CD49d Expression Identifies a Biologically Distinct Subtype of Chronic Lymphocytic Leukemia with Inferior Progression-Free Survival on BTK Inhibitor Therapy.

PURPOSE: To determine the role of CD49d for response to Bruton's tyrosine kinase inhibitors (BTKi) in patients with chronic lymphocytic leukemia (CLL). PATIENTS AND METHODS: In patients treated with acalabrutinib (n = 48), CD49d expression, VLA-4 integrin activation, and tumor transcriptomes of CLL cells were assessed. Clinical responses to BTKis were investigated in acalabrutinib- (n = 48; NCT02337829) and ibrutinib-treated (n = 73; NCT01500733) patients. RESULTS: In patients treated with acalabrutinib, treatment-induced lymphocytosis was comparable for both subgroups but resolved more rapidly for CD49d+ cases. Acalabrutinib inhibited constitutive VLA-4 activation but was insufficient to block BCR and CXCR4-mediated inside-out activation. Transcriptomes of CD49d+ and CD49d- cases were compared using RNA sequencing at baseline and at 1 and 6 months on treatment. Gene set enrichment analysis revealed increased constitutive NF-κB and JAK-STAT signaling, enhanced survival, adhesion, and migratory capacity in CD49d+ over CD49d- CLL that was maintained during therapy. In the combined cohorts of 121 BTKi-treated patients, 48 (39.7%) progressed on treatment with BTK and/or PLCG2 mutations detected in 87% of CLL progressions. Consistent with a recent report, homogeneous and bimodal CD49d-positive cases (the latter having concurrent CD49d+ and CD49d- CLL subpopulations, irrespective of the traditional 30% cutoff value) had a shorter time to progression of 6.6 years, whereas 90% of cases homogenously CD49d- were estimated progression-free at 8 years (P = 0.0004). CONCLUSIONS: CD49d/VLA-4 emerges as a microenvironmental factor that contributes to BTKi resistance in CLL. The prognostic value of CD49d is improved by considering bimodal CD49d expression. See related commentary by Tissino et al., p. 3560.

Select Antitumor Cytotoxic CD8+ T Clonotypes Expand in Patients with Chronic Lymphocytic Leukemia Treated with Ibrutinib.

PURPOSE: In chronic lymphocytic leukemia (CLL), the T-cell receptor (TCR) repertoire is skewed and tumor-derived antigens are hypothesized as drivers of oligoclonal expansion. Ibrutinib, a standard treatment for CLL, inhibits not only Bruton tyrosine kinase of the B-cell receptor signaling pathway, but also IL2-inducible kinase of the TCR signaling pathway. T-cell polarization and activation are affected by ibrutinib, but it is unknown whether T cells contribute to clinical response. EXPERIMENTAL DESIGN: High-throughput TCRß sequencing was performed in 77 longitudinal samples from 26 patients with CLL treated with ibrutinib. TCRß usage in CD4+ and CD8+ T cells and granzyme B expression were assessed by flow cytometric analysis. Antitumor cytotoxicity of T cells expanded with autologous CLL cells or with antigen-independent anti-CD3/CD28/CD137 beads was tested. RESULTS: The clonality of the TCR repertoire increased at the time of response. With extended treatment, TCR clonality remained stable in patients with sustained remission and decreased in patients with disease progression. Expanded clonotypes were rarely shared between patients, indicating specificity for private antigens. Flow cytometry demonstrated a predominance of CD8+ cells among expanded clonotypes. Importantly, bulk T cells from responding patients were cytotoxic against autologous CLL cells in vitro and selective depletion of major expanded clonotypes reduced CLL cell killing. CONCLUSIONS: In patients with CLL, established T-cell responses directed against tumor are suppressed by disease and reactivated by ibrutinib.See related commentary by Zent, p. 4465.

ROR1-targeted delivery of miR-29b induces cell cycle arrest and therapeutic benefit in vivo in a CLL mouse model.

