Bispecific Antibody Armed Metabolically Enhanced Headless CAR T Cells.
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ABSTRACT:
SUBMITTER: Thakur A
PROVIDER: S-EPMC8288104 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:PurposeAlthough adoptive cell therapy with chimeric antigen receptor (CAR)-engineered T cells has shown durable clinical efficacy in patients with CD19+ B cell malignancies, the application of this approach to solid tumors is challenging. The goal of this proof-of-concept study was to investigate whether loading of CD19-CAR T cells (CART19) with anti-HER2 or anti-EGFR bispecific antibodies (BiAb) will target HER2+/EGFR+ CD19- targets and signal the intracellular domain of CAR without engaging antigen-specific CD19 ScFv of CAR T cells.MethodsWe used CART19 armed with anti-CD3 (OKT3) × anti-HER2 BiAb (HER2Bi) or anti-CD3 (OKT3) × anti-EGFR BiAb (EGFRBi) to evaluate the cytotoxicity directed at HER2 or EGFR expressing cancer cell lines compared with unarmed CART19 measured by short-term 51Cr release assay and long-term real-time cell analysis using xCelligence. We also determined the differences in exhaustion or effector phenotypes and cytokine profiles during the short- and long-term cytotoxicity assays.ResultsSpecific cytotoxicity was exhibited by CART19 armed with HER2Bi or EGFRBi against multiple tumor cell lines. Armed CART19 and armed activated T cells (ATC) showed comparable specific cytotoxicity that ranged between 10 and 90% against breast, pancreatic, ovarian, prostate, and lung cancer cell lines at 10:1 E/T ratio. Serial killing (repeated killing) by HER2Bi-armed CART19 ranged between 80 and 100% at 10:1 E/T ratio against MCF-7 cells up to 19 days (up to 4th round of repeated killing) measured by a real-time cell analysis without CART19 becoming exhausted.ConclusionsHER2Bi- or EGFRBi-armed CART19 exhibited specific cytotoxicity against multiple HER2+/EGFR+/CD19- tumor targets in overnight and long-term serial killing assays. CART19 showed improved survival and were resistant to exhaustion after prolonged repeated exposure to tumor cells.
| S-EPMC7375514 | biostudies-literature
Project description:In this study, we investigated the ability of bispecific antibody armed activated T cells to target drug resistant pancreatic cancer cells and whether or not "priming" these resistant cancer cells with bispecific antibody armed activated T cells could enhance subsequent responsiveness to chemotherapeutic drugs. Chemotherapeutic responses for pancreatic cancer are either limited or the tumors develop resistance to chemotherapy regimens. The impetus for this study was the remarkable clinical response seen in our earlier phase I/II clinical trial: a pancreatic cancer patient with drug resistant tumors who showed progression of disease following three infusions of anti-CD3 x anti-EGFR bispecific antibody armed activated T cells (EGFR BATs) was restarted on the initial low dose of 5-fluorouracil showed complete response, suggesting that BATs infusions may have sensitized patient's tumor for chemoresponsiveness. In the current study, we tested the hypothesis that BATs can sensitize tumors for chemoresponsiveness. Gemcitabine or cisplatin-resistant MiaPaCa-2 and L3.6 cell lines were effectively targeted by EGFR BATs. Priming of drug sensitive or resistant cells with EGFR BATs followed by retargeting with lower concentrations of 50% inhibitory concentration of gemcitabine or cisplatin showed enhanced cytotoxicity. Gemcitabine or cisplatin-resistant cell lines show an increased proportion of CD44+/CD24+/EpCAM+ cancer stem like cells as well as an increased number of ABC transporter ABCG2 positive cells compared to the parental cell lines. These data suggest that bispecific antibody armed activated T cells can target and kill chemo-resistant tumor cells and also markedly augment subsequent chemotherapeutic responsiveness, possibly by modulating the expression of ABC transporters.
