ALBERT

Description: Immunotherapy targeting individual antigens in acute myeloid leukemia (AML) has shown promise. However, in view of leukemia heterogeneity and the loss of tumor antigen expression by AML, it is unlikely that any single antigen will be consistently expressed by all leukemia cells. This highlights the need to identify additional antigens in AML that can be targeted. Azurophil granule proteases have been shown to be effective immunotherapeutic targets. Proteinase 3 and neutrophil elastase, the parent proteins for the HLA-A2 restricted peptide PR1, have been targeted successfully in myeloid leukemia using immunotherapy. We recently discovered the HLA-A2 restricted peptide CG1 (FLLPTGAEA), which is derived from the azurophil granule protease cathepsin G (CG). We showed that CG is highly expressed by AML blasts and leukemia stem cells in a limited number of AML samples and showed that CG1 can be targeted in vitro using CG-specific cytotoxic T lymphocytes (CTL). We sought to determine whether CG1 can be targeted in vivo and to characterize the expression of CG in a large cohort of AML patients. To assess the efficacy of targeting CG1 in vivo, we used a NOD scid gamma (NSG) mouse model engrafted with the human HLA-A2+ AML cell line U937 (U937-A2), which is known to express CG. Mice were injected with U937-A2 (0.5 x 106) cells and on the following day were treated with either CG1-CTL (0.25 x 106), negative control HIV-CTL expanded from the same donor or were left untreated. Mice treated with CG1-CTL demonstrated a significantly greater reduction in U937-A2 in the bone marrow (BM) (8% residual AML; P

Description: Abstract 3030 Poster Board II-1006 Introduction NK cells have therapeutic potential for a wide variety of human malignancies. The major obstacle for adoptive NK cell immunotherapy is obtaining sufficient cell numbers, as these cells represent a small fraction of peripheral white blood cells, expand poorly ex vivo, and have limited life spans in vivo. Common gamma-chain cytokines are important in NK cell activation, maturation, and proliferation. Others have described improved ex vivo expansion of NK cells using soluble cytokines, when cocultured with stimulated peripheral blood mononuclear cells (PBMC) or Epstein Barr Virus (EBV) lymphoblastioid cell lines, or with artificial antigen presenting cells (aAPC) engineered with costimulatory molecules and/or membrane-bound IL-15 (mIL-15). Expansion of NK cells by these methods has been limited by senescence from telomere shortening. To generate clinical-grade T cells for adoptive transfer, our group developed aAPC derived from K562 retrovirally transduced to express the costimulatory molecules CD86 and CD137L. These aAPC were produced as a master cell bank and further genetically modified to express membrane-bound cytokines. Since IL-21 signals via STAT3, and STAT3 is a known activator of telomerase transcription, we investigated whether NK cell expansion with mIL-21 would provide a sustained proliferative advantage over or in combination with mIL-15. Methods K562 aAPC were retrovirally transduced to express CD64, CD86, CD137L, CD19 (Clone 9), and mIL-15 (Clone 4). These clones were further modified by Sleeping Beauty integration of mIL-21 (Clone 9+IL-21 and Clone 4+IL-21). Freshly isolated PBMC from 5 donors were co-cultured with irradiated K562 aAPC (Clone 4, Clone 4+mIL-21, and Clone 9+mIL-21) at a ratio of 2:1 (aAPC:PBMC) in the presence of 50 IU/ml of rhIL-2. Half of the media was changed every two days and cells were re-stimulated with aAPC every seven days at ratio of 2:1. Cells were counted and phenotyped on day 0, 7, 14, and 21 for CD3, CD16, CD56, NKG2D, KIR (2DL1, 2DL2/3, and 3DL1), and NCR (NKp30, NKp44, NKp46). A preclinical SOP to expand PBMC from a 20 mL blood draw was established and additional donors of known HLA type were expanded with Clone 9+mIL-21 for up to 7 weeks. Cytotoxicity function against K562, 721.221, Raji, and AML targets was measured using the Calcien-AM assay (Invitrogen). Telomere length of expanded and fresh NK cells was measured with the FlouFish assay using the telomere specific FITC conjugated (C3TA2)3 PNA probe. Results By day 14, aAPCs bearing mIL-21 induced greater total cell expansion than those with mIL-15 alone (188, 2900, and 2281-fold for Clone 4, Clone 4+mIL-21, and Clone 9+mIL-21, respectively). However, PBMC cultured without mIL-15 contained far fewer co-expanding T cells. Exponential expansion continued for up to 7 weeks without evidence of senescence when mIL-21 was present, reaching a mean of 91,566-fold expansion of the CD3−CD16/56+ population at 4 weeks. NK cells expanded with mIL-21 