Chronic lymphocytic leukemia (CLL) occurs in 2 major forms: aggressive and indolent. Low miR-29b expression in aggressive CLL is associated with poor prognosis. Indiscriminate miR-29b overexpression in the B-lineage of mice causes aberrance, thus warranting the need for selective introduction of miR-29b into B-CLL cells for therapeutic benefit. The oncofetal antigen receptor tyrosine kinase orphan receptor 1 (ROR1) is expressed on malignant B-CLL cells, but not normal B cells, encouraging us with ROR1-targeted delivery for therapeutic miRs. Here, we describe targeted delivery of miR-29b to ROR1+ CLL cells leading to downregulation of DNMT1 and DNMT3A, modulation of global DNA methylation, decreased SP1, and increased p21 expression in cell lines and primary CLL cells in vitro. Furthermore, using an Eµ-TCL1 mouse model expressing human ROR1, we report the therapeutic benefit of enhanced survival via cellular reprograming by downregulation of DNMT1 and DNMT3A in vivo. Gene expression profiling of engrafted murine leukemia identified reprogramming of cell cycle regulators with decreased SP1 and increased p21 expression after targeted miR-29b treatment. This finding was confirmed by protein modulation, leading to cell cycle arrest and survival benefit in vivo. Importantly, SP1 knockdown results in p21-dependent compensation of the miR-29b effect on cell cycle arrest. These studies form a basis for leukemic cell-targeted delivery of miR-29b as a promising therapeutic approach for CLL and other ROR1+ B-cell malignancies.

Siglec-6 on Chronic Lymphocytic Leukemia Cells Is a Target for Post-Allogeneic Hematopoietic Stem Cell Transplantation Antibodies.

Although the 5-year survival rate of chronic lymphocytic leukemia (CLL) patients has risen to >80%, the only potentially curative treatment is allogeneic hematopoietic stem cell transplantation (alloHSCT). To identify possible new monoclonal antibody (mAb) drugs and targets for CLL, we previously developed a phage display-based human mAb platform to mine the antibody repertoire of patients who responded to alloHSCT. We had selected a group of highly homologous post-alloHSCT mAbs that bound to an unknown CLL cell surface antigen. Here, we show through next-generation sequencing of cDNAs encoding variable heavy-chain domains that these mAbs had a relative abundance of ∼0.1% in the post-alloHSCT antibody repertoire and were enriched ∼1,000-fold after three rounds of selection on primary CLL cells. Based on differential RNA-seq and a cell microarray screening technology for discovering human cell surface antigens, we now identify their antigen as Siglec-6. We verified this finding by flow cytometry, ELISA, siRNA knockdown, and surface plasmon resonance. Siglec-6 was broadly expressed in CLL and could be a potential target for antibody-based therapeutic interventions. Our study reaffirms the utility of post-alloHSCT antibody drug and target discovery. Cancer Immunol Res; 6(9); 1008-13. ©2018 AACR.

A CD19/CD3 bispecific antibody for effective immunotherapy of chronic lymphocytic leukemia in the ibrutinib era.

The Bruton tyrosine kinase inhibitor ibrutinib induces high rates of clinical response in chronic lymphocytic leukemia (CLL). However, there remains a need for adjunct treatments to deepen response and to overcome drug resistance. Blinatumomab, a CD19/CD3 bispecific antibody (bsAb) designed in the BiTE (bispecific T-cell engager) format, is approved by the US Food and Drug Administration for the treatment of relapsed or refractory B-cell precursor acute lymphoblastic leukemia. Because of its short half-life of 2.1 hours, blinatumomab requires continuous intravenous dosing for efficacy. We developed a novel CD19/CD3 bsAb in the single-chain Fv-Fc format (CD19/CD3-scFv-Fc) with a half-life of ∼5 days. In in vitro experiments, both CD19/CD3-scFv-Fc and blinatumomab induced >90% killing of CLL cells from treatment-naïve patients. Antileukemic activity was associated with increased autologous CD8 and CD4 T-cell proliferation, activation, and granzyme B expression. In the NOD/SCID/IL2Rγnull patient-derived xenograft mouse model, once-weekly treatment with CD19/CD3-scFv-Fc eliminated >98% of treatment-naïve CLL cells in blood and spleen. By contrast, blinatumomab failed to induce a response, even when administered daily. We next explored the activity of CD19/CD3-scFv-Fc in the context of ibrutinib treatment and ibrutinib resistance. CD19/CD3-scFv-Fc induced more rapid killing of CLL cells from ibrutinib-treated patients than those from treatment-naïve patients. CD19/CD3-scFv-Fc also demonstrated potent activity against CLL cells from patients with acquired ibrutinib-resistance harboring BTK and/or PLCG2 mutations in vitro and in vivo using patient-derived xenograft models. Taken together, these data support investigation of CD19/CD3 bsAb's and other T cell-recruiting bsAb's as immunotherapies for CLL, especially in combination with ibrutinib or as rescue therapy in ibrutinib-resistant disease.

