A Comparison of the Anti-Tumor Effects of a Chimeric versus Murine Anti-CD19 Immunotoxins on Human B Cell Lymphoma and Pre-B Acute Lymphoblastic Leukemia Cell Lines

Precursor B cell acute lymphoblastic leukemia (pre-B ALL) affects five to six thousand adults and almost three thousand children every year. Approximately 25% of the children and 60% of the adults die from their disease, highlighting the need for new therapies that complement rather than overlap chemotherapy and bone marrow transplantation. Immunotherapy is a class of therapies where toxicities and mechanisms of action do not overlap with those of chemotherapy. Because CD19 is a B cell- restricted membrane antigen that is expressed on the majority of pre-B tumor cells, a CD19-based immunotherapy is being developed for ALL. In this study, the anti-tumor activities of immunotoxins (ITs) constructed by conjugating a murine monoclonal antibody (MAb), HD37, or its chimeric (c) construct to recombinant ricin toxin A chain (rRTA) were compared both in vitro using human pre-B ALL and Burkitt’s lymphoma cell lines and in vivo using a disseminated human pre-B ALL tumor cell xenograft model. The murine and chimeric HD37 IT constructs were equally cytotoxic to pre-B ALL and Burkitt’s lymphoma cells in vitro and their use in vivo resulted in equivalent increases in survival of SCID mice with human pre-B ALL tumors when compared with control mice

The Role of Regulatory B Cell-Like Malignant Cells and Treg Cells in the Mouse Model of BCL1 Tumor Dormancy.

Cancer dormancy is a clinical state in which residual tumor cells persist for long periods of time but do not cause detectable disease. In the mouse B cell lymphoma model (BCL1), dormancy can be induced and maintained by immunizing mice with a soluble form of the IgM expressed on the surface of the tumor cells. Immunization induces an anti-idiotype antibody response that maintains dormancy. Mice with dormant tumor have low numbers of BCL1 cells in their spleens that divide and are killed at the same rate. When the anti-Id antibodies wane, the tumor cells grow rapidly and kill the host. Spleens from tumor-bearing mice contain both effector (CD4+ and CD8+) and regulatory T cells (Tregs). In other tumor models, it has been reported that Tregs promote tumor progression by preventing effector cells from killing the tumor. In this report, we demonstrate that the tumor site with rapidly dividing BCL1 cells has fewer Tregs than the tumor site harboring dormant BCL1 cells. In both cases, the Tregs were equally suppressive in vitro. In spleens from mice with actively growing tumor, CD8+ but not CD4+ T cells were virtually absent. In vitro analysis demonstrated a tumor-mediated elimination of CD8+ T cells that was contact dependent and involved the caspase-3 pathway. Most importantly, we found that the BCL1 cells expressed characteristics of B10 regulatory B cells, i.e., they were CD1dhiCD5+ and secreted high levels of IL-10. These BCL1 tumor cells can inhibit anti-tumor immune responses by depleting CD8+ effector T cells

T_regs from the dormant BCL1 tumor microenvironment maintain their capacity to suppress CD4⁺ and CD8⁺ T cells.

<p>T_reg suppression assays were performed using CD4⁺ T cells purified from spleens of (A) naive mice, or (B) BCL1 tumor-inoculated mice. Suppression by T_regs was calculated relative to the samples containing CD4⁺T cells only (0:1). The data are shown as mean ± SEM from three experiments (* <i>p</i> < 0.05). (C) T_regs were sorted from spleens of mice harboring dormant BCL1 tumor cells and co-cultured with CFSE-labeled CD8⁺T cells purified from naive mice. Histogram plots are gated on CD8⁺ cells and the values represent the percent CFSE dilution. Representative plots of 3 independent experiments yielding similar results.</p

BCL1 tumor cells do not impact regulatory and conventional T cells in the draining lymph nodes.

<p>(A) The total number and (B) percentage of T_regs (CD4⁺Foxp3⁺) in the inguinal lymph nodes were examined within each group of mice 60 days after tumor challenge. Healthy control mice were age-matched to the experiment groups. The total number of (C) CD4⁺ T cells and (D) CD8⁺ T cells within the inguinal lymph nodes were determined within each group at 60 days after tumor challenge. The values represent a compilation of 1–3 different experiments with 4–13 mice per group. Data are shown as mean ± SEM (* <i>p</i> < 0.05, **<i>p</i> < 0.005, *** <i>p</i> < 0.0005, ****<i>p</i> < 0.0001; student’s t-test).</p

BCL1 tumor cells suppress the proliferation of CD8⁺ T cells in a contact-dependent manner.

<p>The proliferation of CFSE-labeled CD8⁺ T cells was determined (A) alone, or (B) with equal numbers of BCL1 tumor cells (1:1) or (C) High ratios of BCL1 tumor cells to CD8⁺ T cells, as observed in the tumor milieu. (D) BCL1 tumor cells were prevented from making contact with CD8⁺ T cells by using Trans-well inserts. <i>E</i>, Addition of large numbers of spleen cells from naïve mice did not inhibit the proliferation of CD8⁺ T cells. <i>F</i>, Assessment of caspase-3 levels in CD8⁺ T cells following co-culture with graded doses of BCL1 tumor cells. Values in FACS plots represent the percentage of CD8⁺ T cells undergoing proliferation. Representative plots of 3 independent experiments are shown.</p

Increased BCL1 tumor cell burdens leads to the depletion of CD8⁺ T cells.