had increased expression of KIR and NCR, and expressed very high CD16 and NKG2D levels. These NK cells showed much higher cytotoxicity against all targets than fresh NK cells, retained KIR inhibition, and demonstrated enhanced killing via ADCC. Furthermore, telomere lengths of NK cells expanded with Clone 9+mIL-21 were longer than that of fresh NK cells or those expanded without mIL-21, perhaps explaining the continued expansion without senescence. Thus, NK cell expansion is improved using aAPCs expressing mIL-21 rather than mIL-15. We are currently establishing a GMP-grade working cell bank of Clone 9+mIL-21 for use in clinical trials. Funding: Brenda and Howard Johnson Fund, UT MD Anderson Physician Scientist Program Disclosures No relevant conflicts of interest to declare.

Description: Key Points Fc-engineered mAb promotes NK cell ADCC via better activation, serial killing, and kinetic boosting at higher target cell densities. Enhanced target killing also increased frequency of NK cell apoptosis, but this effect is donor-dependent.

Description: Abstract 3629 Acute myeloid leukemia (AML) is an aggressive malignancy for which current therapy fails to provide durable remission in approximately half of cases. Natural killer (NK) cells, as a key component of innate immunity, have recently shown clinical potential for adoptive immunotherapy against AML, particular when the donor and recipient are KIR mismatched. In addition to patients who do not have a suitable related donor, approximately 30% of patients bear all three families of KIR ligands and therefor cannot benefit from KIR mismatch. Thus, finding a related donor with predicted KIR mismatch is a major obstacle for adoptive NK cell immunotherapy. The majority of peripheral blood NK cells express CD16a (FcγRIIIa), which is the most potent receptor among the activating receptors that NK cells posses. NK cells express CD16a in association with disulflde-linked homo- or hetero-dimers of FcRγ or CD3ζ. Clustering of CD16a initiated by binding to the Fc-portion of IgG1 or IgG3 that opsonize target cells induces signals strong enough to overcome KIR inhibition. Thus, combining NK cell adoptive immunotherapy with Abs against tumor antigens could help overcome the limitations of KIR mismatching. Indeed, many promising anticancer Abs have failed in clinical trials because of insufficient efficacy, which, at least in part, may result from low affinity CD16a binding. Indeed, it was shown that the affinity between Fc and FcγRs correlates with cytotoxicity in cell-based assays and that the Abs with optimized FcγR affinity induced strong cytotoxicity against targeted tumor cells. CD33 is expressed on the blast cells of most cases of AML and represents a suitable antigen for antibody-based therapies. Lintuzumab, an unconjugated, humanized anti-CD33 mAb (HuM195), failed to improve patient outcomes in two randomized trials when combined with conventional chemotherapy. Gemtuzumab ozogamicin, an anti-CD33 mAb conjugated to the calicheamicin, in combination with chemotherapy, improved survival in a subset of AML patients, but has been withdrawn from US market by safety concerns. We optimized the FcγR affinity of HuM195 mAb (mNuM195) by cloning into pMaz-IgH Herceptin recipient vector containing S239D, A330L, I332E mutations that, as previously shown, leads to significant improvement of IgG1 binding to CD16a. To generate control wild type variant (wHuM195) we cloned the variable domains of HuM195 into pMaz-IgH Herceptin. Plasmids were transfected into HEK293F, and Abs were purified from cell culture supernatant with protein A resin, eluted with glycine HCL, and then the samples were buffer exchanged into PBS pH 7.4 for long-term storage. This S239D-A330L-I332E triple mutation in Fc portion of IgG1 did not affect antigen-biding affinity for CD33 target protein but showed more than 14-fold higher binding to CD16a than the wild type variant. The mHuM195 Abs increased cytotoxic activity of expanded human NK cells in Calcein AM-release assay when used in concentration as low as 0.01 μg/ml to pretreat murine thymoma EL-4 cells gene-modified to express human CD33 (ADCC, Mean±SD: 38.7±2.25% vs 11.7±3.49% for optimized vs wild type HuM195, and 5±3.15% without Abs, E:T ratio 2:1). We obtained the similar results when using K562 as targets, which naturally express CD33. K562 cells pretreated with mHuM195 Abs induced degranulation in 34±5.25% of NK cells where wHuM195 did so only in 17±4.6% of NK cells. Thus, optimization of HuM195 Ab to improve CD16a affinity results in dramatic increases NK cell cytotoxic activity. Disclosures: No relevant conflicts of interest to declare.

