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Phenotype of T-cells used in Xenograft Studies

Summary of MOR209/ES414 and scFv-scFv Effect on Survival of Mice from C4-2B Xenograft Study

Serum levels of human PSA from day 56 of the MDA-PCa-2b xenograft study (as shown in Figures 5C, 5D).

Supplementary Methods and Supplementary Figure Legends

A. Binding of MOR209/ES414 or human IgG1 control on representative CHO cell lines expressing different Fcγ receptors monitored by flow cytometry and plotted as median fluorescence intensity (MFI). B. Cell divisions observed in proliferating CD4+ and CD8+ T cells induced by MOR209/ES414 (0, 1, 10 nM) in the presence of target (C4-2B cells) visualized by loss of CFSE fluorescence. C. T-cell proliferation induced by 1 nM MOR209/ES414 in the presence of C4-2B cells with the number of cell divisions (0-7) indicated.

Estimated PSMA Receptor Densities and Relative Potency of MOR209/ES414

A. Relative cytokine release observed after \ 24 hours from T cells in the presence of bispecific molecules (5 nM) and C4-2B target cells. Data plotted is within the limit of detection of the assay. B, C. Activation and cytokine release induced by the anti-PSMA x I2C ADAPTIR control molecule. PBMC were stimulated for 20 hrs in the presence of serial dilutions of C4-2B with tandem scFv-scFv (scFv-scFv), the hybrid ADAPTIR molecule or MOR209/ES414 as described before. B. Activation was assessed by measuring the fraction of CD4+ or CD8+ T cells upregulating CD69. C. Cytokine secretion was measured in the culture supernatants. The graphs show levels of IL-2, and IFN-γ; similar trends were observed for TNFα, GM-CSF, IL-4, IL-5 IL-10, IL-13 and IL-17. The figure shows representative results obtained from two independent experiments, each including 2 different human PBMC donors.

Summary of Median Survival Statistics from C4-2B Xenograft Study

Tumor associated FOXP3+ regulatory T cells (Tregs) suppress antitumor immunity, thereby hindering protective immunosurveillance of tumors and hampering effective antitumor immune responses in tumor-bearing hosts. Eliminating the suppressive effects of tumor associated Tregs could, therefore, enhance antitumor activity of existing immunotherapies. CCR8 is a seven transmembrane G-coupled protein whose expression is highly upregulated on tumor associated Tregs in several types of tumors, but not on peripheral Tregs. The specific expression on tumor associated Tregs makes CCR8 an ideal target for treating cancers. The chemokine CCL1 interacts with the N terminus and second extracellular domain of CCR8. CCL1 produced by tumor associated macrophages can induce Tregs migration to the tumors. PSB114, a humanized anti-hCCR8 IgG1 antibody, was developed to block CCL1 mediated Tregs chemotaxis and deplete tumor associated CCR8-expressing Tregs. PSB114 was affinity matured by yeast display and glyco-modified to enhance its ADCC activity. PSB114 has high binding affinity for human CCR8 without significant binding to other membrane proteins, resulting in a favorable PK in hFcRn transgenic mice (T1/2 > 13 days). PSB114 is highly specific for the activated Tregs with low picomolar EC50 of depletion activity. When tested in bioassays, PSB114 enhances T cell activation and TNFα production from activated PBMCs. In a humanized mouse model, PSB114 preferentially depletes tumor infiltrating Tregs and increases NK and CD8 T cell infiltration within the tumor. Moreover, PBS114 significantly inhibited MC38 tumor growth in hCCR8 knock-in mice. These results demonstrate the potential of PSB114 for treating cancers in human. Citation Format: Jamil A. Haque, Cristina Domeier, Paul A. Algate, Zhi Liu, Wei Yan, YuFeng Peng. An ADCC enhanced anti-CCR8 antibody, which preferentially depletes tumor regulatory T cells and inhibits tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2068.

