Dataset Information

Identification and Targeting of Thomsen-Friedenreich and IL1RAP Antigens on Chronic Myeloid Leukemia Stem Cells Using Bi-Specific Antibodies.

ABSTRACT:

Introduction

Quiescent leukemia stem cells (LSCs) play a major role in therapeutic resistance and disease progression of chronic myeloid leukemia (CML). LSCs belong to the primitive population; CD34+CD38-Lin-, which does not distinguish normal hematopoietic stem cells (HSC) from CML LSCs. Because Thomsen-Friedenreich/CD176 antigen is expressed on CD34+ HSC and IL1RAP is tightly correlated to BCR-ABL expression, we sought to increase the specificity towards LSC by using additional biomarkers.

Methods

We evaluated the co-expression of both antigens on CD34+ peripheral blood mononuclear cells (PBMCs) from both healthy volunteers and CML patients, using flow cytometry. Then, we used site-directed mutagenesis to induce knob-in-hole mutations in the human IgG heavy chain and the human lambda light chain to generate the bi-specific antibody (Bis-Ab) TF/RAP that binds both antigens simultaneously. We measured complement-directed cytotoxicity (CDC) in CML samples with the Bis-Ab by flow cytometry.

Results

In contrast to healthy volunteers, CML samples displayed a highly significant co-expression of CD176 and IL1RAP. When either a double-positive cell line or CML samples were treated with increasing doses of Bis-Ab, increased binding and CDC was observed indicating co-operative binding of the Bis-Ab as compared to monoclonal antibodies.

Discussion

These results show that the bi-specific antibody is capable of targeting IL1RAP+ and CD176+ cell population among CML PBMCs, but not corresponding normal cells in CDC assay. We hereby offer a novel strategy for the depletion of CML stem cells from the bulk population in clinical hematopoietic stem cell transplantation.

SUBMITTER: Eldesouki RE

PROVIDER: S-EPMC7837560 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Publications

Identification and Targeting of Thomsen-Friedenreich and IL1RAP Antigens on Chronic Myeloid Leukemia Stem Cells Using Bi-Specific Antibodies.

Eldesouki Raghda E RE Wu Chengxiang C Saleh Fayez M FM Mohammed Eman Abdel-Moemen EA Younes Soha S Hassan Naglaa Elsayed NE Brown Theresa C TC Alt Eckhard U EU Robinson James E JE Badr Fouad Mohamed FM Braun Stephen E SE

OncoTargets and therapy 20210122

<h4>Introduction</h4>Quiescent leukemia stem cells (LSCs) play a major role in therapeutic resistance and disease progression of chronic myeloid leukemia (CML). LSCs belong to the primitive population; CD34+CD38-Lin-, which does not distinguish normal hematopoietic stem cells (HSC) from CML LSCs. Because Thomsen-Friedenreich/CD176 antigen is expressed on CD34+ HSC and IL1RAP is tightly correlated to BCR-ABL expression, we sought to increase the specificity towards LSC by using additional biomark ...[more]

