potent in vitro and in vivo. While we previously focused on these "nanoCARs" in KMT2A/MLLr B-ALL, in this follow-up study we aimed to 1) further expand our nanoCAR indications to other CD72-expressing B-cell malignancies; 2) biophysically characterize our synthetic nanobodies; 3) evaluate the potential for further humanization of the nanobody binder amino acid sequence while retaining anti-tumor efficacy; and 4) characterize the potency and T-cell immunophenotypes in the context of our lead nanobody binder ("NbD4") placed on different CAR backbones. Methods: Flow cytometry of primary patient samples for CD72 was performed in a CLIA-certified laboratory. NbD4 nanobody was recombinantly expressed in E. coli and biolayer interferometry was used to determine the binding affinity to recombinantly-expressed CD72 extracellular domain. CAR-T cells were generated from peripheral blood donor CD4+ and CD8+ cells (1:1) ratio via lentiviral transduction. In vitro cytotoxicity assays were performed at a range of effector:tumor ratios. In vivo studies were performed in human cell line orthotopic xenografts in NSG mice. 1e6 luciferase-labeled Jeko cells were implanted at Day 0 followed by administration of 4e6 CAR-T cells at Day 6. Tumor burden was assessed by bioluminescence. Results: Flow cytometry on primary non-Hodgkin B-cell lymphoma obtained from fine needle aspiration biopsy (n = 5) confirmed CD72 surface expression (not shown), consistent with RNA-seq across larger cohorts. Biolayer interferometry demonstrated that NbD4 bound with surprisingly low affinity to recombinant CD72 (K D ~800 nM) (Fig. 1A), with both slow on rate (k on 8.38e4 M -1s -1) and rapid off rate (k off 6.82e-2 s -1). This affinity stands in contrast to that reported for FMC63 single chain variable fragment (scFv) used in clinically approved CD19-targeting CAR-T cells (K D 0.3-5 nM), despite similar in vitro and in vivo efficacy of both products. Our NbD4 framework region shows ~82% homology to a human IgG variable heavy domain, significantly higher than FMC63 (~59% homology). We made additional substitutions in the framework domain to increase human homology up to 89%. In vitro cytotoxicity assays with SEM B-ALL cells showed several humanized variants with similar efficacy to NbD4 (Fig. 1B). We further evaluated the impact of placing NbD4 on different CAR backbones, including combinations of CD28 or 4-1BB costimulatory domains and CD8 or IgG4-based transmembrane and hinge regions (Fig. 1C). In vivo, CD72 nanoCARs with Backbone 3 showed significantly increased potency (Fig. 1D). Indeed, tumors treated with Backbone 3 CAR-Ts showed complete tumor clearance and did not develop new tumors even after re-challenge with 1e6 Jeko cells at Day 52 (Fig. 1D). Preliminary characterization of effector and memory CAR-T cell phenotypes before exposure to tumor suggested that Backbone 3 had an increased number of naïve T cells compared to empty CAR and CD19 CAR-T cells (data not shown). Conclusions: Our results demonstrate that our fully synthetic CD72 nanoCARs can effectively eliminate CD72-expressing B-cell malignancy models despite low nanobody binding affinity. Humanized NbD4 variants may serve as clinical candidates with even further reduction in possible immunogenicity of the llama amino acid framework. Alterations to the CAR backbone have a major impact on anti-tumor efficacy and phenotypes of our synthetic nanobodies. CD72-targeting therapies may be effective therapeutics not only KMT2A/MLLr B-ALL but also across a broader spectrum of refractory B-cell malignancies. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Tri-Specific CD19xCD20xCD22 VHH CAR-T Cells (LCAR-AIO) Eradicate Antigen-Heterogeneous B Cell Tumors, Enhance Expansion, and Prolong Persistence in Preclinical In Vivo Models

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1700-1700
Author(s):  
Zhe (joy) Zhou ◽  
Yue Han ◽  
Hong-Bo Pan ◽  
Cai-Jun Sang ◽  
Dong-Lin Shi ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Treatment Efficacy ◽  
Cytolytic Activity ◽  
Publicly Traded ◽  
Car T Cells ◽  
Car T ◽  
Ifn Γ

