Superior Antibody-dependent Cellular Cytotoxicity Research Articles

Comprehensive Exploration of NK Cell Education in ADCC Triggered By a Monoclonal Antibody or a Bispecific Killer Engager Molecule (BiKE)

The responsiveness of NK cells to malignant cells that lack self-HLA class I (HLA-I) expression is controlled by NK cell education governed by the interaction between selected inhibitory NK cell receptors and their cognate HLA-I ligands. However, it remains unclear to what extent education impacts the ability of NK cells to perform antibody-dependent cellular cytotoxicity (ADCC) as previous studies show inconsistent results and are limited in resolution. Moreover, no study has yet addressed if NK cell education impacts ADCC triggered by novel CD16-binding therapeutics such as bispecific killer engagers (BiKEs). In this study we systematically characterized NK cell degranulation against a panel of tumor cell lines with defined HLA-I expression in the presence of either the monoclonal antibody rituximab or a recently developed CD34xCD16 BiKE. Using high-resolution flow cytometry, our data revealed that NK cells educated by one or two KIRs did not have significantly enhanced ADCC capacity over uneducated KIR+ or KIR- NK cells, especially when increasing the antibody concentration. Contrasting co-education by KIRs, NKG2A was consistently associated with improved responsiveness and superior ADCC. Surprisingly, these results were despite that KIR-education correlated with higher CD16 expression that was markedly lower on NKG2A+ NK cells compared to both KIR-educated and uneducated subsets. An extended analysis uncovered that the inhibitory KIR2DL1-C245 receptor, reported to mediate weak NK cell education compared to the KIR2DL1-R245 variant, further repressed the ADCC capacity of NKG2A+ NK cells from donors expressing its ligand HLA C2. This highlights that also inhibitory KIRs can dampen the ADCC potential of NK cells similar to that of activating KIRs which also were discovered to restrict ADCC even when stimulated by its ligand. Finally, our study unveiled that BiKE-triggered ADCC has a similar relationship to NK cell education and its hierarchies as that triggered by a monoclonal antibody. In conclusion, the ADCC capacity of an NK cell is more regulated by restrictive mechanisms of education conferred by activating or selected inhibitory KIRs in individuals carrying the cognate KIR ligand than that of inhibitory KIRs conferring higher education potential. Hence, this study establishes that NK cell education does not enhance the ADCC capacity of an NK cell per se while providing new insights that also inhibitory KIRs can dampen the ADCC potential even when the target cell lacks its ligand. Our results add to the fundamental understanding of NK cell biology as well as to avenues of therapeutic applications for NK cells.

885TiP The I-PACE study: Imgatuzumab in PAtients with advanCEd cutaneous squamous cell carcinoma (aCSCC)

Imgatuzumab (formerly known as GA201, RO5083945) is a humanized monoclonal antibody against the epidermal growth factor receptor (EGFR), glycoengineered for enhanced antibody-dependent cellular cytotoxicity (ADCC). It exhibited superior ADCC compared with cetuximab which translated into prolonged survival in several murine xenograft models. Its clinical anti-tumor activity and manageable safety profile as monotherapy and in combination with chemotherapy was demonstrated in previous trials that enrolled 296 patients with advanced solid tumors, including colorectal, head and neck and non-small cell lung cancers.

Phenotypic and Functional Plasticity of CXCR6+ Peripheral Blood NK Cells.

Human NK cells are comprised of phenotypic subsets, whose potentially unique functions remain largely unexplored. C-X-C-motif-chemokine-receptor-6 (CXCR6) + NK cells have been identified as phenotypically immature tissue-resident NK cells in mice and humans. A small fraction of peripheral blood (PB)-NK cells also expresses CXCR6. However, prior reports about their phenotypic and functional plasticity are conflicting. In this study, we isolated, expanded, and phenotypically and functionally evaluated CXCR6+ and CXCR6– PB-NK cells, and contrasted results to bulk liver and spleen NK cells. We found that CXCR6+ and CXCR6– PB-NK cells preserved their distinct phenotypic profiles throughout 14 days of in vitro expansion (“day 14”), after which phenotypically immature CXCR6+ PB-NK cells became functionally equivalent to CXCR6– PB-NK cells. Despite a consistent reduction in CD16 expression and enhanced expression of the transcription factor Eomesodermin (Eomes), day 14 CXCR6+ PB-NK cells had superior antibody-dependent cellular cytotoxicity (ADCC) compared to CXCR6– PB-NK cells. Further, bulk liver NK cells responded to IL-15, but not IL-2 stimulation, with STAT-5 phosphorylation. In contrast, bulk splenic and PB-NK cells robustly responded to both cytokines. Our findings may allow for the selection of superior NK cell subsets for infusion products increasingly used to treat human diseases.