Autocrine Signaling by Wnt-5a Deregulates Chemotaxis of Leukemic Cells and Predicts Clinical Outcome in Chronic Lymphocytic Leukemia.

PURPOSE: ROR1, a receptor in the noncanonical Wnt/planar cell polarity (PCP) pathway, is upregulated in malignant B cells of chronic lymphocytic leukemia (CLL) patients. It has been shown that the Wnt/PCP pathway drives pathogenesis of CLL, but which factors activate the ROR1 and PCP pathway in CLL cells remains unclear. EXPERIMENTAL DESIGN: B lymphocytes from the peripheral blood of CLL patients were negatively separated using RosetteSep (StemCell) and gradient density centrifugation. Relative expression of WNT5A, WNT5B, and ROR1 was assessed by quantitative real-time PCR. Protein levels, protein interaction, and downstream signaling were analyzed by immunoprecipitation and Western blotting. Migration capacity of primary CLL cells was analyzed by the Transwell migration assay. RESULTS: By analyzing the expression in 137 previously untreated CLL patients, we demonstrate that WNT5A and WNT5B genes show dramatically (five orders of magnitude) varying expression in CLL cells. High WNT5A and WNT5B expression strongly associates with unmutated IGHV and shortened time to first treatment. In addition, WNT5A levels associate, independent of IGHV status, with the clinically worst CLL subgroups characterized by dysfunctional p53 and mutated SF3B1. We provide functional evidence that WNT5A-positive primary CLL cells have increased motility and attenuated chemotaxis toward CXCL12 and CCL19 that can be overcome by inhibitors of Wnt/PCP signaling. CONCLUSIONS: These observations identify Wnt-5a as the crucial regulator of ROR1 activity in CLL and suggest that the autocrine Wnt-5a signaling pathway allows CLL cells to overcome natural microenvironmental regulation.

ROR1-targeted delivery of OSU-2S, a nonimmunosuppressive FTY720 derivative, exerts potent cytotoxicity in mantle-cell lymphoma in vitro and in vivo.

Mantle-cell lymphoma (MCL) remains incurable despite numerous therapeutic advances. OSU-2S, a novel nonimmunosuppressive FTY720 (Fingolimod) derivative, exhibits potent cytotoxicity in MCL cell lines and primary cells. OSU-2S increased the surface expression of CD74, a therapeutic antibody target in MCL cells. OSU-2S, in combination with anti-CD74 antibody milatuzumab, enhanced cytotoxicity in MCL. Moreover, MCL tumor antigen receptor tyrosine kinase-like orphan receptor 1 (ROR1) targeted immunonanoparticle-carrying OSU-2S (2A2-OSU-2S-ILP)-mediated selective cytotoxicity of MCL in vitro, as well as activity in a xenografted mouse model of MCL in vivo. The newly developed OSU-2S delivery using ROR1-directed immunonanoparticles provide selective targeting of OSU-2S to MCL and other ROR1(+) malignancies, sparing normal B cells.

Antibody-based therapeutics for the treatment of human B cell malignancies.