Treatment of metastatic, castration-resistant prostate cancer (mCRPC) remains a highly unmet medical need and current therapies ultimately result in disease progression. Immunotherapy is a rapidly growing approach for treatment of cancer but has shown limited success to date in the treatment of mCRPC. We have developed a novel humanized bispecific antibody, MOR209/ES414, built on the ADAPTIR (modular protein technology) platform, to redirect T-cell cytotoxicity toward prostate cancer cells by specifically targeting T cells through CD3ϵ to prostate cancer cells expressing PSMA (prostate-specific membrane antigen). In vitro cross-linking of T cells with PSMA-expressing tumor cells by MOR209/ES414 triggered potent target-dependent tumor lysis and induction of target-dependent T-cell activation and proliferation. This activity occurred at low picomolar concentrations of MOR209/ES414 and was effective at low T-effector to tumor target cell ratios. In addition, cytotoxic activity was equivalent over a wide range of PSMA expression on target cells, suggesting that as few as 3,700 PSMA receptors per cell are sufficient for tumor lysis. In addition to high sensitivity and in vitro activity, MOR209/ES414 induced limited production of cytokines compared with other bispecific antibody formats. Pharmacokinetic analysis of MOR209/ES414 demonstrated a serum elimination half-life in NOD/SCID γ (NSG) mice of 4 days. Administration of MOR209/ES414 in murine xenograft models of human prostate cancer significantly inhibited tumor growth, prolonged survival, and decreased serum prostate-specific antigen levels only in the presence of adoptively transferred human T cells. On the basis of these preclinical findings, MOR209/ES414 warrants further investigation as a potential therapeutic for the treatment of CRPC. Mol Cancer Ther; 15(9); 2155–65. ©2016 AACR.

Unlike HER2-positive breast cancer, there are limited treatment options for patients diagnosed with triple negative and endocrine resistant HER2-negative breast cancer, and as such HER2-negative breast cancer represents a significant unmet medical need. The goal of this work is to use Theraclone’s proprietary I-STAR platform to mine the memory B cell immune repertoire of breast cancer patients for the discovery of therapeutically relevant monoclonal antibodies (mAbs) and targets that may be exploited as candidates for treatment of HER2-negative breast cancer. Matched serum and PBMC samples were collected from breast cancer patients at multiple clinical sites. The selected patient populations included, but were not limited to, patients who were treatment naïve, those who had received immunotherapy or adjuvant chemotherapy, and patients who had an exceptional clinical response to treatment and/or disease. Serum antibody binding to a diverse panel of 5 well characterized breast cancer-derived cell lines of luminal and basal sub-types was determined. Utilizing this approach, patients with a robust serological profile across multiple breast cancer cell lines were identified and prioritized for memory B cell repertoire analysis via the I-STAR platform. Using a high throughput and miniaturized, multiplex flow cytometry assay, the secreted IgG antibodies from over 85,000 individually enriched and expanded B cell clones were screened for binding to the tumor cell line panel and a large number of positive B cell clones were identified. The screening hits can be binned into several unique binding profiles, many of which were confirmed to be shared across multiple patient samples. Deep sequence analysis of the variable regions of the antibodies produced by the B cell clones demonstrated that several of the screening hits were derived from clonally related B cells; the majority of the screening hits represented antibodies derived from unique B cell clones. A representative set of these antibodies was expressed recombinantly for further in vitro characterization. Citation Format: Christy Boozer, Paul Algate, Aurelio Bonavia, Mark Branum, Po-Ying Chan-Hui, Alison Fitch, Brad Greenfield, Claire Sutherland, Kristine Swiderek. Discovery of fully human monoclonal antibodies as therapeutic candidates for the treatment of HER2-negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-16-01.