Humanized Anti-CD26 Monoclonal Antibody Has Anti-Myeloma Efficacy for the Treatment of High-Risk Multiple Myeloma

Background: Despite remarkable progress in treatment options of multiple myeloma (MM) over the last decade with proteasome inhibitors and immunomodulatory drugs (IMiDs), most of patients with high-risk multiple myeloma (MM) relapse and ultimately become refractory. Recently, novel monoclonal antibody directly targeting CS1(elotuzumab) or CD38 (daratuzumab) induced durable responses in patients with multidrug-refractory MM. CD26, a 110-kDa transmembrane glycoprotein which is expressed on several tumor cells including malignant lymphoma, has been implicated in tumorigenesis, whereas its role in plasma cell malignancies has not been characterized yet. Our recent work has revealed that CD26 is intensely expressed on activated osteoclasts (OCs) in MM. In addition, in vitro studies showed that although CD26 expression is only slightly detected on MM cell lines cultured alone, it is intensely and uniformly expressed on MM cell lines co-cultured with OCs. In this study, we clarify the direct on-tumor and immune mechanism of action with huCD26mAb, a humanized monoclonal IgG1 antibody specifically targeting CD26, alone or in combination with approved agents in high-risk MM.Methods and Results: Immunostaining on bone marrow biopsy specimens showed that CD26 is expressed on plasma cells in several MM patients, but not in those of healthy individuals. So, we first analyzed the effect of huCD26mAb on survival of MM cell lines (KMS18, KMS26, KMS27, KMS28, KMS34, U266). HuCD26mAb had no direct effect on proliferation in any of tested CD26- MM cell lines cultured alone, but it inhibited growth of CD26+ MM cell lines co-cultured with OCs at higher concentrations (>10μg/ml), as measured by the MTT assay. Next, we examined the ability of huCD26mAb to lyse CD26 positive MM cell lines by antibody-dependent cellular cytotoxicity (ADCC) using the calcein-AM release assay. HuCD26mAb, but not isotype control IgG1, triggered ADCC against CD26+ KMS18, KMS26, KMS28, KMS34 and U266 with t(4;14) by natural killer (NK) effector cells in a dose-dependent manner with lytic activity starting at 0.0001μg/ml and maximum lysis at 10μg/ml and in an effector to target (E/T) ratio-dependent manner. Moreover, huCD26mAb dose-dependently induced lysis of lenalidomide-resistant MM.1R via ADCC. We further asked whether pretreatment with conventional or novel agents is able to induce superior ADCC activity by huCD26mAb. CD26+ KMS18, KMS26 and KMS28 were pretreated with bortezomib (3nM), lenamlidomide (0.5μM) or dexamethasone (25nM) overnight. Indeed, subsequent MM cell lysis triggered by huCD26mAb (10μg/ml) was significantly augumented. Treatment with lenalidomide (0.5μM) followed by huCD26mAb (10μg/ml) also markedly reduced the viable MM.1R in the presence of NK effector cells. The synergistic ADCC efficacy of huCD26mAb in combination with lenalidomide resulted in 2.0-fold increase in MM.1R lysis. In addition, pretreatment of NK effector cells with lenalidomide also potentiated ADCC against MM.1R mediated by huCD26mAb in a dose-dependent fashion. Complement dependent cytotoxicity (CDC) of CD26+ MM cells by huCD26mAb was also tested using human serum as a source of complement. However, in the presence of human serum, the incubation with huCD26mAb or isotype IgG1 exerted no effect on MM cell lysis. These results indicate that huCD26mAb (≦10μg/ml) kills CD26+ MM cells by inducing ADCC but not CDC, regardless of sensitivity or resistance to conventional or novel agents. Finally, to further determine whether huCD26mAb alone or combination with lenalidomide affects side population (SP) fractions of drug-resistant MM cells, we first examined CD26 expression in SP and main population (MP) fractions by flow cytometry. Both CD26+ RPMI8226 and KMS11 exhibited SP fractions which equally expressed CD26 at high levels as MP fractions. So, we examined ADCC activity by huCD26mAb (10μg/ml) against these SP fractions, mixed with NK effector cells. Interestingly, although lenalidomide alone did not decrease the ratio of SP fractions, huCD26mAb substantially reduced their ratio and its further reduction was observed with both in combination against CD26+ RPMI8226 and KMS11.Conclusions: HuCD26mAb induced significant anti-MM efficacy chiefly through ADCC. CD26 may be a novel valuable target for antibody therapy and huCD26mAb, alone or in combination, may be a promising immunotherapeutic strategy for the treatment of high-risk MM. DisclosuresMorimoto:Y's Therapeutics: Consultancy, Research Funding. Yamada:Y's therapeutics: Consultancy, Research Funding.