| S-EPMC8172155 | biostudies-literature
Project description:BackgroundHumanized 3F8-bispecific antibody (hu3F8-BsAb) using the IgG(L)-scFv format (where scFv is single-chain variable fragment), where the anti-CD3 huOKT3 scFv is fused with the carboxyl end of the hu3F8 light chain, has potent antitumor cytotoxicity against GD2(+) tumors. To overcome the insufficient number and function of T cells in cancer patients, they can be rejuvenated and expanded ex vivo before arming with hu3F8-BsAb for adoptive transfer, potentially reducing toxic side effects from direct BsAb administration.ProcedureT cells from normal volunteers were expanded and activated ex vivo using CD3/CD28 beads for 8 days. Activated T cells (ATCs) were harvested and co-incubated with a Good Manufacturing Practice grade hu3F8-BsAb at room temperature for 20 min. These armed ATCs were tested for cytotoxicity in vitro and in vivo against human GD2(+) cell lines and patient-derived xenografts in BALB-Rag2-/- IL-2R-γc-KO mice.ResultsHu3F8-BsAb armed ATCs showed robust antigen-specific tumor cytotoxicity against GD2(+) tumors in vitro. In vivo, T cells armed with hu3F8-BsAb were highly cytotoxic against GD2(+) melanoma and neuroblastoma xenografts in mice, accompanied by T-cell infiltration without significant side effects. Only zeptomole (10-21 ) quantities of BsAb per T cell was required for maximal antitumor effects. Tumor response was a function of T-cell dose.ConclusionBsAb armed T cells may have clinical utility as the next generation of cytotherapy combined with recombinant BsAb against human tumors for both adult and pediatrics, if autologous T cells can be activated and expanded ex vivo.
| S-EPMC9347186 | biostudies-literature
Project description:BackgroundThe survival benefit observed in children with neuroblastoma (NB) and minimal residual disease who received treatment with anti-GD2 monoclonal antibodies prompted our investigation into the safety and potential clinical benefits of anti-CD3×anti-GD2 bispecific antibody (GD2Bi) armed T cells (GD2BATs). Preclinical studies demonstrated the high cytotoxicity of GD2BATs against GD2+cell lines, leading to the initiation of a phase I/II study in recurrent/refractory patients.MethodsThe 3+3 dose escalation phase I study (NCT02173093) encompassed nine evaluable patients with NB (n=5), osteosarcoma (n=3), and desmoplastic small round cell tumors (n=1). Patients received twice-weekly infusions of GD2BATs at 40, 80, or 160×106 GD2BATs/kg/infusion complemented by daily interleukin-2 (300,000 IU/m2) and twice-weekly granulocyte macrophage colony-stimulating factor (250 µg/m2). The phase II segment focused on patients with NB at the dose 3 level of 160×106 GD2BATs/kg/infusion.ResultsOf the 12 patients enrolled, 9 completed therapy in phase I with no dose-limiting toxicities. Mild and manageable cytokine release syndrome occurred in all patients, presenting as grade 2-3 fevers/chills, headaches, and occasional hypotension up to 72 hours after GD2BAT infusions. GD2-antibody-associated pain was minimal. Median overall survival (OS) for phase I and the limited phase II was 18.0 and 31.2 months, respectively, with a combined OS of 21.1 months. A phase I NB patient had a complete bone marrow response with overall stable disease. In phase II, 10 of 12 patients were evaluable: 1 achieved partial response, and 3 showed clinical benefit with prolonged stable disease. Over 50% of evaluable patients exhibited augmented immune responses to GD2+targets post-GD2BATs, as indicated by interferon-gamma (IFN-γ) EliSpots, Th1 cytokines, and/or chemokines.ConclusionsThis study demonstrated the safety of GD2BATs up to 160×106 cells/kg/infusion. Coupled with evidence of post-treatment endogenous immune responses, our findings support further investigation of GD2BATs in larger phase II clinical trials.