Epidemiological studies have shown that consumption of red meat increases the risk of developing colon cancer. An enzyme, α-methylacyl CoA racemase (AMACR), also known as P504S, plays an important role in peroxisomal β-oxidation of branched-chain fatty acids from red meat and dairy products. High expression of AMACR was recently found in prostate cancer. In this study, we investigated expression of AMACR in 242 cases of colonic tumors including 176 colorectal carcinomas, 38 colon adenomas and 28 hyperplastic (non-neoplastic) polyps by immunohistochemical analysis. The mRNA levels of AMACR expression in normal and colon cancer tissues were assessed by real-time PCR. Significant up-regulation of AMACR mRNA was found in colon carcinomas compared to normal tissue. There was very low or no expression of AMACR protein in normal colon, but AMACR was highly expressed in 76 and 75% of well and moderately differentiated colon carcinomas, respectively, and in 79% of adenomas. In contrast, only 4% of hyperplastic polyps expressed AMACR. Since this enzyme is involved in the metabolism of branched-chain fatty acids from beef, milk and dairy products, our results provide important molecular information regarding a possible link between diet and development of colon cancer. AMACR may also serve as a molecular marker for colon cancers and its precursor lesions.

The SH2 domain containing inositol 5'-phosphatase (SHIP) was initially described as a 145-kDa protein phosphorylated on tyrosines upon growth factor and cytokine stimulation. It was shown to be phosphorylated after Fc and B cell receptor activation and plays a role in negative signaling. Different isoforms of the SHIP protein result from alternative mRNA splicing, proteolysis, or a combination of both. The expression of discrete SHIP isoforms changes with the potential developmental-dependent maturation state of myeloid cells, suggesting mechanisms for the regulation of SHIP interactions with other signaling molecules. A p135 (SHIPbeta) spliced isoform is known to be expressed in developing myeloid cells. Now we have identified a new SHIP isoform, SHIPdelta, which is the product of an out-of-frame splice with a deletion of 167 nucleotides in the C-terminal region, resulting in an approximately 110-kDa protein. Biochemically, SHIPdelta differs from SHIPalpha by exhibiting little or no tyrosine phosphorylation or association with the signaling protein Shc after M-CSF activation of FD-Fms cells. In addition, we have characterized the structure of the entire SHIP genomic locus, which provides a basis for understanding the alternative splicing events. SHIP is expressed in hematopoiesis and spermatogenesis, and we also describe the promoter for the SHIP gene, which has potential for explaining the tissue-specific expression pattern.

The macrophage colony-stimulating factor receptor and several other hematopoietic growth factor receptors induce the tyrosine phosphorylation of a 145- to 150-kD protein in murine cells. We have previously cloned a cDNA for the murine 150-kD protein, SHIP, and found that it encodes a unique signaling intermediate that binds the SHC PTB domain through at least one tyrosine phosphorylated (NPXY) site in the carboxyl-terminal region. SHIP also contains several potential SH3 domain-binding sites, an SH2 domain for binding other tyrosine phosphorylated proteins, and an enzymatic activity that removes the phosphate from the 5 position of phosphatidylinositol 3,4,5-phosphate or from inositol 1,3,4,5-phosphate. SHIP has a negative effect on cell growth and therefore loss or modification may have profound effects on hematopoietic cell development. In this study, we have cloned a cDNA for human SHIP and examined mRNA and protein expression of SHIP and related species in bone marrow and blood cells. Flow cytometry indicates that at least 74% of immature CD34+ cells express SHIP cross-reacting protein species, whereas within the more mature population of CD33+ cells, only 10% of cells have similar expression. The majority of T cells react positively with the anti-SHIP antibodies, but significantly fewer B cells are positive. Immunoblotting detects up to seven different cross-reacting SHIP species, with peripheral blood mononuclear cells exhibiting primarily a 100-kD protein and a CD34+ acute myeloblastic leukemia expressing mainly 130-kD and 145-kD forms of SHIP. Overall, these results indicate that there is an enormous diversity in the size of SHIP or SHIP-related mRNA and protein species. Furthermore, the expression of these protein species changes according to both the developmental stage and differentiated lineage of the mature blood cell.