Generation of Multiplexed Engineered, Off-the-Shelf CAR T Cells Uniformly Carrying Multiple Anti-Tumor Modalities to Prevent Tumor Relapse

The development of chimeric antigen receptor (CAR) T cell therapeutics is widely recognized as a significant advancement for the treatment of cancer. However, several obstacles currently impede the broad use of CAR T cells, including the inherent process variability, cost of manufacturing, the absolute requirement for precise and uniform genetic editing in the allogeneic setting, and the challenge to keep pace with clonal heterogeneity and tumor growth. Utilizing our previously described induced pluripotent stem cell (iPSC)-derived T (iT) cell platform, we illustrate here the unique ability to address these challenges by creating a consistent CAR iT cell product that can be repeatedly manufactured in large quantities from a renewable iPSC master cell bank that has been engineered to mitigate the occurrence of graft versus host disease (GvHD), antigen escape and tumor relapse. Utilizing our proprietary cellular reprogramming and engineering platform and stage-specific T cell differentiation protocol, we demonstrate that iPSC can be engineered at the single cell level to generate a fully characterized clonal iPSC line, which can then be accessed routinely to yield CAR iT cells in a highly scalable manufacturing process (>100,000 fold expansion). Through bi-allelic targeting of a CAR into the T cell receptor alpha constant (TRAC) region, we generated CAR iT cells with uniform CAR expression (99.0 ± 0.5% CAR+) and complete elimination of T cell receptor (TCR) expression to avoid GvHD in the allogeneic setting. We elected to utilize the 1XX-CAR configuration, which has demonstrated superior anti-tumor performance relative to other CAR designs and when introduced into iT cells displayed enhanced antigen specificity (% specific cytotoxicity at E:T=10:1, antigen positive group: 86.4 ± 7.8; antigen null group: 8.9 ± 3.5). To enhance persistence without reliance on exogenous cytokine support, we engineered signaling-fusion complexes, including IL-7 receptor fusion (RF), into iPSC and studied its impact on iT phenotype, persistence, and efficacy. In vitro, IL-7RF clones demonstrated improved anti-tumor activity in a serial antigen dependent tumor challenge assay (Day 10, relative tumor counts, IL-7RF group: 1.95 ± 0.01; control group: 57.56 ± 4.55, P<0.000001). In a preclinical in vivo model of disseminated leukemia, IL-7RF clones demonstrate enhanced tumor growth inhibition (Day 34, Log [BLI], IL-7RF group: 6.68 ± 1.93; control group: 9.99 ± 0.23, P=0.0143). We next investigated a unique strategy to incorporate multi-antigen targeting potential into anti-CD19 1XX CAR iT cells with the addition of a high-affinity non-cleavable CD16 (hnCD16) Fc receptor. The combination of hnCD16 with anti-CD19 1XX CAR culminated in iT cells capable of multi-antigen specificity through combinatorial use with monoclonal antibodies to tackle antigen escape. Utilizing CD19 negative leukemia cells as targets, superior antibody-dependent cellular cytotoxicity (ADCC) was demonstrated by the combination of hnCD16 CAR iT and Rituximab (% specific cytotoxicity at E:T=1:1, hnCD16 group + Rituximab: 75.64 ± 2.12; control group + Rituximab: 16.98 ± 3.87, P<0.001). To address T cell fitness, the role of CD38 knockout (KO) in T cells was investigated, which we have previously shown to mediate NK cell resistance to oxidative stress induced apoptosis. CD38 gene was disrupted at the iPSC stage to generate 1XX-CAR T cells that lack CD38 expression (% CD38+ population, CD38WT group: 69.67 ± 24.34; CD38KO group: 0.12 ± 0.11) and upon antigen mediated stimulation, CD38KO CAR iT cells showed higher percentages of degranulation (2.3-fold increase in CD107a/b), and IFNγ (4.1-fold increase) and TNFα (2.5-fold increase) production. Antigen specific in vitro tumor killing also was enhanced in CD38KO CAR iT cells (EC50, 3.2-fold decrease). Lastly, to avoid the potential host-mediated rejection, the inclusion of allogeneic defense receptor (ADR) which has been shown to significantly reduce host-mediated rejection will be discussed. Collectively, the described studies demonstrate that iPSCs are an ideal cellular source to generate large-quantities of uniformly multi-edited off-the-shelf CAR T cell products that include a best-in-class CAR design, enhanced product modalities, and complete elimination of TCR expression to avoid the potential of GvHD while maintaining high anti-tumor efficacy in allogeneic setting. Disclosures Hsia: Fate Therapeutics Inc.: Current Employment. Clarke:Fate Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Lee:Fate Therapeutics, Inc.: Current Employment. Robbins:Fate Therapeutics, Inc.: Current Employment. Denholtz:Fate Therapeutics, Inc: Current Employment. Hanok:Fate Therapeutics, Inc.: Current Employment. Carron:Fate Therapeutics, Inc.: Current Employment. Navarrete:Fate Therapeutics, Inc.: Current Employment. ORourke:Fate Therapeutics, Inc.: Current Employment. Sung:Fate Therapeutics, Inc.: Current Employment. Gentile:Fate Therapeutics, Inc.: Current Employment. Nguyen:Fate Therapeutics, Inc.: Current Employment. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company.