| S-EPMC10961524 | biostudies-literature
Project description:T cells expressing chimeric antigen receptors, especially CD19 CAR-T cells have exhibited effective antitumor activities in B cell malignancies, but due to several factors such as antigen escape effects and tumor microenvironment, their curative potential in hepatocellular carcinoma has not been encouraging. To reduce the antigen escape risk of hepatocellular carcinoma, this study was to design and construct a bispecific CAR targeting c-Met and PD-L1. c-Met/PD-L1 CAR-T cells were obtained by lentiviral transfection, and the transfection efficiency was monitored by flow cytometry analysis. LDH release assays were used to elucidate the efficacy of c-Met/PD-L1 CAR-T cells on hepatocellular carcinoma cells. In addition, xenograft models bearing human hepatocellular carcinoma were constructed to detect the antitumor effect of c-Met/PD-L1 CAR-T cells in vivo. The results shown that this bispecific CAR was manufactured successfully, T cells modified with this bispecific CAR demonstrated improved antitumor activities against c-Met and PD-L1 positive hepatocellular carcinoma cells when compared with those of monovalent c-Met CAR-T cells or PD-L1 CAR-T cells but shown no distinct cytotoxicity on hepatocytes in vitro. In vivo experiments shown that c-Met/PD-L1 CAR-T cells significantly inhibited tumor growth and improve survival persistence compared with other groups. These results suggested that the design of single-chain, bi-specific c-Met/PD-L1 CAR-T is more effective than that of monovalent c-Met CAR-T for the treatment of hepatocellular carcinoma., and this bi-specific c-Met/PD-L1 CAR is rational and implementable with current T-cell engineering technology.
| S-EPMC7987916 | biostudies-literature
Project description:The success of chimeric antigen receptor (CAR) T-cell therapy against hematologic malignancies has altered the treatment paradigm for patients with these diseases. Nevertheless, the occurrence of relapse due to antigen escape or heterogeneous antigen expression on tumors remains a challenge for first-generation CAR T-cell therapies as only a single tumor antigen can be targeted. To address this limitation and to add a further level of tunability and control to CAR T-cell therapies, adapter or universal CAR T-cell approaches use a soluble mediator to bridge CAR T cells with tumor cells. Adapter CARs allow simultaneous or sequential targeting of multiple tumor antigens, control of immune synapse geometry, dose control, and the potential for improved safety. Herein, we described a novel CAR T-cell adapter platform that relies on a bispecific antibody (BsAb) targeting both a tumor antigen and the GGGGS (G4S) linker commonly used in single-chain Fv (ScFv) domains expressed on CAR T-cell surfaces. We demonstrated that the BsAb can bridge CAR T cells to tumor cells and potentiate CAR T-cell activation, proliferation, and tumor cell cytolysis. The cytolytic activity of CAR T-cells was redirected to different tumor antigens by changing the BsAb in a dose-dependent manner. This study highlights the potential of G4S-displaying CAR T cells to be redirected to engage alternative tumor-associated antigens (TAA).SignificanceNew approaches are needed to address relapsed/refractory disease and manage potential toxicities associated with CAR T-cell therapy. We describe an adapter CAR approach to redirect CAR T cells to engage novel TAA-expressing cells via a BsAb targeting a linker present on many clinical CAR T-cell therapeutics. We anticipate the use of such adapters could increase CAR T-cell efficacy and reduce potential CAR-associated toxicities.