Abstract Introduction: Anti-CD19 CAR-T therapy has achieved remarkable treatment efficacy in B cell lymphoma. However, targeting CD19 antigen alone can only benefit about half of patients with B cell malignancies. The FDA-approved CD19 CAR-T therapies all use same binder, which is murine FMC63 scFv targeting CD19 and up to 39%-88% of patients have relapsed. Possible mechanisms of relapse include mutations or downregulation of the targeted antigen, CD19, however, the targetable expression of CD20 and CD22 is preserved. In addition, immunogenicity against murine FMC63 scFv could have a negative impact on possible re-dosing regimen. To overcome these limitations, we designed and developed a novel tri-specific VHH CAR-T, targeting three antigens that include CD19, CD20 and CD22, for treating patients who relapsed from prior CAR-T therapies. Methods: We engineered mono-, bi-, or tri-specific VHH CAR constructs targeting CD19, CD20 and/or CD22 respectively in a lentiviral vector. The mono-, bi- or tri-specific CAR-T cells were tested against tumor lines expressing single, dual or triple antigens in an in vitro cytotoxicity assay. In addition, we evaluated the contribution of different CAR backbones, and possible combinations of scFv, VH or VHH to CAR design. We hypothesized that our lead tri-specific VHH CAR-T, LCAR-AIO, would potently inhibit tumors with heterogeneous Ag expression and prevent Ag escape. To validate this, we compared in vitro cytolytic activity and cytokine production of LCAR-AIO CAR-T to anti-CD19 FMC63 CAR-T against CD19 +CD20 +CD22 + Raji.Luc and CD19KOCD20 +CD22 +Raji.Luc cells . In vivo treatment efficacy and CAR-T persistence were also investigated in NCG murine model xenografted with Raji tumor line. 0.3x10 6 CAR +T cells or dose-matched untransduced T cells were given to NCG mice four days post i.v. implantation of Raji.Luc tumor cells. Tumor growth was monitored weekly by bioluminescence imaging until achieved endpoint (55 days), and CAR-T persistence was determined using genomic DNA level. Results: Tri-specific VHH CAR-T cells can mediate dose-dependent cytotoxicity against Raji tumor lines. Compared to mono- or bi-specific VHH or scFv CAR-T, tri-specific VHH CAR-T demonstrated equal or better cytolytic activity. Our lead tri-VHH CAR-T, LCAR-AIO, was able to specifically lyse K652 over-expressing single target such as CD19, CD20 or CD22, at the similar level to mono-specific CD19, CD20 or CD22 VHH CAR-T. Since no blocking effect of recognition against these three antigens was observed, our result suggested that all three VHHs in LCAR-AIO are functional. In comparison to anti-CD19 FMC63 scFv CAR-T, LCAR-AIO exhibited higher lytic activity and IFN-γ production against Raji.Luc tumor lines in vitro. In addition, LCAR-AIO retained its robust lytic activity and IFN-γ production when co-cultured with CD19KO-Raji.Luc cells while anti-CD19 FMC63 scFv CAR-T could not, suggesting LCAR-AIO may prevent tumor escape due to loss of CD19. Furthermore, comparison of LCAR-AIO to mono-scFv CAR-T (anti-CD19 FMC63-BBz, anti-CD20 Leu16-BBz or anti-CD22 m971-BBz) was performed in NCG mice xenografted with Raji cell line, LCAR-AIO exhibited better T cell expansion, longer persistence, and superior efficacy in eliminating tumors. Conclusions: Based on in vitro and in vivo preclinical data, tri-specific CD19xCD20xCD22 VHH CAR-T can be effective targeting tumors lack of CD19 expression, therefore, it has the potential of treating relapsed patients with prior CD19 CAR-T therapy. The feasibility of making tri-specific CAR-T would help to extend this technology to solid cancers where heterogeneity poses a major challenge at current stage. Figure 1 Figure 1. Disclosures Zhou: Legend Biotech: Current Employment, Current equity holder in publicly-traded company. Han: Legend Biotech: Current Employment, Current equity holder in publicly-traded company. Pan: Legend Biotech: Current Employment, Current equity holder in publicly-traded company. Sang: Legend Biotech: Current Employment, Current equity holder in publicly-traded company. Shi: Legend Biotech: Current Employment. Feng: Legend Biotech: Current Employment. Xiao: Legend Biotech: Current Employment. Zhuang: Legend Biotech: Current Employment, Current equity holder in publicly-traded company. Wang: Legend Biotech: Current Employment, Current equity holder in publicly-traded company. Fan: Legend Biotech: Current Employment, Current equity holder in publicly-traded company.

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