Characterization of structurally defined epitopes recognized by monoclonal antibodies produced by chronic lymphocytic leukemia B cells

AbstractDespite a wealth of information about the structure of surface membrane immunoglobulin (smIg) on chronic lymphocytic leukemia (CLL) cells, little is known about epitopes reacting with their binding sites. Probing phage-displayed peptide libraries, we identified and characterized mimetopes for Igs of 4 patients with IGHV mutated CLL (M-CLL) and 4 with IGHV unmutated CLL (U-CLL). Six of these mAbs were representatives of stereotyped B-cell receptors characteristic of CLL. We found that mimetic epitopes for U- and M-CLL Igs differed significantly. M-CLL–derived peptides exhibited better amino acid motifs, were more similar to each other, aligned more easily, and formed tighter clusters than U-CLL–derived peptides. Mono-, oligo-, and polyreactivity of peptides correlated with structural changes within antigen-binding sites of selecting M-CLL mAbs. Although M-CLL–isolated peptides and certain U-CLL mAbs bound more effectively to the selecting mAb, others were not as specific, reacting with M-CLL and U-CLL mAbs; these data suggest that in vivo structurally diverse epitopes could bind smIgs of distinct CLL clones, thereby altering survival and growth. Finally, an M-CLL–derived peptide inhibited, in a dose-dependent manner, binding of its homologous mAb to human B lymphocytes; therefore peptides that inhibit or alter the consequences of antigen-smIg interactions may represent therapeutic modalities in CLL.

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Functional and clinical relevance of VLA-4 (CD49d/CD29) in ibrutinib-treated chronic lymphocytic leukemia

Journal of Experimental Medicine ◽

10.1084/jem.20171288 ◽

2018 ◽

Vol 215 (2) ◽

pp. 681-697 ◽

Cited By ~ 21

Author(s):

Erika Tissino ◽

Dania Benedetti ◽

Sarah E.M. Herman ◽

Elisa ten Hacken ◽

Inhye E. Ahn ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Progression Free Survival ◽

Cell Receptor ◽

Lymphocytic Leukemia ◽

Clinical Activity ◽

Independent Manner ◽

Btk Inhibitor

The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib, which antagonizes B cell receptor (BCR) signals, demonstrates remarkable clinical activity in chronic lymphocytic leukemia (CLL). The lymphocytosis experienced by most patients under ibrutinib has previously been attributed to inhibition of BTK-dependent integrin and chemokine cues operating to retain the tumor cells in nodal compartments. Here, we show that the VLA-4 integrin, as expressed by CD49d-positive CLL, can be inside-out activated upon BCR triggering, thus reinforcing the adhesive capacities of CLL cells. In vitro and in vivo ibrutinib treatment, although reducing the constitutive VLA-4 activation and cell adhesion, can be overcome by exogenous BCR triggering in a BTK-independent manner involving PI3K. Clinically, in three independent ibrutinib-treated CLL cohorts, CD49d expression identifies cases with reduced lymphocytosis and inferior nodal response and behaves as independent predictor of shorter progression-free survival, suggesting the retention of CD49d-expressing CLL cells in tissue sites via activated VLA-4. Evaluation of CD49d expression should be incorporated in the characterization of CLL undergoing therapy with BCR inhibitors.

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Docosahexaenoic acid induces apoptosis in primary chronic lymphocytic leukemia cells

Hematology Reports ◽

10.4081/hr.2015.6043 ◽

2015 ◽

Vol 7 (4) ◽

Cited By ~ 3

Author(s):

Romain Guièze ◽

Emmanuel Gyan ◽

Olivier Tournilhac ◽

Christelle Halty ◽

Richard Veyrat-Masson ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Docosahexaenoic Acid ◽

