Jiehua Zhou

CTLA-4 Blockade following Relapse of Malignancy after Allogeneic Stem Cell Transplantation Is Associated with T Cell Activation But Not with Increased Levels of T Regulatory Cells

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a key negative regulator of T cell activation and proliferation. Ipilimumab is a human monoclonal antibody that specifically blocks the binding of CTLA-4 to its ligand. To test the hypothesis that blockade of CTLA-4 by ipilimumab could augment graft-versus-malignancy (GVM) effects without a significant impact on graft-versus-host disease (GVHD), we conducted a phase I clinical trial of ipilimumab infusion in patients with relapsed malignancy following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Here, we report the analysis of peripheral blood T lymphocyte reconstitution, T regulatory cell (Treg) expression, and T cell activation markers after a single dose of ipilimumab in 29 patients. Peripheral blood samples were collected from all patients before and after ipilimumab infusion. Lymphocyte immunophenotyes, including levels of CD4(+)CD25(high) cells and T cell activation markers, were analyzed in all cases. Levels of CD4(+)CD25(high)Foxp3(+) cells and intracellular CTLA-4 in CD4(+) T cells also were evaluated in the last 11 cases. We found lower baseline levels of CD4(+) and CD45RO(+) T cells in patients compared with normal controls. More than 50% of the patients had abnormally low lymphocyte counts (CD4 or/and CD8 T cells), and some had no circulating B lymphocytes. The percentages of both CD4(+)CD25(high) and CD4(+)CD25(high)Foxp3(+) T cells were significantly higher in patients before ipilimumab infusion than in healthy donors. Twenty of 29 patients exhibited an elevated level of CD4(+)CD25(low) activated T cells at baseline, compared with only 3 of 26 healthy donors. Both CD4(+) and CD8(+) T lymphocyte counts were significantly increased after ipilimumab infusion. There was no consistent change in absolute lymphocyte count or in the number of T cells expressing the activation marker CD69. However, increases in CD4(+)CD25(low) T cells were seen in 20 of 29 patients and increases in CD4(+)HLA-DR(+) T cells were seen in the last 10 patients in the first 60 days after ipilimumab infusion. Although the percentages of both CD4(+)CD25(high) and CD4(+)CD25(high)Foxp3(+) T cells decreased significantly during the observation period, the absolute cell counts did not change. Intracellular CTLA-4 expression in CD4(+)CD25(lo/-) T cells increased significantly after ipilimumab infusion. We conclude that CTLA-4 blockade by a single infusion of ipilimumab increased CD4(+) and CD4(+)HLA-DR(+) T lymphocyte counts and intracellular CTLA-4 expression at the highest dose level. There was no significant change in Treg cell numbers after ipilimumab infusion. These data demonstrate that significant changes in T cell populations occur on exposure to a single dose of ipilimumab. Further studies with multiple doses are needed to explore this phenomenon further and to correlate changes in lymphocyte subpopulations with clinical events.

Targeting gastrin-releasing peptide receptors for cancer treatment.

Growth factor receptors play critical roles in cancer cell proliferation and progression. A number of such receptors have been targeted for cancer treatment by either a monoclonal antibody or a specifically designed small molecule to inhibit the receptor function. Bombesin/gastrin-releasing peptide receptors (BN/GRP-Rs) are expressed in a variety of cancer cells and have limited distribution in normal human tissue. Inhibition of BN/GRP-Rs has been shown to block small cell lung cancer growth in vitro. Early phase clinical trials targeting human GRP-R showed anti-cancer activity. This review will focus on the study of the distribution of BN/GRP-Rs in normal and malignant tissues, and various approaches to targeting BN-GRP-Rs for cancer diagnosis and treatment.

Targeting Gastrin-Releasing Peptide Receptors on Small Cell Lung Cancer Cells with a Bispecific Molecule that Activates Polyclonal T Lymphocytes

Purpose: Gastrin-releasing peptide (GRP) is a growth factor for small cell lung cancer (SCLC). GRP belongs to the bombesin peptide family and has significant homology to bombesin. We constructed a bispecific molecule, OKT3xAntag2, by conjugating a monoclonal antibody OKT3 (anti-CD3) with a bombesin/GRP antagonist (Antag2) and evaluated cytotoxicity against SCLC cells. Experimental Design: We tested binding of the bispecific molecule to SCLC cell lines and T cells by flow cytometry, antibody-dependent cellular cytotoxicity (ADCC) of SCLC cells in vitro and in a murine SCLC xenograft model. We studied SCLC apoptosis and necrosis during ADCC and the activity and cleavage of caspase-3, caspase-9, and poly(ADP-ribose) polymerase (PARP). Results: The bispecific molecule functions as a cross-linker between T cells and SCLC cells, induces T cell activation, and mediates ADCC of SCLC cells; 40% to 80% growth inhibition of SCLC cells mediated by the bispecific molecule at low effector to target cell ratios was achieved. Activation of T cells by the bispecific molecule resulted in significant increases in IFNγ production and apoptosis and necrosis of SCLC cells associated with cleavage of PARP and caspase-3. Targeted immunotherapy with the bispecific molecule–armed human T cells significantly reduced SCLC tumor burdens in a mouse model. Conclusion: The bispecific molecule OKT3xAntag2 mediates growth inhibition and apoptosis of SCLC cells by activated T cells through activation and cleavage of caspase-3 and PARP in vitro and in vivo. Clinical trials of this bispecific molecule through adoptive transfer of ex vivo activated T cells in GRP receptor–positive tumors, such as SCLC, are warranted.

VX-322: A Novel Dual Receptor Tyrosine Kinase Inhibitor for the Treatment of Acute Myelogenous Leukemia

In acute myelogenous leukemia (AML), the FLT3 receptor tyrosine kinase (RTK) is highly expressed with 30% of patients expressing a mutated, constitutively active form of this protein. To inhibit this receptor, VX-322 was developed and found to be very potent against both the FLT3 and c-KIT RTKs with enzyme K(i) values of <1 nM and a cellular IC(50) between 1 and 5 nM. It was efficacious in a FLT3-ITD dependent myeloproliferative mouse model, doubling survival compared to other FLT3 inhibitors, with 25% of the mice cured. Upon treatment of primary AML patient blast cells, the dual inhibition of FLT3 and c-KIT was superior to inhibitors targeting a single RTK. Thus, this compound may represent an improved pharmacologic and selectivity profile that could be effective in the treatment of AML.

Characterization of a New Monoclonal Antibody 6G7 That Recognizes a Unique Antigen on Myeloid and Lymphoid Cells

We generated a monoclonal antibody (mAb) 6G7, which recognizes a 220-kD antigen on selected subpopulations of normal myeloid and lymphoid cells and their malignant counterparts. 6G7 reacts with 90-95% of peripheral blood B cells, 70-80% of CD8(+) cells, 30-35% of CD4(+) cells, 20-40% of monocytes, and 20-40% of CD34(+) cells from bone marrow. 6G7 reacts with leukemic blasts in acute myeloid leukemia (14/16), adult acute lymphoblastic leukemia (ALL) (5/5), pediatric ALL (5/9), chronic lymphocytic leukemia (8/8), follicular lymphoma (7/7), and Burkitts lymphoma (1/1). Long-term bone marrow culture of 6G7(+/-) cells showed the majority of clonogenic hematopoietic cells were in mAb 6G7 subpopulation. An immunotoxin of 6G7 and ricin A chain was cytotoxic to 6G7(+) leukemia cell lines. mAb 6G7 has potential clinical applications for targeted immunotherapy of both leukemia and lymphoma.