Phase Ib Study of Ianalumab (VAY736) and Ibrutinib in Patients with Chronic Lymphocytic Leukemia (CLL) on Ibrutinib Therapy

Phase Ib Study of Ianalumab (VAY736) and Ibrutinib in Patients with Chronic Lymphocytic Leukemia (CLL) on Ibrutinib Therapy

Natural Killer (NK) Cells: The Missing Link between Antibody-Mediated Rejection (ABMR) and Chronic Lung Allograft Dysfunction (CLAD)?

The long-term success of lung transplantation (LTx) is limited by CLAD. The complement-independent pathway of ABMR, driven by antibody-dependent cellular cytotoxicity (ADCC) may contribute to CLAD. ADCC is mediated through the CD16 Fc receptor expressed on NK cells, however a role for different NK cell subsets in mediating ABMR is yet to be elucidated. In this pilot study, we investigated whether the presence of donor-specific antibodies (DSA) following LTx effects NK cell ADCC and whether particular NK cell subsets exhibited superior ADCC activity. Peripheral blood mononuclear cells (PBMC) were isolated from LTx recipients at 3 and 12-months post-LTx. NK cell ADCC activity (degranulation (CD107a) and the production of cytokines from PBMC (interferon gamma (IFN-γ) and tumor necrosis alpha (TNF-α))) was measured by flow cytometry following antibody-dependent activation (ADA). The detection of anti-human leukocyte antigen (HLA) antibodies at the same time points was determined by Luminex. LTx recipients were classified as DSA positive (DSA+, presence of anti-donor HLA antibodies, n=12), anti-HLA antibody positive (HLA+, presence of anti-HLA antibodies but not to donor HLA, n=12) or no HLA antibodies (Ab-, no anti-HLA antibodies detected, n=7). Surprisingly, a higher proportion of NK cells expressed the activating receptor NKG2C in the DSA+ group (p=0.0078). Furthermore, NKG2C+ NK cells isolated from recipients with HLA antibodies were significantly more activated than NKG2A+ cells. Following ADA, a higher proportion of NKG2C+ NK cells produced cytokines in DSA+ (IFNγ, p=0.0095) and HLA+ (IFNγ: p=0.0317; TNFα: p=0.0159) recipients, compared to NKG2A+ cells. In addition, CD16 expression was more down-regulated following ADA in DSA+ recipients (p=0.0084). Our data shows that the presence of DSA impacts on the phenotype and function of NK cells following LTx. Moreover, as previous research has shown an association between NKG2C expression, cytomegalovirus (CMV) infection and CLAD, our study potentially provides a missing link between CMV infection and CLAD. Future confirmation of NKG2C+ NK cells as mediators of ABMR will lead to development of targeted diagnostic criteria and specific therapies to offer early therapeutic interventions to prevent CLAD, thereby improving LTx outcomes.

Increased, Durable B-Cell and ADCC Responses Associated with T-Helper Cell Responses to HIV-1 Envelope in Macaques Vaccinated with gp140 Occluded at the CD4 Receptor Binding Site.

ABSTRACTStrategies are needed to improve the immunogenicity of HIV-1 envelope (Env) antigens (Ag) for more long-lived, efficacious HIV-1 vaccine-induced B-cell responses. HIV-1 Env gp140 (native or uncleaved molecules) or gp120 monomeric proteins elicit relatively poor B-cell responses which are short-lived. We hypothesized that Env engagement of the CD4 receptor on T-helper cells results in anergic effects on T-cell recruitment and consequently a lack of strong, robust, and durable B-memory responses. To test this hypothesis, we occluded the CD4 binding site (CD4bs) of gp140 by stable cross-linking with a 3-kDa CD4 miniprotein mimetic, serving to block ligation of gp140 on CD4+ T cells while preserving CD4-inducible (CDi) neutralizing epitopes targeted by antibody-dependent cellular cytotoxicity (ADCC) effector responses. Importantly, immunization of rhesus macaques consistently gave superior B-cell (P < 0.001) response kinetics and superior ADCC (P < 0.014) in a group receiving the CD4bs-occluded vaccine compared to those of animals immunized with gp140. Of the cytokines examined, Ag-specific interleukin-4 (IL-4) T-helper enzyme-linked immunosorbent spot (ELISpot) assays of the CD4bs-occluded group increased earlier (P = 0.025) during the inductive phase. Importantly, CD4bs-occluded gp140 antigen induced superior B-cell and ADCC responses, and the elevated B-cell responses proved to be remarkably durable, lasting more than 60 weeks postimmunization.IMPORTANCE Attempts to develop HIV vaccines capable of inducing potent and durable B-cell responses have been unsuccessful until now. Antigen-specific B-cell development and affinity maturation occurs in germinal centers in lymphoid follicles through a critical interaction between B cells and T follicular helper cells. The HIV envelope binds the CD4 receptor on T cells as soluble shed antigen or as antigen-antibody complexes, causing impairment in the activation of these specialized CD4-positive T cells. We proposed that CD4-binding impairment is partly responsible for the relatively poor B-cell responses to HIV envelope-based vaccines. To test this hypothesis, we blocked the CD4 binding site of the envelope antigen and compared it to currently used unblocked envelope protein. We found superior and durable B-cell responses in macaques vaccinated with an occluded CD4 binding site on the HIV envelope antigen, demonstrating a potentially important new direction in future design of new HIV vaccines.