The dynamic expression of various phenotypic markers during B cell development not only defines the particular stage in ontogeny but also provides the necessary growth, differentiation, maturation and survival signals. When a B cell at any given stage becomes cancerous, these cell surface molecules, intracellular signaling molecules, and the over-expressed gene products become favorite targets for potential therapeutic intervention. Various adaptive and adoptive immunotherapeutic approaches induce T cell and antibody responses against cancer cells, and successful remission leading to minimal residual disease has been obtained. Nonetheless, subsequent relapse and development of resistant clones prompted further development and several novel strategies are evolving. Engineered monoclonal antibodies with high affinity and specificity to target antigens have been developed and used either alone or in combination with chemotherapeutic drugs. They are also used as vehicles to deliver cytotoxic drugs, toxins, or radionuclides that are either directly conjugated or encapsulated in liposomal vesicles. Likewise, genetically engineered T cells bearing chimeric antigen receptors are used to redirect cytotoxicity to antigen-positive target cells. This review describes recent advancements in some of these adoptive immunotherapeutic strategies targeting B cell malignancies.

Targeting malignant B cells with an immunotoxin against ROR1.

The selective cell surface expression of receptor tyrosine kinase-like orphan receptor 1 (ROR1) in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) has made ROR1 a novel and promising target for therapeutic monoclonal antibodies (mAbs). Four mouse mAbs generated by hybridoma technology exhibited specific binding to human ROR1. Epitope mapping studies showed that two mAbs (2A2 and 2D11) recognized N-terminal epitopes in the extracellular region of ROR1 and the other two (1A1 and 1A7) recognized C-terminal epitopes. A ROR1- immunotoxin (BT-1) consisting of truncated Pseudomonas exotoxin A (PE38) and the VH and VL fragments of 2A2-IgG was made recombinantly. Both 2A2-IgG and BT-1 showed dose-dependent and selective binding to primary CLL and MCL cells and MCL cell lines. Kinetic analyses revealed 0.12-nM (2A2-IgG) to 65-nM (BT-1) avidity/affinity to hROR1, depicting bivalent and monovalent interactions, respectively. After binding to cell surface ROR1, 2A2-IgG and BT-1 were partially internalized by primary CLL cells and MCL cell lines, and BT-1 induced profound apoptosis of ROR1-expressing MCL cell lines in vitro (EC 50 = 16 pM-16 nM), but did not affect ROR1-negative cell lines. Our data suggest that ROR1-immunotoxins such as BT-1 could serve as targeted therapeutic agents for ROR1-expressing B cell malignancies and other cancers.

Restricted cell surface expression of receptor tyrosine kinase ROR1 in pediatric B-lineage acute lymphoblastic leukemia suggests targetability with therapeutic monoclonal antibodies.

BACKGROUND: Despite high cure rates for pediatric B-lineage acute lymphoblastic leukemia (B-ALL), short-term and long-term toxicities and chemoresistance are shortcomings of standard chemotherapy. Immunotherapy and chemoimmunotherapy based on monoclonal antibodies (mAbs) that target cell surface antigens with restricted expression in pediatric B-ALL may offer the potential to reduce toxicities and prevent or overcome chemoresistance. The receptor tyrosine kinase ROR1 has emerged as a candidate for mAb targeting in select B-cell malignancies. METHODOLOGY AND PRINCIPAL FINDINGS: Using flow cytometry, Western blotting, immunohistochemistry, and confocal immunofluorescence microscopy, we analyzed the cell surface expression of ROR1 across major pediatric ALL subtypes represented by 14 cell lines and 56 primary blasts at diagnosis or relapse as well as in normal adult and pediatric tissues. Cell surface ROR1 expression was found in 45% of pediatric ALL patients, all of which were B-ALL, and was not limited to any particular genotype. All cell lines and primary blasts with E2A-PBX1 translocation and a portion of patients with other high risk genotypes, such as MLL rearrangement, expressed cell surface ROR1. Importantly, cell surface ROR1 expression was found in many of the pediatric B-ALL patients with multiply relapsed and refractory disease and normal karyotype or low risk cytogenetics, such as hyperdiploidy. Notably, cell surface ROR1 was virtually absent in normal adult and pediatric tissues. CONCLUSIONS AND SIGNIFICANCE: Collectively, this study suggests that ROR1 merits preclinical and clinical investigations as a novel target for mAb-based therapies in pediatric B-ALL. We propose cell surface expression of ROR1 detected by flow cytometry as primary inclusion criterion for pediatric B-ALL patients in future clinical trials of ROR1-targeted therapies.