The normal proto-oncogene c-fms encodes the macrophage growth factor (M-CSF) receptor involved in growth, survival, and differentiation along the monocyte-macrophage lineage of hematopoietic cell development. A major portion of our research concerns unraveling the temporal, molecular, and structural features that determine and regulate these events. Previous results indicated that c-fms can transmit a growth signal as well as a signal for differentiation in the appropriate cells. To investigate the role of the Fms tyrosine autophosphorylation sites in proliferation vs. differentiation signaling, four of these sites were disrupted and the mutant receptors expressed in a clone derived from the myeloid FDC-P1 cell line. These analyses revealed that: (1) none of the four autophosphorylation sites studied (Y697, Y706, Y721, and Y807) are essential for M-CSF-dependent proliferation of the FDC-P1 clone; (2) Y697, Y706, and Y721 sites, located in the kinase insert region of Fms, are not necessary for differentiation but their presence augments this process; and (3) the Y807 site is essential for the Fms differentiation signal: its mutation totally abrogates the differentiation of the FDC-P1 clone and conversely increases the rate of M-CSF-dependent proliferation. This suggests that the Y807 site may control a switch between growth and differentiation. The assignment of Y807 as a critical site for the reciprocal regulation of growth and differentiation may provide a paradigm for Fms involvement in leukemogenesis, and we are currently investigating the downstream signals transmitted by the tyrosine-phosphorylated 807 site. In Fms-expressing FDC-P1 cells, M-CSF stimulation results in the rapid (30 sec) tyrosine phosphorylation of Fms on the five cytoplasmic tyrosine autophosphorylation sites, and subsequent tyrosine phosphorylation of several host cell proteins occurs within 1-2 min. Complexes are formed between Fms and other signal transduction proteins such as Grb2, Shc, Sos1, and p85. In addition, a new signal transduction protein of 150 kDa is detectable in the FDC-P1 cells. The p150 is phosphorylated on tyrosine, and forms a complex with Shc and Grb2. The interaction with Shc occurs via a protein tyrosine binding (PTB) domain at the N-terminus of Shc. The p150 is not detectable in Fms signaling within fibroblasts, yet the PDGF receptor induces the tyrosine phosphorylation of a similarly sized protein. In hematopoietic cells, this protein is involved in signaling by receptors for GM-CSF, IL-3, KL, MPO, and EPO. We have now cloned a cDNA for this protein and found at least one related family member. The related family member is a Fanconia Anemia gene product, and this suggests potential ways the p150 protein may function in Fms signaling.

Previously, we characterized the transposition of an intracisternal type A particle (IAP) to the 32 untranslated region (UTR) of the interleukin-3 (IL-3) gene, which displaced two of the six AUUUA motifs associated with mRNA stability in an IL-3-secreting clone. To determine whether this rearrangement was involved in the autocrine transformation of the parental IL-3-dependent FL5.12 cell line, the germline (gIL-3) and rearranged IL-3 (rIL-3) genes were isolated and subcloned into a gene transfer vector. Moreover, the IAP-long terminal repeat (LTR) and the IL-3 32 UTR AUUUA motifs were deleted (rIL-3 + delta LTR and gIL-3 + delta AUUUA) in some IL-3 constructs to ascertain their role in the transformation process. The IAP-LTR was also added to these constructs (rIL-3 + delta LTR + IAP-LTR, gIL-3 + delta AUUUA + IAP-LTR, and gIL-3 + IAP-LTR), to determine whether it was necessary for autocrine transformation. The ability of the modified IL-3 genes to abrogate the IL-3 dependency of FL5.12 cells had the following rank order: rIL-3 was greater than rIL-3 + delta LTR + IAP-LTR, which was greater than gIL-3 + delta AUUUA + IAP-LTR, which was greater than gIL-3 + delta AUUUA, which was equal to rIL-3 + delta LTR, which was greater than gIL-3. The half-life of IL-3 mRNA was 20-fold longer in cells containing a mutated as opposed to a wild-type AUUUA region. All of the factor-independent cells that expressed the IL-3 transgenes secreted IL-3 and were tumorigenic after injection into BALB/c nude mice. These results indicated that two events could synergize in the autocrine transformation of hematopoietic cells: (1) addition of a transcriptional enhancer present in a retroviral LTR, and (2) disruption of an mRNA stability region.