Producing defucosylated antibodies with enhanced in vitro antibody‐dependent cellular cytotoxicity via FUT8 knockout CHO‐S cells

To engineer a host cell line that produces defucosylated mAbs with superior antibody-dependent cellular cytotoxicity, we disrupted α-1, 6 fucosyltransferase (FUT8) gene in CHO-S (CHO is Chinese hamster ovary) cells by clustered regularly interspaced short palindromic repeats-CRISPR associated nuclease 9. The gene knockout cell line was evaluated for growth, stability, and product quality. The growth profile of FUT8 gene knockout CHO-S (FUT8 -/-) cells was comparable with wild type CHO-S cells. FUT8 catalyzes the transfer of a fucose residue from GDP-fucose to N-glycans residue. Defucosylated IgG1 antibodies produced by FUT8 -/- cells showed increased binding affinities to human FcγRIIIa and higher activities in mediating antibody-dependent cellular cytotoxicity, comparing with conventional fucosylated IgG1. Our results demonstrated the potential of using the clustered regularly interspaced short palindromic repeats-CRISPR associated nuclease 9 technology in cell line engineering for biopharmaceutical industrial applications.

P2-18-06: Conventional Trastuzumab Is an Antagonist of Natural Killer Cells: Making the Case for Fucose-Depleted Trastuzumab.

Abstract The discovery of the HER2 receptor and later the development of trastuzumab (an anti-HER2 antibody) were major advances leading toward the treatment of a significant proportion of human breast cancers. Trastuzumab (Herceptin®) is a humanized monoclonal IgG1 whose discrete antitumor effects are exerted through the 2 main components of the antibody. Trastuzumab's FAB portion binds to the HER2 extracellular domain, interrupting HER2 signaling. The FC portion then engages the activating receptor (FcγRIIIa) on an effector cell, usually a natural killer (NK) cell and instigates a lytic attack via antibody-dependent cellular cytotoxicity (ADCC). The value of trastuzumab in breast cancer therapy is well established, but, there are limitations to the drug's effectiveness. These include: (1) unresponsive tumors with low or equivocal HER2 expression; (2) de novo (primary) resistance to therapy in over 50% of patients with HER2−positive tumors; (3) frequent emergence of secondary resistance often after short exposure periods; (4) competition of plasma IgG proteins with antibodies for binding to the activating FC receptors on NK cells; and (5) the high cost of trastuzumab treatment, raising concerns for the economic sustainability of this form of therapy. Trastuzumab and other therapeutic antibodies share a structural feature that is critical to their effectiveness: a specific oligosaccharide which is covalently linked to the FC region at asparagine 297 (Asn297) in each of the 2 heavy chains. This oligosaccharide, a mannosyl-chitobiose core, is “core-fucosylated” with a single α-L-fucose (fucose) sugar attached via an α(1,6) linkage. In 2002, Shields et al. developed a novel form of trastuzumab which lacked core fucose in the Fc region [J Biol Chem 2002; 277: 26733–40]. When compared to conventional trastuzumab, the fucose-depleted trastuzumab was 43-fold more potent in provoking ADCC against HER2−expressing human breast cancer cells in vitro. Subsequent studies with several monoclonal antibodies have elucidated the role of core fucose in inhibiting ADCC, by reducing the affinity of trastuzumab's Fc portion for the activating FC receptors located on effector cells. The superior ADCC effect of fucose-depleted trastuzumab has now been demonstrated in vivo [Junttila TT et al., Cancer Res 2010; 70: 4481–4489]. We review both in vitro and in vivo evidence from multiple laboratories (working with trastuzumab, rituximab and other therapeutic antibodies) to argue that fucose-depleted trastuzumab offers several potential advantages over conventional trastuzumab. Such advantages in clinical use, we postulate, include (1) treatment of patients with trastuzumab-resistant tumors, (2) treatment of patients with equivocal or low expression of HER2, and (3) decreased cost of treatment. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-18-06.