Lymphocytic Leukemia ◽

Dependent Manner ◽

Omega 3 ◽

Infectious Risk ◽

Highly Active ◽

Primary Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia is an indolent disorder with an increased infectious risk remaining one of the main causes of death. Development of therapies with higher safety profile is thus a challenging issue. Docosahexaenoic acid (DHA, 22:6) is an omega-3 fatty acid, a natural compound of normal cells, and has been shown to display antitumor potency in cancer. We evaluated the potential in vitro effect of DHA in primary CLL cells. DHA induces high level of in vitro apoptosis compared to oleic acid in a dose-dependent and time-dependent manner. Estimation of IC50 was only of 4.813 μM, which appears lower than those reported in solid cancers. DHA is highly active on CLL cells in vitro. This observation provides a rationale for further studies aiming to understand its mechanisms of action and its potent in vivo activity.

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In Vitro Activity of a Novel Fully Human Anti-CD40 Antibody CHIR-12.12 in Chronic Lymphocytic Leukemia: Blockade of CD40 Activation and Induction of ADCC.

Blood ◽

10.1182/blood.v104.11.2504.2504 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2504-2504 ◽

Cited By ~ 2

Author(s):

Xia Tong ◽

Georgios V. Georgakis ◽

Long Li ◽

O’Brien Susan ◽

Younes Anas ◽

...

Keyword(s):

Monoclonal Antibody ◽

Cell Death ◽

Chronic Lymphocytic Leukemia ◽

Cell Line ◽

Blood Donors ◽

Lymphocytic Leukemia ◽

Dependent Manner ◽

Dose Dependent

Abstract B-cell chronic lymphocytic leukemia (CLL) is characterized by in vivo accumulation of long-lived CD5+ B cells. However when cultured in vitro CLL cells die quickly by apoptosis. Protection from apoptosis in vivo is believed to result from supply of survival signals provided by cells in the microenvironment. We and others have previously reported that CLL cells express CD40 receptor, and that CD40 stimulation of CLL cells may rescue CLL cells from spontaneous and drug-induced apoptosis in vitro. These observations suggested that blocking CD40-CD40L pathway might deprive CLL cells from survival signals and induce apoptosis. To test this hypothesis, we have generated a fully human anti-CD40 blocking monoclonal antibody in XenoMousemice (Abgenix, Inc.). The antibody CHIR-12.12 was first evaluated for its effect on normal human lymphocytes. Lymphocytes from all 10 healthy blood donors did not proliferate in response to CHIR-12.12 at any concentration tested (0.0001 mg/ml to 10 mg/ml range). In contrast, activating CD40 on normal B-lymphocytes by CD40L induced their proliferation in vitro. Importantly, CHIR-12.12 inhibited CD40L- induced proliferation in a dose dependent manner with an average IC50 of 51 ± 26 pM (n=10 blood donors). The antagonistic activity of CHIR-12.12 was then tested in primary CLL samples from 9 patients. CHIR-12.12 alone did not induce CLL cell proliferation. In contrast, primary CLL cells incubated with CD40L, either resisted spontaneous cell death or proliferated. This effect was reversed by co-incubation with CHIR-12.12 antibody, restoring CLL cell death (n=9). CHIR-12.12 was then examined for its ability to lyse CLL cell line EHEB by antibody dependent cell mediated cytotoxicity (ADCC). Freshly isolated human NK cells from normal volunteer blood donors were used as effector cells. CHIR-12.12 showed lysis activity in a dose dependent manner and produced maximum lysis levels at 0.1 mg/ml. When compared with rituximab, CHIR-12.12 mediated greater maximum specific lysis (27.2 % Vs 16.2 %, p= 0.007). The greater ADCC by CHIR-12.12 was not due to higher density of CD40 molecules on CLL cell line compared to CD20 molecules. The CLL target cells expressed 509053 ±13560 CD20 molecules compared to 48416 ± 584 CD40 molecules. Collectively, these preclinical data suggest that CHIR-12.12 monoclonal antibody may have a therapeutic role in patients with CLL.