The IgG+ memory B cell repertoire of cancer patients demonstrating durable responses to immune checkpoint blockade therapy has been interrogated to identify tumor specific antibodies. Matched serum and PBMC samples have been collected from 10 melanoma patients with durable responses to treatment with ipilimumab. A panel of 7 well characterized melanoma cell lines with a diverse set of oncogenic driver mutations has been employed to assess durable responder patient serum reactivity to cell surface determinants in a live-cell flow cytometry assay. IgG+ memory B cells obtained from several prioritized durable responder melanoma patients have been plated out on 384 well plates at monoclonal density. The memory B cells have been activated in a short term culture system optimized to expand the clonal B cells and induce secretion of IgG. Antibodies secreted from clonally expanded B cells have been screened using live cell high throughput multiplex flow cytometry assays against a panel of melanoma cell lines. A prioritized subset of antibodies, selected on the basis of tumor cell line binding profiles and antibody v-region sequences, has been cloned and expressed as recombinant IgG1. A cell surface target for a prioritized antibody has been identified and the in vitro analysis and characterization of the antibody including functional activity and immunohistochemistry staining on tumor and normal tissues has been carried out in preparation for in vivo testing of efficacy. Citation Format: Mark Branum, Laura Smith, Heather Brett, Paul Algate, Brad Greenfield, William Brown, Kristine M. Swiderek. Derivation and characterization of antibodies from immune checkpoint blockade treated cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 870.

To determine the effects of a cytokine on cognate receptor expression in normal and neoplastic cells, the interleukin-3 receptor (IL-3R) complex was examined in the parental IL-3-dependent line FL5.12, which was isolated from fetal liver, and in autocrine- and v-abl-transformed derivative lines. IL-3 decreased the amount of the IL-3R alpha and beta chains detected on the cell surface of the parental IL-3-dependent cells. In contrast, high levels of IL-3R beta were constitutively detected on the autocrine-transformed lines in the absence and presence of exogenous IL-3. Only low levels of IL-3R beta were observed in the two v-abl-transformed derivative cell lines examined, which no longer required IL-3 for growth. The levels of the IL-3R alpha chain detected were similar in these transformed cells and were not regulated by IL-3. These results were substantiated further by RNA analysis, because IL-3 decreased the levels of IL-3R transcripts in the parental factor- dependent FL5.12 line. The pattern of IL-3R gene expression was opposite to that of other receptors or proto-oncogenes, because RNA transcripts for all other genes examined were induced by IL-3. We conclude that IL-3 tightly controls IL-3R expression in the IL-3- dependent FL5.12 cells, whereas steady-state mRNA levels were not altered in the two v-abl-transformed derivative cell lines examined in this study.

SummaryTetraspanins are commonly believed to act only as “molecular facilitators,” with no direct role in signal transduction. We herein demonstrate that upon ligation, CD37, a tetraspanin molecule expressed on mature normal and transformed B cells, becomes tyrosine phosphorylated, associates with proximal signaling molecules, and initiates a cascade of events leading to apoptosis. Moreover, we have identified two tyrosine residues with opposing regulatory functions: one lies in the N-terminal domain of CD37 in a predicted “ITIM-like” motif and mediates SHP1-dependent death, whereas the second lies in a predicted “ITAM motif” in the C-terminal domain of CD37 and counteracts death signals by mediating phosphatidylinositol 3-kinase-dependent survival.