Abstract C221: Glycoengineered anti-EGFR (GA201) elicits enhanced ADCC responses by NK cells from colorectal cancer patients despite tumor-associated impairments to natural cytotoxicity.

Abstract Background: One component of the therapeutic efficacy of IgG1 monoclonal antibodies (mAb) is their ability to induce antibody dependent cellular cytotoxicity (ADCC) by NK cells and other immune effectors bearing the low-affinity Fc RIIIa (CD16) receptor. Clinical studies establish the relevance of ADCC finding widespread polymorphisms that encode a very low affinity CD16 (158F/F) predict limited clinical responses to Cetuximab or Rituximab. Now, mAbs with increased affinity for CD16 can be produced using GlycoMab technology. The altered glycosylation pattern in the Fc domain of these antibodies enhances CD16-binding and ADCC irrespective of genotype. Yet, the potential for enhanced ADCC in patients with advanced colorectal carcinoma (CRC) has not been demonstrated. Considering the potential for tumor-associated immune impairments and treatments-associated leukopenia, we enumerated and characterized NK cells from patients prior to chemotherapy, on active therapy, or following two-lines of standard treatment and compared responsiveness to standard and glycoengineered anti-EGFR mAbs. Methodology: PBMCs were isolated from blood of age-matched healthy donors and 100 patients: either at presentation with metastatic disease; during chemotherapy with FOLFOX, FOLFIRI, or Irinotecan; or at disease progression &amp;gt;4 weeks following second line failure. Laboratory tests included: immunophenotyping for CD3,CD8,CD45,CD4 and CD3,CD56/16,CD45,CD19 markers; measurements of CD16- and NKG2D-expressing NK cells; CD16 158 genotype; and assessment of K562-killing (natural cytotoxicity) and degranulation (CD107+) of NK cells among cryopreserved PBMC in response to K562 or EGFR+ A431 cells with 10 g/ml GlycoMAb anti-EGFR (GA201), wild-type GA201, Panitumumab or Cetuximab. Results: For each group of patients, the proportion of NK cells among lymphocytes remained similar to controls. However, NK cells counts were reduced about half in patients (affected primarily by disease and only slightly by treatment). Per NK cell killing capacity of K562 was substantially impaired in patients prior to therapy and most profoundly among the post-chemotherapy group. ADCC responses were similarly impaired but to lesser degrees. In all cohorts tested, GlycoMAb anti-EGFR was superior to cetuximab in activating more NK cells. The superior ADCC activation of NK cells by GlycoMAb was most pronounced in the patients with chemotherapy-resistant disease. Conclusion: Chemotherapy does not induce gross alterations in the immune phenotype of CRC patients. At all stages of treatment, patients retain CD16+ NK cells capable of ADCC. The impairments in the NK functional responses tested are likely due to host/tumor interaction and not a consequence of treatment. Despite impairments, the best activation of NK cells was always in response to GlycoMAb GA201 for all individuals tested. These findings support testing for enhanced ADCC by GA201 in clinical trials of CRC patients following two lines of chemotherapy or in combination with chemotherapy. Acknowledgements: Simon Hollingsworth, current affiliation Astra Zeneca UK Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C221.

GlycoVariant Anti-CD37 Small Modular Immuno-Pharmaceutical Exhibits Superior Natural Killer Cell Mediated Cytotoxicity Against Chronic Lymphocytic Leukemia Cells at Low Concentrations and Low Antigen Density