| S-EPMC9980914 | biostudies-literature
Project description:BackgroundSuccess of T cell immunotherapy hinges on the tumor microenvironment (TME), and abnormal tumor vasculature is a hallmark of most solid tumors and associated with immune evasion. The efficacy of T cell engaging bispecific antibody (BsAb) treatment relies on the successful trafficking and cytolytic activity of T cells in solid tumors. Normalization of tumor vasculature using vascular endothelial growth factor (VEGF) blockades could improve efficacy of BsAb-based T cell immunotherapy.MethodsAnti-human VEGF (bevacizumab, BVZ) or anti-mouse VEGFR2 antibody (DC101) was used as VEGF blockade, and ex vivo armed T cells (EATs) carrying anti-GD2, anti-HER2, or anti-glypican3 (GPC3) IgG-(L)-scFv platformed BsAb were used. BsAb-driven intratumoral T cell infiltration and in vivo antitumor response were evaluated using cancer cell line-derived xenografts (CDXs) or patient-derived xenografts (PDXs) carried out in BALB-Rag2 -/-IL-2R-γc-KO (BRG) mice. VEGF expression on human cancer cell lines was analyzed by flow cytometry, and VEGF levels in mouse serum were measured using VEGF Quantikine ELISA Kit. Tumor infiltrating lymphocytes (TILs) were evaluated using flow cytometry and by bioluminescence; both TILs and tumor vasculature were studied using immunohistochemistry.ResultsVEGF expression on cancer cell lines increased with seeding density in vitro. BVZ significantly reduced serum VEGF levels in mice. BVZ or DC101 increased high endothelial venules (HEVs) in the TME and substantially enhanced (2.1-8.1 fold) BsAb-driven T cell infiltration into neuroblastoma and osteosarcoma xenografts, which was preferential for CD8(+) TILs versus CD4(+) TILs, leading to superior antitumor effects in multiple CDX and PDX tumor models without added toxicities.ConclusionsVEGF blockade using specific antibodies against VEGF or VEGFR2 increased HEVs in the TME and cytotoxic CD8(+) TILs, significantly improving the therapeutic efficacy of EAT strategies in preclinical models, supporting the clinical investigation of VEGF blockades to further enhance BsAb-based T cell immunotherapies.
| S-EPMC10069597 | biostudies-literature
Project description:BackgroundSince treatment of neuroblastoma (NB) with anti-GD2 monoclonal antibodies provides a survival benefit in children with minimal residual disease and our preclinical study shows that anti-CD3 x anti-GD2 bispecific antibody (GD2Bi) armed T cells (GD2BATs) were highly cytotoxic to GD2+ cell lines, we conducted a phase I/II study in recurrent/refractory patients to establish safety and explore the clinical benefit of GD2BATs.MethodsThe 3+3 dose escalation study (NCT02173093) phase I involved 9 evaluable patients with NB (n=5), osteosarcoma (OST) (n=3), and desmoplastic small round cell tumors (DSRCT) (n=1) with twice weekly infusions of GD2BATs at 40, 80, or 160 x 106 GD2BATs/kg/infusion with daily interleukin 2 (300,000 IU/m2) and twice weekly granulocyte-macrophage colony stimulating factor (250 μg/m2). Phase II portion of the trial was conducted in patients with NB at the dose 3 level of 160 x 106 GD2BATs/kg/infusion but failed to enroll the planned number of patients.ResultsNine of 12 patients in the phase I completed therapy. There were no dose limiting toxicities (DLTs). All patients developed mild and manageable cytokine release syndrome (CRS) with grade 2-3 fevers/chills, headaches, and occasional hypotension up to 72 hours after GD2BAT infusions. GD2-antibody associated pain was not significant in this study. The median OS for patients in the Phase I and limited Phase II was 18.0 and 31.2 months, respectively, whereas the combined OS was 21.1 months. There was a complete bone marrow response with overall stable disease in one of the phase I patients with NB. Ten of 12 phase II patients were evaluable for response: 1 had partial response. Three additional patients were deemed to have clinical benefit with prolonged stable disease. More than 50% of evaluable patients showed augmented immune responses to GD2+ targets after GD2BATs as measured by interferon-gamma (IFN-γ) EliSpots, Th1 cytokines, and/or chemokines.ConclusionsOur study demonstrated safety of up to 160 x 106 cells/kg/infusion of GD2BATs. Combined with evidence for the development of post treatment endogenous immune responses, this data supports further investigation of GD2 BATs in larger Phase II clinical trials.