Although insulin-like growth factor (IGF-1) stimulated 3H-thymidine incorporation upon addition to the interleukin-3 (IL-3)-dependent cell line FDC-P1, IGF-1 did not relieve IL-3 dependency for growth. To further examine the effects of IGF-1 on hematopoietic cells, FDC-P1 cells were infected with a retroviral construct (LISN) containing the human IGF-1 receptor (hIGF-1R) and neo genes. IL-3-independent cells were readily isolated after LISN infection when either IGF-1 or supraphysiologic concentrations of insulin were included in the culture medium. These cells were transformed to IL-3 independence by a ligand- dependent mechanism because their growth was dependent on the presence of either IGF-1 or insulin and growth factors capable of supporting autocrine growth were not detected. Furthermore, a monoclonal antibody (MoAb) directed against the human IGF-1R (alpha IR-3) inhibited IGF-1 but not IL-3-induced proliferation and these cells contained 20- to 200- fold more IGF-1 receptors than uninfected FDC-P1 cells. In contrast, when LISN-infected cells were plated in medium without exogenously supplied IGF-1 or insulin, factor-independent cells were rarely isolated. Growth of these cells was also inhibited by the alpha IR-3 MoAb and they expressed 100- to 400-fold more IGF-1 receptors than uninfected FDC-P1 cells. The endogenous IGF-1 and/or insulin present in the calf serum may have enabled their growth because these cells, unlike the parental cells, would proliferate in serum-free defined media and their growth was again inhibited by the alpha IR-3 MoAb. These results demonstrate that IGF-1 can replace IL-3 for growth when FDC-P1 cells overexpress the IGF-1R. Given the fairly ubiquitous expression of the IGF-1 receptor, these and additional experiments might help to determine whether increased expression of endogenous receptors by cells can lead to leukemogenesis and tumorigenesis. Moreover, hIGF-1R-infected cells will be useful in investigating the mechanisms of IGF1-mediated signal transduction because they are now known to proliferate in response to IGF-1.

The BCR/ABL oncogene causes chronic myelogenous leukemia (CML), a myeloproliferative disorder characterized by clonal expansion of hematopoietic progenitor cells and granulocyte lineage cells. The SH2-containing inositol-5-phosphatase SHIP is a 145-kDa protein which has been shown to regulate hematopoiesis in mice. Targeted disruption of the murine SHIP gene results in a myeloproliferative syndrome characterized by a dramatic increase in numbers of granulocyte-macrophage progenitor cells in the marrow and spleen. Also, hematopoietic progenitor cells from SHIP(-/-) mice are hyperresponsive to certain hematopoietic growth factors, a phenotype very similar to the effects of BCR/ABL in murine cells. In a series of BCR/ABL-transformed hematopoietic cell lines, Philadelphia chromosome (Ph)-positive cell lines, and primary cells from patients with CML, the expression of SHIP was found to be absent or substantially reduced compared to untransformed cell lines or leukemia cells lacking BCR/ABL. Ba/F3 cells in which expression of BCR/ABL was under the control of a tetracycline-inducible promoter showed rapid loss of p145 SHIP, coincident with induction of BCR/ABL expression. Also, an ABL-specific tyrosine kinase inhibitor, CGP57148B (STI571), rapidly caused reexpression of SHIP, indicating that BCR/ABL directly, but reversibly, regulates the expression of SHIP protein. The estimated half-life of SHIP protein was reduced from 18 h to less than 3 h. However, SHIP mRNA also decreased in response to BCR/ABL, suggesting that SHIP protein levels could be affected by more than one mechanism. Reexpression of SHIP in BCR/ABL-transformed Ba/F3 cells altered the biological behavior of cells in culture. The reduction of SHIP due to BCR/ABL is likely to directly contribute to the pathogenesis of CML.