Description: NK Cell subpopulations express tremendous diversity through polymorphisms, haplotypes, differential expression, and licensing of the Killer Immunoglobulin-like Receptors (KIR). KIR diversity affects both the predisposition to cancer, and the response to therapies such as hematopoietic stem cell transplantation. Clinical trials that take advantage of the anti-cancer properties of NK cells have been limited to choosing donors on the basis of KIR genotypes and/or HLA haplotypes. Moreover, adoptive immunotherapy approaches have been limited by low NK cell doses. The latter hurdle has been recently mitigated by methods for expanding clinical grade NK cells ex vivo. These approaches for growing large numbers of cells now enable investigation into selecting more potent NK cell subsets for increased therapeutic efficacy. We hypothesized that the desired KIR repertoire could be molded through inhibition of undesirable KIR populations by crosslinking with relevant anti-KIR antibodies during expansion with our previously described method, which produces a mean 30,000-fold expansion of NK cells in 3 weeks. First, we determined that maximum inhibition was obtained when anti-KIR antibodies were applied to previously activated NK cells, crosslinked with secondary antibody, and then restimulated for proliferation. Robust reduction of targeted KIR-positive populations could be achieved for each inhibitory KIR (Fig. 1). When pre-activated with anti-KIR2DL1 for one stimulation cycle, NK cells expressing this KIR were decreased by a median of 70.4% ± 19.3%. Similarly, KIR2DL2/3+ NK cells could be reduced by 56% ± 17.5%, and KIR3DL1+ NK cells could be reduced by 53.5% ± 16.3%. When anti-KIR antibodies were combined, similar suppression of multiple-KIR subpopulations was observed. Other NK cell receptors were not significantly affected during targeted KIR inhibition. We then assessed the resulting NK cell populations for degranulation responses to targets with selected HLA as KIR ligands. Inhibition of KIR-expressing subpopulations during expansion resulted in NK cell populations with enhanced degranulation against tumor cells expressing the HLA ligand of the targeted KIR. Importantly, the cytotoxicity of the bulk NK cell population against HLA-negative targets remained. These results indicate that KIR crosslinking during NK cell propagation enables significant reduction in the targeted KIR subpopulations, resulting in an NK cell population with a selective decrease in KIR inhibition. By utilizing antibody-controlled expansion for molding of the KIR repertoire according to patient HLA type, a personalized NK cell product may be produced with enhanced potency, improving NK cell immunotherapy. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points STAT3 directly regulates expression of NKG2D in NK cells. Defects in STAT3 signaling result in deficient NKG2D responses to cytokine.