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Dual B and T markers in acute and chronic lymphocytic leukemia

Blood ◽

10.1182/blood.v55.1.16.bloodjournal55116 ◽

1980 ◽

Vol 55 (1) ◽

pp. 16-20

Author(s):

KA Foon ◽

RJ Billing ◽

PI Terasaki

Keyword(s):

T Cell ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

B Lymphocyte ◽

Surface Membrane ◽

Lymphocytic Leukemia ◽

Rosette Formation ◽

Leukemic Cells ◽

Cell Markers ◽

Membrane Immunoglobulin

Leukemic cells from 20 patients with chronic lymphocytic leukemia (CLL) and 60 patients with acute lymphocytic leukemia (ALL) were studied for T- and B-lymphocyte cell surface membrane markers. B-cell markers included surface membrane immunoglobulin, erythrocyte-antibody complement rosette formation, and B-cell (a-like or HLA-DR) antigens detected by a B-cell antiserum. T-cell markers included spontaneous sheep red blood cell rosette formation and a cytotoxic reaction to a specific T-cell antiserum. Seven patients with CLL and two with ALL had dual B and T markers. We propose that dual B- and T-cell markers are more common in CLL and ALL patients than previously reported. With newer and more sensitive tests for identification of B and T cells, this observation may be recognized more frequently.

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Human interleukin-7 induces proliferation of neoplastic cells from chronic lymphocytic leukemia and acute leukemias

Blood ◽

10.1182/blood.v78.3.753.bloodjournal783753 ◽

1991 ◽

Vol 78 (3) ◽

pp. 753-759 ◽

Cited By ~ 2

Author(s):

W Digel ◽

M Schmid ◽

G Heil ◽

P Conrad ◽

S Gillis ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Binding Sites ◽

Lymphocytic Leukemia ◽

Membrane Receptors ◽

Receptor Expression ◽

Leukemic Cells ◽

Dependent Manner ◽

Neoplastic Cells ◽

Acute Leukemias ◽

Interleukin 7

The biologic effects of interleukin-7 (IL-7) and the expression of specific IL-7 membrane receptors on isolated neoplastic cells from previously untreated patients with chronic lymphocytic leukemia as well as acute leukemias were investigated in vitro. Leukemic cells were incubated for up to 6 days with various concentrations of IL-7 (0.01 to 2,000 U/mL). Neoplastic cells of the T- or B-phenotype from chronic as well as from acute leukemias proliferated in a dose-dependent manner. Cells from acute myeloid leukemias also proliferated in response to IL- 7. An optimal proliferative effect was achieved between 96 and 120 hours with 200 U/mL IL-7. Combinations of IL-7 with IL-2 and tumor necrosis factor-alpha showed an additive effect on [3H]TdR incorporation. IL-7 binding assays gave a value of approximately 33 to 180 high-affinity (kd approximately 20 pmol/L) binding sites/cell and approximately 241 to 3,280 low-affinity (kd approximately 600 pmol/L) binding sites/cell. Receptor expression correlated with the proliferation in response to IL-7. These data indicate that IL-7 can induce proliferation of relatively mature tumor cells, and that this effect is not restricted to the lymphoid lineage.

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Characterization of chronic lymphocytic leukemia (CLL) cells with low density of surface membrane bound immunoglobulins (smIg)

Medical Oncology and Tumor Pharmacotherapy ◽

10.1007/bf02934912 ◽

1985 ◽

Vol 2 (4) ◽

pp. 261-267

Author(s):

E. Kimby ◽

B. Axelsson ◽

M. Björkholm ◽

J. Gordon ◽

G. Holm ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Surface Membrane ◽

Lymphocytic Leukemia ◽

Low Density ◽

Membrane Bound

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A6 Peptide Is Selectively Cytotoxic For Chronic Lymphocytic Leukemia Cells

Blood ◽

10.1182/blood.v122.21.5303.5303 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5303-5303

Author(s):

Suping Zhang ◽

Hsien Lai ◽

Grace Liu ◽

Laura Rassenti ◽

Michael Y. Choi ◽

...