Abstract 622 CD37 is a tetraspanin selectively expressed on normal and transformed B-cells with unknown function. A role for CD37 in signal transduction pathways affecting cell development and activation has been proposed. However, the direct involvement of CD37 in signaling has always been excluded due to short cytoplasmic tails that lack canonic signaling motifs. Given its B-cell selectivity CD37 is a candidate therapeutic target for B-cells malignancies including chronic lymphocytic leukemia (CLL). Small Modular ImmunoPharmaceutical proteins targeting CD37 such as SMIP-016 have already demonstrated clinical activity and yet their mechanism of killing has not been described. Despite the common belief that tetraspanins only act as “molecular facilitators”, with no direct role in signal transduction, we demonstrate a completely new role for CD37. Analysis of CD37 sequence revealed the presence of two tyrosine residues in cytoplasmic tails, Tyr13 within a predicted weak ITIM-motif, (known to recruit inhibitory signaling effectors such as SHP1) and Tyr274 within a single tyrosine ITAM-like motif. To study the relevance of each of these tyrosine residues to SMIP-016 induced cell death we generated 697 cell lines expressing wild type or mutant Flag-tagged CD37 [Tyr13(−) or Tyr274(−)] and demonstrated by biochemical and proteomic analysis that upon ligation CD37 becomes phosphorylated, associates to the membrane lipid rafts, recruits pLyn and pSHP1 at the Tyr13 and initiates a cascade of events leading to mitochondrial membrane depolarization (MMD) and apoptosis. Down regulation of SHP1 by siRNA or Sodium Stibogluconate (10ug/ml) resulted in almost complete loss of SMIP-016-induced cytotoxicity (p Disclosures: Algate: emergent biosolutions: Employment. Stromatt:Emergent Product Development Seattle, LLC, Seattle, WA: Employment.

Abstract 3931 Background: CD37 is a 50–55 kDa heavily glycosylated member of the tetraspanin superfamily of molecules. This cell surface protein is expressed on normal and transformed B-cells, and has been implicated in diverse processes including cellular activation and proliferation, cell motility, and cell-cell adhesion. TRU-016 is a novel humanized anti-CD37 SMIP™ protein. Pre-clinical studies have demonstrated that anti-CD37 SMIP™ protein mediates caspase-independent direct killing of normal and malignant B-cells, a mechanism of action that appears to be different than CD20 therapies. In addition, TRU-016 results in indirect killing through NK cell mediated SMIP-protein directed cellular cytotoxicity (SDCC). The therapeutic potential of TRU-016 against several subsets of B-cell malignancies is currently being investigated in the clinic. Methods: The ability of TRU-016 to interact and increase cell killing with established therapeutics rituximab (anti-CD20 antibody), bendamustine (bi-functional alkylating agent/nucleoside analog), LY294002 (PI3K inhibitor) and temsirolimus (mTOR inhibitor) was investigated in vitro using the Rec-1 (mantle cell lymphoma) and SU-DHL-6 (diffuse large B cell lymphoma) cell lines. Individual drugs were tested in combination with TRU-016 as well as in a multiple drug cocktail. Combination index analyses were performed for drug combinations over the 20–90% effect levels. To determine whether in vitro synergy could be recapitulated in vivo, DoHH-2 (follicular lymphoma) xenografts were treated with TRU-016, bendamustine, and the combination of TRU-016 and bendamustine with or without rituximab. Furthermore, the effect of the dosing schedule with the combination of TRU-016 and rituximab was explored by comparing the treatment over a short time period to an extended (maintenance) dosing regimen. CD37 expression on the tumor xenografts was evaluated post different treatment by immunohistochemistry. Results: Combination index analyses determined that the killing effects of TRU-016 was synergistic with rituximab, bendamustine and temsirolimus in NHL models. Furthermore, TRU-016 provided additional efficacy when added to the combination of rituximab and bendamustine. In vivo results demonstrated that the in vitro synergy results were applicable to a more complex in vivo disease model. The combination of TRU-016 with bendamustine or rituximab resulted in increased tumor growth delay compared to that attained with the individual drugs. The addition of TRU-016 to the combination of bendamustine and rituximab resulted in increased tumor growth delay compared to the two drugs alone. The observed efficacy of the combination of TRU-016 and rituximab could be extended with repeated (maintenance) dosing with tumor free survival being observed beyond the 35 days of dosing. The combination of TRU-016 with temsirolimus also resulted in a reduction of tumor growth compared to either molecule alone. CD37 target expression was detected in the xenograft tumors post-treatment with all drugs tested. Conclusions: TRU-016 in combination with rituximab, bendamustine or temsirolimus increased cell killing of NHL cells in vitro over that observed for each agent alone. Furthermore, the triple combination of TRU-016 with rituximab, bendamustine or temsirolimus displayed greater anti-tumor activity in vivo than each of the agents alone against a follicular lymphoma tumor model. The addition of TRU-016 to a combination of rituximab and bendamustine resulted in increased killing in vitro and in vivo. The combinatorial activity of TRU-016 and rituximab in vivo was increased when the drugs were administered over a longer period. These results provide preclinical rationale for the potential different combinations of TRU-016 with several established therapeutics for the treatment of NHL and related B-cell malignancies. Disclosures: Algate: Trubion Pharmaceuticals: Employment. Wiens:Trubion Pharmaceuticals: Employment. Nilsson:Trubion Pharmaceuticals: Employment. Sho:Facet/Abbott: Employment. Chao:Facet/Abbott: Employment. Starling:Facet/Abbott: Employment. Gordon:Trubion Pharmaceuticals: Employment.

