Therapeutic Potential and Challenges of Targeting Receptor Tyrosine Kinase ROR1 with Monoclonal Antibodies in B-Cell Malignancies

Based on its selective cell surface expression in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), receptor tyrosine kinase ROR1 has recently emerged as a promising target for therapeutic monoclonal antibodies (mAbs). To further assess the suitability of ROR1 for targeted therapy of CLL and MCL, a panel of mAbs was generated and its therapeutic utility was investigated.A chimeric rabbit/human Fab library was generated from immunized rabbits and selected by phage display. Chimeric rabbit/human Fab and IgG1 were investigated for their capability to bind to human and mouse ROR1, to mediate antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and internalization, and to agonize or antagonize apoptosis using primary CLL cells from untreated patients as well as MCL cell lines. A panel of mAbs demonstrated high affinity and specificity for a diverse set of epitopes that involve all three extracellular domains of ROR1, are accessible on the cell surface, and mediate internalization. The mAb with the highest affinity and slowest rate of internalization was found to be the only mAb that mediated significant, albeit weak, ADCC. None of the mAbs mediated CDC. Alone, they did not enhance or inhibit apoptosis.Owing to its relatively low cell surface density, ROR1 may be a preferred target for armed rather than naked mAbs. Provided is a panel of fully sequenced and thoroughly characterized anti-ROR1 mAbs suitable for conversion to antibody-drug conjugates, immunotoxins, chimeric antigen receptors, and other armed mAb entities for preclinical and clinical studies

Autologous lymphoma vaccines induce human T cell responses against multiple, unique epitopes

The clonotypic surface Ig receptor expressed by malignant B cells, idiotype, is a tumor-specific antigen and an attractive target for active immunotherapy. While Ab’s specific for tumor idiotype have been well described in patients with B cell malignancies, the precise antigenic epitopes in human idiotype recognized by autologous T cells remain largely unknown. We report here that T cell lines generated from lymphoma patients actively immunized with idiotype protein specifically recognized multiple, unique immunodominant epitopes in autologous tumor idiotype. Synthetic peptides corresponding to hypervariable, but not framework, regions of Ig heavy chain specifically stimulated CD4(+) and CD8(+) T cells to proliferate and secrete proinflammatory cytokines in an MHC-associated manner. Detailed analysis revealed a minimal determinant of an immunodominant epitope, comprising critical residues at the amino terminus that may be a product of somatic hypermutation. Association of idiotype-specific T cell responses with previously documented molecular remissions in idiotype-vaccinated patients suggests that the newly identified T cell epitopes may be clinically relevant. Such antigenic epitopes may serve as candidates for novel peptide-vaccine strategies, and as tools to selectively expand tumor antigen–specific T cells for adoptive immunotherapy and for monitoring T cell immunity in vaccinated patients

Specificity of cell surface binding.

<p>Flow cytometry was used to identify PBMC subpopulations from patients CLL-2 (<b>A</b>), CLL-3 (<b>B</b>), CLL-4 (<b>C</b>), and CLL-5 (<b>D</b>) as NK cells (CD16+ CD3−), T cells (CD16− CD3+ and CD19− CD5+), and CLL cells (CD19+ CD5+). The binding of biotinylated IgG1 R12 (blue; 1 µg/mL) to total PBMC is shown at the bottom. The gray shade indicates the background observed with PE-streptavidin alone. The <i>x</i> and <i>y</i> axes in the top and middle row and the <i>x</i> axis in the bottom row depict the fluorescence intensity, the <i>y</i> axis in the bottom row the number of events.</p

ADCC.

<p>A bioluminescent intracellular protease detection assay was used to quantify the ADCC potency of IgG1 R11, R12, and Y31 in comparison to IgG1 TT11 (negative control) and rituximab (RTX; positive control; all at 5 µg/mL) toward (<b>A</b>) MCL lines JeKo-1 and HBL-2 and (<b>B</b>) PBMC from patients CLL-2, CLL-3, and CLL-4. NK cells isolated from PBMC of a healthy volunteer were used as effector cells at an effector-to-target cell ratio of 25∶1. In (<b>A</b>), specific cytotoxicity is shown as columns with error bars to indicate mean and standard deviation values of side-by-side triplicates. Stars indicate significant differences (p<0.05) to both the negative control (IgG1 TT11) and the background (none). In (<b>B</b>), specific cytotoxicity toward PBMC from the three individual patients is shown with mean values indicated by horizontal bars. (<b>C</b>) Using the same assay and mAbs, ADCC potency toward HBL-2 cells was compared over a concentration range of 0.02 to 20 µg/mL. NK cells isolated from PBMC of a different healthy volunteer were used at an effector-to-target cell ratio of 20∶1. Columns indicate mean values and error bars indicate standard deviation values of side-by-side triplicates.</p

Flow cytometry binding of IgG1 R11, R12, Y31, and control antibodies to PBMC from patient CLL-1.

1<p>Not determined.</p>2<p>Mean Fluorescence Intensity (MFI).</p

Epitope mapping studies by surface plasmon resonance.

<p>Shown are Biacore ×100 sensorgrams obtained for the binding of IgG1 R11, R12, and Y31 to immobilized Fc-hROR1. IgG1 were injected in different orders and mixtures to identify independent and overlapping epitopes. Resonance unit (<i>y</i> axis) increases that exceeded the values found for IgG1 R11, R12, and Y31 alone indicated independent epitopes that allow simultaneous binding. For example, the increase found for the combination of IgG1 R11 and R12 (top left and middle left graph) exceeded the values found for R11 and R12 alone, indicating that R11 and R12 can bind simultaneously to human ROR1. By contrast, the increase found for the combination of IgG1 Y31 and R11 (top right and bottom left graph) did not exceed the values found for R11 and Y31 alone, indicating overlapping epitopes. The simultaneous binding of IgG1 R12 and Y31 can be seen in the middle right graph. The <i>x</i> axis depicts the time in seconds (s).</p

Internalization.

<p>Shown is the internalization of IgG1 R11, R12, and Y31 by PBMC from patients CLL-6 (top), CLL-7 (middle), and CLL-8 (bottom). Biotinylated IgG1 R11 (10 µg/mL), R12 (1 µg/mL), and Y31 (10 µg/mL) were incubated at 37°C and analyzed at the indicated time points with PE-streptavidin. The percentage of MFI reduction was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021018#s4" target="_blank">Materials and Methods</a>. The data set on the right shows MFI reduction in the presence of endocytosis inhibitor phenylarsine oxide (PAO).</p