<p>Groups of mice immunized with the BCL1-Id along with non-immunized groups were inoculated with BCL1 tumor cells. Sixty days after tumor challenge, immunophenotyping was performed on spleen cells. (A) The total number of spleen cells from mice that were challenged with BCL1 tumor. (B) The total number of BCL1 tumor cells in the spleen. The total number of (C) CD4⁺ T cells, and (D) CD8⁺ T cells in the spleen from all experiment groups. Each group represents a mean of four to eight mice from at least 3 experiments. Data are shown as mean ± SEM (* <i>p</i> < 0.05, **<i>p</i> < 0.005, *** <i>p</i> < 0.0005, ****<i>p</i> < 0.0001; student’s t-test).</p

T_regs suppress CD4⁺ T cell proliferation by cell contact but do not suppress BCL1 tumor cells.

<p>(A) T_regs from mice harboring dormant BCL1 tumor cells were cultured in equal numbers with CFSE-labeled CD4⁺ T cells. (B) T_regs were plated onto trans-well inserts and placed in wells containing CFSE-labeled CD4⁺ T cells in an equal ratio. (C) CFSE-labeled CD4⁺ T cells were assessed for proliferation in the absence of T_regs. Plots are representative of 3 or more experiments yielding similar results. (D) Graded numbers of T_regs from mice with dormant BCL1 tumor cells were co-cultured with BCL1.3B3 tumor cells. Proliferation of BCL1.3B3 cells in the absence of T_regs (0:1) was considered to be the baseline values. Data represent the average of 3 experiments. <i>E</i>, BCL1-Id⁺ IgM secreted by BCL1.3B3 cells as measured by ELISA. Data represent the average of 4 experiments.</p

BCL1 tumor cells express the characteristics of B10 B_regs.

<p>Examination of a B10 B_reg phenotype performed on spleen cells from mice injected with (A) BCL1 tumor cells only, (B) pre-immunized with BCL1-Id⁺ IgM and challenged with BCL1 tumor cells, and (C) <i>in</i> vitro-adapted BCL1 tumor cells. The analysis shows 3 distinct subsets, of which the B10 B_reg phenotype (CD1d^hiCD5⁺) is unique to the BCL1-Id⁺ tumor cells. Representative plots of at least 3 or more independent experiments are shown. (D) Intracellular cytokine staining of spleen cells from mice challenged with BCL1 tumor cells (solid line) or naïve mice (dashed line). Histogram plots are representative of 3 independent experiments. (E) The cytokine profile of supernatants from BCL1.3B3 tumor cells following a 72 h culture period as determined by ELISA. Total values are shown as mean ± SEM from 2–3 experiments. ND, below detection limits.</p

Immunization results in an increase in T_regs while BCL1 tumor cell growth reduces T_regs at the tumor site.

<p>(A) Spleen cells from mice immunized with BCL1-Id and inoculated with BCL1 tumor cells were examined for T_regs (CD4⁺Foxp3⁺) 60 days after tumor challenge. (B) total number, and (C) percentage of T_regs in the spleen were determined based on the total viable spleen cells. The values are a compilation of at least 3 different experiments with 4–7 mice per group. Representative plots include 8 or more mice per group. Data are shown as mean ± SEM (* <i>p</i> < 0.05, **<i>p</i> < 0.005, *** <i>p</i> < 0.0005, ****<i>p</i> < 0.0001; student’s t-test).</p

Failure of vaccination with idiotypic protein or DNA, (+/-IL-2), the depletion of regulatory T cells, or the blockade of CTLA-4 to prolong dormancy in mice with BCL1 lymphoma

Immunization of mice with the idiotype (Id) immunoglobulin from the murine B cell lymphoma, BCL1, before inoculating tumor cells can induce tumor dormancy. In this model, the tumor cells grow for a short period of time and then regress. The mice live for months or years with approximately 1 million tumor cells in their spleens. Some mice relapse due to decreases in the anti-Id antibody titers or the development of mutations in the residual tumor cells which render them refractory to negative signaling by the anti-Id antibody. In this study we determined whether we could eliminate the residual dormant cells by using a DNA vaccine against the Id or by immunomodulation of T-cell subsets in vivo. Our results demonstrate that dormancy can be maintained by further immunizations with either the BCL1 Id protein or DNA vaccine encoding its single-chain Fv fragment. We also found that a cytotoxic T-cell response was not induced by either in vivo administration of vaccine alone or by the vaccine plus interleukin-2. In addition the injection of anti-cytotoxic T-lymphocyte-associate antigen did not prolong dormancy. Finally, the in vivo administration of anti-CD25 to deplete regulatory T cells did not prolong dormancy. Dormancy in this model is dependent primarily upon anti-Id antibodies, our results suggest that other strategies to target residual dormant BCL1 cells are warranted. They also suggest that the elimination of dormant tumor may represent a greater challenge than the elimination of primary tumors