| S-EPMC10659559 | biostudies-literature
Project description:Natural killer group 2D (NKG2D) is an activating receptor of natural killer (NK) cells and other lymphocytes that mediates lysis of malignant cells through recognition of stress-induced ligands such as MICA and MICB. Such ligands are broadly expressed by cancer cells of various origins and serve as targets for adoptive immunotherapy with effector cells endogenously expressing NKG2D or carrying an NKG2D-based chimeric antigen receptor (CAR). However, shedding or downregulation of NKG2D ligands (NKG2DL) can prevent NKG2D activation, resulting in escape of cancer cells from NKG2D-dependent immune surveillance. To enable tumor-specific targeting of NKG2D-expressing effector cells independent of membrane-anchored NKG2DLs, we generated a homodimeric recombinant antibody which harbors an N-terminal single-chain fragment variable (scFv) antibody domain for binding to NKG2D, linked via a human IgG4 Fc region to a second C-terminal scFv antibody domain for recognition of the tumor-associated antigen ErbB2 (HER2). The ability of this molecule, termed NKAB-ErbB2, to redirect NKG2D-expressing effector cells to ErbB2-positive tumor cells of different origins was investigated using peripheral blood mononuclear cells, ex vivo expanded NK cells, and NK and T cells engineered with an NKG2D-based chimeric receptor. On its own, bispecific NKAB-ErbB2 increased lysis of ErbB2-positive breast carcinoma cells by peripheral blood-derived NK cells endogenously expressing NKG2D more effectively than an ErbB2-specific IgG1 mini-antibody able to induce antibody-dependent cell-mediated cytotoxicity via activation of CD16. Furthermore, NKAB-ErbB2 synergized with NK-92 cells or primary T cells engineered to express an NKG2D-CD3ζ chimeric antigen receptor (NKAR), leading to targeted cell killing and greatly enhanced antitumor activity, which remained unaffected by soluble MICA known as an inhibitor of NKG2D-mediated natural cytotoxicity. In an immunocompetent mouse glioblastoma model mimicking low or absent NKG2DL expression, the combination of NKAR-NK-92 cells and NKAB-ErbB2 effectively suppressed outgrowth of ErbB2-positive tumors, resulting in treatment-induced endogenous antitumor immunity and cures in the majority of animals. Our results demonstrate that combining an NKAB antibody with effector cells expressing an activating NKAR receptor represents a powerful and versatile approach to simultaneously enhance tumor antigen-specific as well as NKG2D-CAR and natural NKG2D-mediated cytotoxicity, which may be particularly useful to target tumors with heterogeneous target antigen expression.
| S-EPMC8488744 | biostudies-literature
Project description:BackgroundTaking advantage of nanobodies (Nbs) in immunotherapy, we investigated the cytotoxicity of Nb-based chimeric antigen receptor T cells (Nb CAR-T) against lymphoma cells.MethodsCD19 Nb CAR-T, CD20 Nb CAR-T, and Bispecific Nb CAR-T cells were generated by panning anti-human CD19- and CD20-specific nanobody sequences from a natural Nb-expressing phage display library, integrating Nb genes with a lentiviral cassette that included other CAR elements, and finally transducing T cells that were expanded under an optimization system with the above generated CAR lentivirus. Prepared Nb CAR-T cells were cocultured with tumour cell lines or primary tumour cells for 24 h or 5 days to evaluate their biological function.ResultsThe nanobodies that we selected from the natural Nb-expressing phage display library had a high affinity and specificity for CD19 and CD20. CD19 Nb CAR-T, CD20 Nb CAR-T and Bispecific Nb CAR-T cells were successfully constructed, and these Nb CAR-T cells could strongly recognize Burkitt lymphoma cell lines (Raji and Daudi), thereby leading to activation, enhanced proliferation, and specific killing of target cells. Furthermore, similar results were obtained when using patient samples as target cells, with a cytotoxicity of approximately 60%.ConclusionsNanobody-based CAR-T cells can kill both tumour cell lines and patient-derived tumour cells in vitro, and Nb-based CAR-T cells may be a promising therapeutic strategy in future immunotherapy.
| S-EPMC8386010 | biostudies-literature