AbstractAbstract 1847CD37 is a tetraspanin transmembrane family protein that is strongly expressed on the surface of mature human B cells and transformed mature B cell lymphoma and leukemia cells, including CLL cells. It is absent or minimally expressed on normal T cells, NK cells, monocytes, and granulocytes. Predominant expression of CD37 on CLL cells makes it an ideal candidate to target with potential agents for treatment of CLL. TRU-016, a small modular immunopharmaceutical protein (SMIP) that specifically binds to an extracellular region of CD37, is presently in clinical trials in CLL patients. TRU-016 includes humanized immunoglobulin variable regions (scFv) fused to a human IgG1 Fc region. We have previously reported that SMIP-016, the chimeric version of the humanized TRU-016, induced apoptosis in CLL B cells in the presence of goat anti-human Fc antibody cross-linker through a novel, caspase-independent pathway. Furthermore, SMIP-016 showed potent in vivo activity in a SCID xenograft mouse model. Aside from direct cytotoxicity, SMIP-016 mediates antibody-dependent cellular cytotoxicity (ADCC) by NK cells both in vitro and in vivo. In an attempt to enhance its ADCC function, a new variant of SMIP-016, SMIP-016GV, was designed with a modification of the glycosylation of the Fc portion of the molecule. SMIP-016GV exhibits enhanced binding to both low- and high-affinity molecular variants of human CD16 (FcγRIII) and augments ADCC potency when compared to SMIP-016. In this study, we compared SMIP-016GV and SMIP-016 in direct cytotoxicity and ADCC against CLL B cells. While SMIP-016 and SMIP-016GV mediated comparable direct cytotoxicity at 48 hrs in the presence of goat anti-human Fc crosslinker, the SMIP-016GV resulted in 2 to 4 fold increase in NK cell mediated ADCC function at all effector to target ratios tested. This increased ADCC with SMIP-016GV was observed using NK cell effectors derived from both normal as well as CLL-affected individuals. In addition, this enhanced cytotoxicity was sustained at concentrations of SMIP-016GV as low at 5E-6 μg/ml. These low concentrations of SMIP-016GV were also able to mediate superior ADCC in 697 cells expressing as few as 10,000 molecules of surface CD37 antigen. Furthermore, NK cells stimulated with the glycovariant were potently activated and released 3 to 4 fold more IFNγ compared to SMIP-016. Ongoing studies are aimed at defining other effector cells which may interact with SMIP-016GV via different Fcγ Receptors. Collectively, these results suggest potential use of the SMIP-016GV with enhanced ADCC function as an alternate for TRU-016 in B cell malignancies including CLL therapy. Disclosures:Siadak:Trubion Pharmaceuticals: Employment.

Development of a new fully human anti-CD20 monoclonal antibody for the treatment of B-cell malignancies

Despite the widespread use of rituximab, a chimeric monoclonal antibody with demonstrated efficacy in the treatment of non-Hodgkin's lymphomas, there is a recognized need to develop new agents with improved efficacy. Towards this end, using XenoMouse technology, a fully human IgG1 anti-CD20 monoclonal antibody was generated. This antibody, denoted mAb 1.5.3, evoked enhanced pro-apoptotic activity in vitro, as compared to rituximab, in the Ramos lymphoma cell line. Also, mAb 1.5.3 mediated both complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) similar to rituximab in human B-lymphoma lines. Interestingly, mAb 1.5.3 demonstrated superior ADCC compared to rituiximab when FcgammaRIIIa F/F allotype donors were profiled and superior cytolytic activity across multiple human B-lymphoma and chronic B-cell leukemia lines in an in vitro whole blood assay. Furthermore, mAb 1.5.3 exhibited enhanced anti-tumor activity in Ramos, Daudi, and Namalwa tumour xenograft models. Lastly, mAb 1.5.3 produced a superior B-cell depletion profile in lymph node organs and bone marrow as compared to rituximab in a primate pharmacodynamic (PD) model. These findings underscore the potential of mAb 1.5.3 to exhibit improved clinical activity in the treatment of B-cell malignancies compared to rituximab.

Variable Contribution of Monoclonal Antibodies to ADCC in patients with chronic lymphocytic leukemia

Monoclonal antibodies (mAbs) are increasingly used in treatment protocols for chronic lymphocytic leukemia (CLL). Here we determined (i) the extent of antibody-dependent cellular cytotoxicity (ADCC) of four different mAbs against primary CLL cells, (ii) whether ADCC correlates with antigen density on CLL cells, and (iii) whether allogeneic natural killer (NK) cells display superior ADCC than autologous. Effector cells for ADCC were (i) NK-92 cells not expressing FcR, (ii) NK-92 cells transfected with a high-affinity Fc receptor, (iii) autologous NK cells from patients with CLL, (iv) allogeneic NK cells. Results suggest that ADCC contributes to killing of CLL cells by anti-CD20 antibodies (rituximab and veltuzumab), whereas mAbs against CD22 (epratuzumab) and CD23 (lumiliximab) showed minimal ADCC. The magnitude of anti-CD20 mediated ADCC did not correlate with antigen density of CD20. ADCC was not influenced by the FcR genotype expressed by autologous NK cells. Allogeneic NK cells were superior to autologous NK cells in killing primary CLL cells.

Novel humanized anti-CD20 monoclonal antibodies with unique germline VH and VL gene recruitment and potent effector functions