Therapeutic potential and challenges of targeting receptor tyrosine kinase ROR1 with monoclonal antibodies in B-cell malignancies.

BACKGROUND: 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. METHODOLOGY AND PRINCIPAL FINDINGS: 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. CONCLUSIONS AND SIGNIFICANCE: 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.

A novel adoptive transfer model of chronic lymphocytic leukemia suggests a key role for T lymphocytes in the disease.

Chronic lymphocytic leukemia (CLL) is an incurable adult disease of unknown etiology. Understanding the biology of CLL cells, particularly cell maturation and growth in vivo, has been impeded by lack of a reproducible adoptive transfer model. We report a simple, reproducible system in which primary CLL cells proliferate in nonobese diabetes/severe combined immunodeficiency/γc(null) mice under the influence of activated CLL-derived T lymphocytes. By co-transferring autologous T lymphocytes, activated in vivo by alloantigens, the survival and growth of primary CFSE-labeled CLL cells in vivo is achieved and quantified. Using this approach, we have identified key roles for CD4(+) T cells in CLL expansion, a direct link between CD38 expression by leukemic B cells and their activation, and support for CLL cells preferentially proliferating in secondary lymphoid tissues. The model should simplify analyzing kinetics of CLL cells in vivo, deciphering involvement of nonleukemic elements and nongenetic factors promoting CLL cell growth, identifying and characterizing potential leukemic stem cells, and permitting preclinical studies of novel therapeutics. Because autologous activated T lymphocytes are 2-edged swords, generating unwanted graph-versus-host and possibly autologous antitumor reactions, the model may also facilitate analyses of T-cell populations involved in immune surveillance relevant to hematopoietic transplantation and tumor cytoxicity.

The New Zealand black mouse as a model for the development and progression of chronic lymphocytic leukemia.

BACKGROUND: Similar to a subset of human patients who progress from monoclonal B lymphocytosis (MBL) to chronic lymphocytic leukemia (CLL), New Zealand Black (NZB) mice have an age-associated progression to CLL. The murine disease is linked to a genetic abnormality in microRNA mir-15a/16-1 locus, resulting in decreased mature miR-15a/16. METHODS: Spleens of aging NZB were analyzed for the presence of B-1 cells via flow cytometry and for the presence of a side population (SP) via the ability of cells to exclude Hoechst 33342 dye. The SP was assayed for the presence of hyperdiploid B-1 clones and for the ability to differentiate into B-1 cells in vitro and transfer disease in vivo. In addition, enhanced apoptosis of chemoresistant NZB B-1 cells was examined by restoring miR-16 levels in nutlin-treated cells. RESULTS: Aging NZB mice develop a B-1 expansion and clonal development that evolves from MBL into CLL. An expansion in SP is also seen. Although the SP did contain increased cells with stem cell markers, they lacked malignant B-1 cells and did not transfer disease in vivo. Similar to B-1 cells, splenic NZB SP also has decreased miR-15a/16 when compared with C57Bl/6. Exogenous addition of miR-15a/16 to NZB B-1 cells resulted in increased sensitivity to nutlin. CONCLUSION: NZB serve as an excellent model for studying the development and progression of age-associated CLL. NZB SP cells do not seem to contain cancer stem cells, but rather the B-1 stem cell. NZB B-1 chemoresistance may be related to reduced miR-15a/16 expression.

The B-cell tumor-associated antigen ROR1 can be targeted with T cells modified to express a ROR1-specific chimeric antigen receptor.

Monoclonal antibodies and T cells modified to express chimeric antigen receptors specific for B-cell lineage surface molecules such as CD20 exert antitumor activity in B-cell malignancies, but deplete normal B cells. The receptor tyrosine kinase-like orphan receptor 1 (ROR1) was identified as a highly expressed gene in B-cell chronic lymphocytic leukemia (B-CLL), but not normal B cells, suggesting it may serve as a tumor-specific target for therapy. We analyzed ROR1-expression in normal nonhematopoietic and hematopoietic cells including B-cell precursors, and in hematopoietic malignancies. ROR1 has characteristics of an oncofetal gene and is expressed in undifferentiated embryonic stem cells, B-CLL and mantle cell lymphoma, but not in major adult tissues apart from low levels in adipose tissue and at an early stage of B-cell development. We constructed a ROR1-specific chimeric antigen receptor that when expressed in T cells from healthy donors or CLL patients conferred specific recognition of primary B-CLL and mantle cell lymphoma, including rare drug effluxing chemotherapy resistant tumor cells that have been implicated in maintaining the malignancy, but not mature normal B cells. T-cell therapies targeting ROR1 may be effective in B-CLL and other ROR1-positive tumors. However, the expression of ROR1 on some normal tissues suggests the potential for toxi-city to subsets of normal cells.