Description: Abstract 2467 Poster Board II-444 Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. The main restrictions to developing NK cells for immunotherapy are the limited quantity of cells available for adoptive transfer and their relative resistance to gene transfer by any method. We have developed an efficient method to expand CD3-CD56+ primary NK cells in vitro using K562 artificial APCs expressing membrane-bound IL21. Here we have investigated the potential of these expanded human NK cells to be gene modified through electroporation of DNA and mRNA. Expanded NK cells were electroporated (Amaxa Nucleofector device, program X-01) with DNA or mRNA coding for the GFP reporter gene, and expression of the transgene was evaluated by flow cytometry. Analysis at 48 hours post electroporation revealed that the viability of NK cells electroporated with GFP mRNA was 78% and those electroporated with GFP DNA was 69%. When electroporated with DNA, 32% of the viable NK cells were positive for GFP but had heterogeneous expression level, whereas 98% of viable cells were positive for GFP following mRNA electroporation, with much more homogeneous GFP expression (Figure). Based on this success we further investigated the potential of expanded NK cells to be gene modified with a Sleeping Beauty transposon/transposase vector system carrying the transgene for a second-generation Chimeric Antigen Receptor (CAR) against the GD2 ganglioside antigen, with signaling via the CD28 and CD3z endodomains. The GD2 antigen is abundantly expressed in neuroblastoma and melanoma and is therefore a relevant target for adoptive immunotherapy. Electroporation of expanded NK cells with the GD2-CAR transposon alone yielded 25% electroporation efficiency, with a viability of 55%. Electroporation of expanded NK cells with the GD2-CAR transposon and the transposase plasmid decreased the transfection efficiency to 14%. Nonetheless, expanded NK cells modified with the GD2-CAR showed improved killing of the target cell CHP-134 using the calcein AM release assay, as compared to unelectroporated expanded NK cells from the same donor. While freshly isolated human NK cells are highly resistant to gene modification, in this study we show that expanded human NK cells can be efficiently electroporated with both DNA and mRNA. NK cells modified with DNA to express CAR gain improved cytotoxic function against target cells, but viability and gene transfer efficiency are low. Since electroporation of GFP mRNA resulted in increased transduction efficiency and viability, we are now evaluating electroporation of expanded NK cells with GD2-CAR mRNA. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2463 Poster Board II-440 Acute myeloid leukemia (AML) is an aggressive malignancy for which current therapy fails to provide durable remission in approximately half of cases. Natural killer (NK) cells, as a key component of innate immunty, have recently shown clinical potential for adoptive immunotherapy against AML, particular when the donor and recipient are KIR mismatched. In addition to patients who do not have a suitable related donor, approximately 30% of patients bear all three families of KIR ligands and therefor can not benefit from KIR mismatch. Thus, the major obstacles for adoptive NK cell immunotherapy are 1) obtaining sufficient numbers of NK cells for effective thereapy and 2) finding a related donor with predicted KIR mismatch. Clinical trials with humanized or engineered mAbs against CD33 have validated this antigen as a target for immunotherapy of AML, but are complicated by side effects such as a hepatotoxicity due to CD33 expression on normal hepatocytes. To address the first hurdle, we developed a method to expand CD3-CD56+ primary NK cells in vitro using artificial APCs expressing membrane-bound IL21, and have validated electroporation as an efficient method for gene modification of these NK cells. To address the second hurdle and expand the therapeutic potential of KIR-matched expanded NK cells, we hypothesized that gene transfer of CD33 Chimeric Antigen Receptor (CAR) could provide additional activation signal to increase the lysis of AML blasts by expanded NK cells, and sought to compare signaling endodomains for this purpose. CD3z is a signal adapter molecule for NKp30, NKp46, and CD16 in NK cells. We developed a CD33CAR composed of a CD33 single-chain variable fragment fused with the CD3z transmembrane domain expressed in Sleeping Beauty transposon vector system, and compared a first generation (CD3z only) endodomain with second generation endodomains (CD3z plus either CD28 or CD137). Transient gene transfer of the CD33CAR DNA into NK cells was accomplished using the Amaxa Nucleofector device. Functional expression of the CAR was determined by binding of a Siglec3-IgG fusion protein to the cell surface followed by secondary staining with anti-IgG-FITC. Cytotoxicity of the NK cells against CD33+ AML cells and CD33-transduced HEK293T cells was determined in a 4h lysis assay using Calcein-AM. While the maximum electroporation efficiency was only 15% at 24h, expression levels as low as 4% significantly increased the cytotoxic activity of NK cells compared to unelectroporated NK cells. Each of the CD33CAR constructs harboring different endodomains yielded an equivalent increase in target cell lysis (Figure). This data supports recent observations that signal transduction through CD3z is sufficient to activate cytotoxic activity in NK cells. However, to increase the percentage of CAR-expressing NK cells we are further evaluating the role of endodomain signaling in CAR-dependent proliferation of NK cells electroporated with both transposon and transposase. Disclosures: No relevant conflicts of interest to declare.