Abstract 1744 Poster Board I-770 CD37 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 expressed minimally or is absent on normal T-cells, natural killer 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) targeted towards the extracellular region of CD37, is presently in clinical trials in CLL patients. TRU-016 consists of variable regions (scFv) and engineered constant regions encoding the human IgG1 domains. We have previously reported that SMIP-016, the chimeric precursor of the fully humanized TRU-016, induced apoptosis in CLL B cells in the presence of goat anti-human Fc ab 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. Recently, in an attempt to enhance the ADCC function, a new variant of SMIP-016, Tru-ADhanCe SMIP-016, has been created with a modification of the glycosylation of the Fc portion of the molecule. TRU-ADhanCe SMIP-016 has been shown to exhibit enhanced binding to both low- and high-affinity molecular variants of human CD16 (FcRIII) and augmented ADCC potency when compared to SMIP-016. In this study, we compared TruADhanCe SMIP-016 and SMIP-016 in direct cytotoxicity and ADCC experiments using CLL B-cells. While SMIP-016, and TruADhanCe SMIP-016 mediated comparable direct cytotoxicity at 24, 48 and 72 hrs in the presence of anti-human Fc crosslinker, the TruADhanCe SMIP-016 resulted in 2 to 4 fold increased NK cell mediated ADCC function. Consistent with the comparable direct cytotoxic effects, the early phosphorylation patterns were similar in cells treated with TruADhanCe SMIP-016 or SMIP-016 in the presence of anti-human Fc cross linker. Ongoing studies are aimed to define the mechanistic basis of the enhanced ADCC function by TruADhanCe SMIP-016 and to determine if use of soluble CD16.Fc as a cross-linker, an in vitro model of in vivo Fc receptor binding, may reveal enhanced apoptotic-signaling of TruADhanCe SMIP-016. These results suggest potential use of TruADhanCe versions of TRU-016 with enhanced ADCC function as an alternate for TRU-016 in B cell malignancies including CLL therapy. [This work was supported by D. Warren Brown Foundation, Leukemia and Lymphoma Society and National Cancer Institute.] Disclosures Thompson: Trubion Pharmaceuticals: Employment. Siadak:Trubion Pharmaceuticals: Employment. Algate:Trubion Pharmaceuticals: Employment. Cerveny:Trubion Pharmaceuticals: Employment.