A human monoclonal antibody drug and target discovery platform for B-cell chronic lymphocytic leukemia based on allogeneic hematopoietic stem cell transplantation and phage display.

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is the only potentially curative treatment available for patients with B-cell chronic lymphocytic leukemia (B-CLL). Here, we show that post-alloHSCT antibody repertoires can be mined for the discovery of fully human monoclonal antibodies to B-CLL cell-surface antigens. Sera collected from B-CLL patients at defined times after alloHSCT showed selective binding to primary B-CLL cells. Pre-alloHSCT sera, donor sera, and control sera were negative. To identify post-alloHSCT serum antibodies and subsequently B-CLL cell-surface antigens they recognize, we generated a human antibody-binding fragment (Fab) library from post-alloHSCT peripheral blood mononuclear cells and selected it on primary B-CLL cells by phage display. A panel of Fab with B-CLL cell-surface reactivity was strongly enriched. Selection was dominated by highly homologous Fab predicted to bind the same antigen. One Fab was converted to immunoglobulin G1 and analyzed for reactivity with peripheral blood mononuclear cells from B-CLL patients and healthy volunteers. Cell-surface antigen expression was restricted to primary B cells and up-regulated in primary B-CLL cells. Mining post-alloHSCT antibody repertoires offers a novel route to discover fully human monoclonal antibodies and identify antigens of potential therapeutic relevance to B-CLL and possibly other cancers. Trials described herein were registered at www.clinicaltrials.gov as nos. NCT00055744 and NCT00003838.

Generation, affinity maturation, and characterization of a human anti-human NKG2D monoclonal antibody with dual antagonistic and agonistic activity.

In humans, NKG2D is an activating receptor on natural killer (NK) cells and a costimulatory receptor on certain T cells and plays a central role in mediating immune responses in autoimmune diseases, infectious diseases, and cancer. Monoclonal antibodies that antagonize or agonize immune responses mediated by human NKG2D are considered to be of broad and potent therapeutic utility. Nonetheless, monoclonal antibodies to NKG2D that are suitable for clinical investigations have not been published yet. Here, we describe the generation, affinity maturation, and characterization of a fully human monoclonal antibody to human NKG2D. Using phage display technology based on a newly generated naïve human Fab library in phage display vector pC3C followed by a tandem chain shuffling process designed for minimal deviation from natural human antibody sequences, we selected a human Fab, designated KYK-2.0, with high specificity and affinity to human NKG2D. KYK-2.0 Fab blocked the binding of the natural human NKG2D ligands MICA, MICB, and ULBP2 as potently as a commercially available mouse anti-human NKG2D monoclonal antibody in immunoglobulin G (IgG) format. Conversion of KYK-2.0 Fab to IgG1 resulted in subnanomolar avidity for human NKG2D. KYK-2.0 IgG1 was found to selectively recognize defined subpopulations of human lymphocytes known to express NKG2D, that is, the majority of human CD8+, CD16+, and CD56+ cells as well as a small fraction of human CD4+ cells. In solution, KYK-2.0 IgG1 interfered with the cytolytic activity of ex vivo expanded human NK cells. By contrast, immobilized KYK-2.0 IgG1 was found to strongly induce human NK cell activation. The dual antagonistic and agonistic activity promises a wide range of therapeutic applications for KYK-2.0 IgG1 and its derivatives.

Unique cell surface expression of receptor tyrosine kinase ROR1 in human B-cell chronic lymphocytic leukemia.