The anti-CD20 chimeric monoclonal antibody (mAb) rituximab is the most widely used therapeutic antibody for B-cell malignancies. However, ≈50% of non-Hodgkin's lymphoma (B-NHL) patients respond to treatment with this antibody. Novel humanized antibodies target membrane CD20 with enhanced effector properties should improve treatment for a broader patient population with relapsed and refractory disease. A novel chimerized form of the murine anti-CD20 1K1791 exerts more potent antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities and induces cell death by a non-caspase dependent process. Humanized mAbs derived from 1K1791 were designed using four different humanization techniques and characterized. In contrast to rituximab or 2F2 (human anti-CD20 mAb), several of these exhibited superior ADCC, CDC, inhibition of cell growth and cell death. There was a wide range of functional differences among the humanized forms of 1K1791 despite a modest replacement of amino acid residues in the CDRs. To determine whether the superior activities exhibited by parental murine mAb 1K1791 were due to differences in VH and VL rearrangement, we analyzed its germline and compared it to other anti-CD20 mAbs. A remarkable conservation of VH and Vk (VL kappa) gene usage was observed in the murine anti-CD20 mAbs. 18/23 used the same germline gene J558.42 and 4/23 used closely related genes of the ‘J558’ group. Thus, 22/23 belonged to VH1 family. One exception was the mAb 1K1791, which was derived from the VH9.12 germline gene. 1K1791 was also unique in its use of a Vk19/28 family gene whereas most other mAbs (21/23) used Vk4/5 family genes. A formal relationship between the particular germline gene recruitment and antibody functionality has not been established, however, the present findings identified humanized mAbs with functional activities that were superior to rituximab and 2F2. These in vitro results support future in vivo animal testing and subsequent clinical trials.

Anti-neuroblastoma effect of ch14.18 antibody produced in CHO cells is mediated by NK-cells in mice

Successful treatment of stage 4 neuroblastoma remains a major challenge in pediatric oncology. In order to improve the outcome, passive immunotherapy using human–mouse chimeric monoclonal anti-disialoganglioside GD2 antibody ch14.18 has been evaluated in early phase clinical trials with promising results in progressing stage 4 neuroblastoma patients. In preparation of European phase III clinical trial (HR-NBL-1/ESIOP), the cell line used for production of ch14.18 was changed. Specifically, the plasmid encoding for ch14.18 antibody was recloned into CHO cells. Here, we report the in vitro and in vivo anti-neuroblastoma activity of antibody ch14.18 produced in CHO cells (ch14.18/CHO) compared to that of ch14.18 manufactured from SP2/0 (ch14.18/SP2/0) and NS0 cells (ch14.18/NS0). First, we demonstrate identical binding of ch14.18/CHO to the nominal antigen disialoganglioside GD2 in vitro compared to ch14.18/SP2/0 and ch14.18/NS0. Binding was GD2-specific, since all precursor- and metabolite-gangliosides of GD2 tested were not recognized by ch14.18/CHO. Second, the functional properties of ch14.18/CHO were determined in complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) reactions against GD2 positive neuroectodermal tumor cell lines in vitro. There was no difference in CDC mediated specific tumor cell lysis among the three different ch14.18 antibody preparations. Interestingly, ch14.18/CHO showed superior ADCC activity at low antibody concentrations. Third, the efficacy of ch14.18/CHO was evaluated in the NXS2 neuroblastoma model in vivo. Importantly, the ch14.18/CHO preparation was effective in suppression of experimental liver metastasis in this model. In vivo depletion of NK-cells completely abrogated this effect, suggesting that the mechanism involved in the ch14.18/CHO induced anti-neuroblastoma effect is mediated by NK-dependent ADCC.

Novel humanized anti-CD20 monoclonal antibodies with unique germline VH and VL gene recruitment and potent effector functions

The anti-CD20 chimeric monoclonal antibody (mAb) rituximab is the most widely used therapeutic antibody for B-cell malignancies. However, approximately 50% of non-Hodgkin's lymphoma (B-NHL) patients respond to treatment with this antibody. Novel humanized antibodies target membrane CD20 with enhanced effector properties should improve treatment for a broader patient population with relapsed and refractory disease. A novel chimerized form of the murine anti-CD20 1K1791 exerts more potent antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities and induces cell death by a non-caspase dependent process. Humanized mAbs derived from 1K1791 were designed using four different humanization techniques and characterized. In contrast to rituximab or 2F2 (human anti-CD20 mAb), several of these exhibited superior ADCC, CDC, inhibition of cell growth and cell death. There was a wide range of functional differences among the humanized forms of 1K1791 despite a modest replacement of amino acid residues in the CDRs. To determine whether the superior activities exhibited by parental murine mAb 1K1791 were due to differences in VH and VL rearrangement, we analyzed its germline and compared it to other anti-CD20 mAbs. A remarkable conservation of VH and Vk (VL kappa) gene usage was observed in the murine anti-CD20 mAbs. 18/23 used the same germline gene J558.42 and 4/23 used closely related genes of the 'J558' group. Thus, 22/23 belonged to VH1 family. One exception was the mAb 1K1791, which was derived from the VH9.12 germline gene. 1K1791 was also unique in its use of a Vk19/28 family gene whereas most other mAbs (21/23) used Vk4/5 family genes. A formal relationship between the particular germline gene recruitment and antibody functionality has not been established, however, the present findings identified humanized mAbs with functional activities that were superior to rituximab and 2F2. These in vitro results support future in vivo animal testing and subsequent clinical trials.