PMID: 33519209

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Project description:BackgroundThe interleukin-1 receptor accessory protein (IL1RAP) is highly expressed on acute myeloid leukemia (AML) bulk blasts and leukemic stem cells (LSCs), but not on normal hematopoietic stem cells (HSCs), providing an opportunity to target and eliminate the disease, while sparing normal hematopoiesis. Herein, we report the activity of BIF002, a novel anti-IL1RAP/CD3 T cell engager (TCE) in AML.MethodsAntibodies to IL1RAP were isolated from CD138+ B cells collected from the immunized mice by optoelectric positioning and single cell sequencing. Individual mouse monoclonal antibodies (mAbs) were produced and characterized, from which we generated BIF002, an anti-human IL1RAP/CD3 TCE using Fab arm exchange. Mutations in human IgG1 Fc were introduced to reduce FcγR binding. The antileukemic activity of BIF002 was characterized in vitro and in vivo using multiple cell lines and patient derived AML samples.ResultsIL1RAP was found to be highly expressed on most human AML cell lines and primary blasts, including CD34+ LSC-enriched subpopulation from patients with both de novo and relapsed/refractory (R/R) leukemia, but not on normal HSCs. In co-culture of T cells from healthy donors and IL1RAPhigh AML cell lines and primary blasts, BIF002 induced dose- and effector-to-target (E:T) ratio-dependent T cell activation and leukemic cell lysis at subnanomolar concentrations. BIF002 administered intravenously along with human T cells led to depletion of leukemic cells, and significantly prolonged survival of IL1RAPhigh MOLM13 or AML patient-derived xenografts with no off-target side effects, compared to controls. Of note, BiF002 effectively redirects T cells to eliminate LSCs, as evidenced by the absence of disease initiation in secondary recipients of bone marrow (BM) from BIF002+T cells-treated donors (median survival not reached; all survived > 200 days) compared with recipients of BM from vehicle- (median survival: 26 days; p = 0.0004) or isotype control antibody+T cells-treated donors (26 days; p = 0.0002).ConclusionsThe novel anti-IL1RAP/CD3 TCE, BIF002, eradicates LSCs and significantly prolongs survival of AML xenografts, representing a promising, novel treatment for AML.

Project description:IntroductionChimeric antigen receptor-engineered T cells (CAR-Ts) are investigated in various clinical trials for the treatment of cancer entities beyond hematologic malignancies. A major hurdle is the identification of a target antigen with high expression on the tumor but no expression on healthy cells, since "on-target/off-tumor" cytotoxicity is usually intolerable. Approximately 90% of carcinomas and leukemias are positive for the Thomsen-Friedenreich carbohydrate antigen CD176, which is associated with tumor progression, metastasis and therapy resistance. In contrast, CD176 is not accessible for ligand binding on healthy cells due to prolongation by carbohydrate chains or sialylation. Thus, no "on-target/off-tumor" cytotoxicity and low probability of antigen escape is expected for corresponding CD176-CAR-Ts.MethodsUsing the anti-CD176 monoclonal antibody (mAb) Nemod-TF2, the presence of CD176 was evaluated on multiple healthy or cancerous tissues and cells. To target CD176, we generated two different 2nd generation CD176-CAR constructs differing in spacer length. Their specificity for CD176 was tested in reporter cells as well as primary CD8+ T cells upon co-cultivation with CD176+ tumor cell lines as models for CD176+ blood and solid cancer entities, as well as after unmasking CD176 on healthy cells by vibrio cholerae neuraminidase (VCN) treatment. Following that, both CD176-CARs were thoroughly examined for their ability to initiate target-specific T-cell signaling and activation, cytokine release, as well as cytotoxicity.ResultsSpecific expression of CD176 was detected on primary tumor tissues as well as on cell lines from corresponding blood and solid cancer entities. CD176-CARs mediated T-cell signaling (NF-κB activation) and T-cell activation (CD69, CD137 expression) upon recognition of CD176+ cancer cell lines and unmasked CD176, whereby a short spacer enabled superior target recognition. Importantly, they also released effector molecules (e.g. interferon-γ, granzyme B and perforin), mediated cytotoxicity against CD176+ cancer cells, and maintained functionality upon repetitive antigen stimulation. Here, CD176L-CAR-Ts exhibited slightly higher proliferation and mediator-release capacities. Since both CD176-CAR-Ts did not react towards CD176- control cells, their response proved to be target-specific.DiscussionGenetically engineered CD176-CAR-Ts specifically recognize CD176 which is widely expressed on cancer cells. Since CD176 is masked on most healthy cells, this antigen and the corresponding CAR-Ts represent a promising approach for the treatment of various blood and solid cancers while avoiding "on-target/off-tumor" cytotoxicity.

Dataset Information

Identification and Targeting of Thomsen-Friedenreich and IL1RAP Antigens on Chronic Myeloid Leukemia Stem Cells Using Bi-Specific Antibodies.

Introduction

Methods

Results

Discussion

Publications

Identification and Targeting of Thomsen-Friedenreich and IL1RAP Antigens on Chronic Myeloid Leukemia Stem Cells Using Bi-Specific Antibodies.

Similar Datasets

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