PURPOSE: Gene expression profiling identified receptor tyrosine kinase ROR1, an embryonic protein involved in organogenesis, as a signature gene in B-cell chronic lymphocytic leukemia (B-CLL). To assess the suitability of ROR1 as a cell surface antigen for targeted therapy of B-CLL, we carried out a comprehensive analysis of ROR1 protein expression. EXPERIMENTAL DESIGN: Peripheral blood mononuclear cells, sera, and other adult tissues from B-CLL patients and healthy donors were analyzed qualitatively and quantitatively for ROR1 protein expression by flow cytometry, cell surface biotinylation, Western blotting, and ELISA. RESULTS: ROR1 protein is selectively expressed on the surface of B-CLL cells, whereas normal B cells, other normal blood cells, and normal adult tissues do not express cell surface ROR1. Moreover, cell surface expression of ROR1 is uniform and constitutive, i.e., independent of anatomic niches, independent of biological and clinical heterogeneity of B-CLL, independent of B-cell activation, and found at similar levels in all B-CLL samples tested. The antibody binding capacity of B-CLL cell surface ROR1 was determined to be in the range of 10(3) to 10(4) molecules per cell. A portion of B-CLL cell surface ROR1 was actively internalized upon antibody binding. Soluble ROR1 protein was detectable in sera of <25% of B-CLL patients and a similar fraction of healthy donors at concentrations below 200 ng/mL. CONCLUSIONS: The restricted, uniform, and constitutive cell surface expression of ROR1 protein in B-CLL provides a strong incentive for the development of targeted therapeutics such as monoclonal antibodies.

Human autologous tumor-specific T-cell responses induced by liposomal delivery of a lymphoma antigen.

PURPOSE: The idiotype (Id) of the immunoglobulin on a given B-cell malignancy is a clonal marker that can serve as a tumor-specific antigen. We developed a novel vaccine formulation by incorporating Id protein with liposomal lymphokine that was more potent than a prototype, carrier-conjugated Id protein vaccine in preclinical studies. In the present study, we evaluated the safety and immunogenicity of this vaccine in follicular lymphoma patients. EXPERIMENTAL DESIGN: Ten patients with advanced-stage follicular lymphoma were treated with five doses of this second generation vaccine after chemotherapy-induced clinical remission. All patients were evaluated for cellular and humoral immune responses. RESULTS: Autologous tumor and Id-specific type I cytokine responses were induced by vaccination in 10 and 9 patients, respectively. Antitumor immune responses were mediated by both CD4+ and CD8+ T cells, were human lymphocyte antigen class I and II associated, and persisted 18 months beyond the completion of vaccination. Specific anti-Id antibody responses were detected in four patients. After a median follow-up of 50 months, 6 of the 10 patients remain in continuous first complete remission. CONCLUSIONS: This first clinical report of a liposomal cancer vaccine demonstrates that liposomal delivery is safe, induces sustained tumor-specific CD4+ and CD8+ T-cell responses in lymphoma patients, and may serve as a model for vaccine development against other human cancers and infectious pathogens.

Chemokine receptor-mediated delivery directs self-tumor antigen efficiently into the class II processing pathway in vitro and induces protective immunity in vivo.

Nonimmunogenic antigens can be efficiently rendered immunogenic by targeting them to antigen-presenting cells via differentially expressed chemokine receptors. For example, self-tumor or HIV antigens genetically fused with proinflammatory chemoattractants elicit potent immune responses and protective antitumor immunity in mice. Herein we demonstrate that the mechanism by which chemokine fusions elicit responses is efficient uptake, processing, and presentation of antigens via the major histocompatibility complex class II pathway. Experiments with inhibitors of intracellular trafficking suggest that chemoattractant fusion proteins, but not antigen alone, were processed and presented through early/late endosomal and Golgi compartments and stimulated antigen-specific CD4+ T cells both in vitro and in vivo. Chemokine fusion also facilitated the presentation of antigen by dendritic cells to an autologous human tumor-specific CD4+ T-cell line. Taking advantage of chemokine redundancy, viral chemokine fusions were equally potent in inducing protective immunity in vivo, providing a possible strategy to circumvent hypothetical, vaccine-induced antihost chemokine autoimmunity, for example, by use of viral chemoattractants in humans.