Keyword(s):

Hyaluronic Acid ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Lymphocytic Leukemia ◽

Surface Glycoprotein ◽

Leukemia Cells ◽

Dependent Manner ◽

Cell Surface Glycoprotein

Abstract Chronic lymphocytic leukemia (CLL) cells express high levels of CD44, a cell-surface glycoprotein receptor for hyaluronic acid (HA). We found that a mAb specific for CD44 was directly cytotoxic for leukemia B cells, but had little effect on normal B cells. Moreover, this anti-CD44 mAb could induce CLL cells that expressed the zeta-associated protein of 70 kDa (ZAP-70) to undergo caspase-dependent apoptosis, independent of complement or cytotoxic effector cells (Proc Natl Acad Sci, USA 2013, PMID: 23530247). The cytotoxic effect of this mAb was not mitigated when the CLL cells were co-cultured with mesenchymal stromal cells (MSCs) or hyaluronic acid or when they were stimulated via ligation of the B-cell receptor with anti-µ. A6 (Angstrom Pharmaceuticals) is an 8-amino acid peptide that has marked homology with a linear sequence of CD44. A6 can bind CD44 within a region of the ligand-binding domain, leading to inhibition of the migration and metastatic potential of CD44-expressing cancer cells in vitro and in vivo (Mol Cancer Ther, 2011 PMID: 21885863). We evaluated the cytotoxic activity of A6 against primary leukemia cells of patients with CLL (n = 22). We found that A6 peptide also was directly cytotoxic for CLL cells isolated from different patients in a dose-dependent manner at concentrations that may be achieved in vivo. The A6 peptide appeared less cytotoxic for CLL cells than the intact anti-CD44 mAb, but still had greater direct cytotoxicity for CLL cells that expressed ZAP-70 than for CLL cells that were ZAP-70 negative. Furthermore, the A6 peptide had negligible effect on the viability of lymphocytes isolated from the blood of healthy donors (n = 3). Because clinical studies have found the A6 peptide to be well-tolerated and without dose-limiting toxicity in patients with solid tumors who have been treated to date (N = 40), a clinical study is planned to evaluate the safety and activity of the A6 peptide in the treatment of patients with CLL. Disclosures: Howell: Angstrom Phamaceuticals: Membership on an entity’s Board of Directors or advisory committees. Finlayson:Angstrom Phamaceuticals: Employment.

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Dual B and T markers in acute and chronic lymphocytic leukemia

Blood ◽

10.1182/blood.v55.1.16.16 ◽

1980 ◽

Vol 55 (1) ◽

pp. 16-20 ◽

Cited By ~ 44

Author(s):

KA Foon ◽

RJ Billing ◽

PI Terasaki

Keyword(s):

T Cell ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

B Lymphocyte ◽

Surface Membrane ◽

Lymphocytic Leukemia ◽

Rosette Formation ◽

Leukemic Cells ◽

Cell Markers ◽

Membrane Immunoglobulin

Abstract Leukemic cells from 20 patients with chronic lymphocytic leukemia (CLL) and 60 patients with acute lymphocytic leukemia (ALL) were studied for T- and B-lymphocyte cell surface membrane markers. B-cell markers included surface membrane immunoglobulin, erythrocyte-antibody complement rosette formation, and B-cell (a-like or HLA-DR) antigens detected by a B-cell antiserum. T-cell markers included spontaneous sheep red blood cell rosette formation and a cytotoxic reaction to a specific T-cell antiserum. Seven patients with CLL and two with ALL had dual B and T markers. We propose that dual B- and T-cell markers are more common in CLL and ALL patients than previously reported. With newer and more sensitive tests for identification of B and T cells, this observation may be recognized more frequently.

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