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 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 (SHIPβ) spliced isoform is known to be expressed in developing myeloid cells. Now we have identified a new SHIP isoform, SHIPδ, 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, SHIPδ differs from SHIPα 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.
Abstract 3722
Despite advances in treatments for B-cell leukemias and lymphomas, many patients ultimately relapse and succumb to disease following multiple courses of therapy. Bispecific antibody ...fragments that can simultaneously engage T cells and tumor cells have been shown, in the literature, to destroy tumor cells by effectively redirecting the cytotoxic function of T cells. T-cell engaging bispecific molecules linking anti-CD19 and anti-CD3 binding domains in the context of novel SCORPION™ (multi-specific protein therapeutic) proteins were evaluated both in vitro and in vivo for function and stability.
Redirected T-cell cytotoxicity (RTCC) was measured by combining CD19 positive or negative cell lines with SCORPION proteins in the presence of human T cells. In a similar assay context, CFSE-labeled T cells were monitored for activation and proliferation. Functional RTCC assays were also used to analyze serum stability of SCORPION molecules in vitro and to complete an in vivo pharmacokinetic analysis. In vivo efficacy was assessed by monitoring the rate of tumor outgrowth of Ramos xenografts co-implanted with human peripheral blood mononuclear cells (PBMC) in NOD/SCID mice after treatment with SCORPION molecules.
SCORPION molecules potently mediate target-specific T-cell cytotoxicity toward tumor cell lines presenting cell surface CD19, with EC50 values for cytotoxicity at low pM concentrations. These molecules also demonstrate induction of T-cell activation and proliferation in the presence of target-bearing tumor cells but not in the absence of target expression. SCORPION molecules retain stable function following incubation at 37°C in mouse serum for up to a week in vitro, and pharmacokinetic analysis of SCORPION protein function in BALB/c mouse serum following intravenous administration resulted in half-life estimates of 69–84 hours. In efficacy studies conducted in NOD/SCID mice, SCORPION proteins significantly inhibited the outgrowth of Ramos tumor xenografts in the presence of human effector cells.
SCORPION molecules targeting CD19 and CD3 effectively harness the cytotoxic activity of T cells to kill CD19 positive tumor cells both in vitro and in vivo and show potential for further investigation as possible therapeutic agents for B-cell malignancies.
Chenault:Emergent BioSolutions: Employment. Gottschalk:Emergent BioSolutions: Employment. Hernandez-Hoyos:Emergent BioSolutions: Employment. Wiens:Emergent BioSolutions: Employment. Gordon:Emergent BioSolutions: Employment. Klee:Emergent BioSolutions: Employment, Equity Ownership. Bienvenue:Emergent BioSolutions: Employment. Dasovich:Emergent BioSolutions: Employment. Kumer:Emergent BioSolutions: Employment. Aguilar:Emergent BioSolutions: Employment. Bannink:Emergent BioSolutions: Employment, Equity Ownership. McMahan:Emergent BioSolutions: Employment, Equity Ownership. Natarajan:Emergent BioSolutions: Employment, Equity Ownership. Algate:Emergent BioSolutions: Employment, Equity Ownership. Blankenship:Emergent BioSolutions: Employment, Equity Ownership, Patents & Royalties.
Abstract 3931
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.
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.
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.
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.
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.
Tetraspanins 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.
► CD37 is a tetraspanin directly involved in signal transduction ► CD37 possesses dual noncanonical ITIM and ITAM motifs that regulate cell death ► CD37 ligation mediates BIM-dependent mitochondrial apoptosis of CLL B cells
Understanding how broadly neutralizing antibodies (bnAbs) to HIV envelope (Env) develop during natural infection can help guide the rational design of an HIV vaccine. Here, we described a bnAb ...lineage targeting the Env V2 apex and the Ab-Env co-evolution that led to development of neutralization breadth. The lineage Abs bore an anionic heavy chain complementarity-determining region 3 (CDRH3) of 25 amino acids, among the shortest known for this class of Abs, and achieved breadth with only 10% nucleotide somatic hypermutation and no insertions or deletions. The data suggested a role for Env glycoform heterogeneity in the activation of the lineage germline B cell. Finally, we showed that localized diversity at key V2 epitope residues drove bnAb maturation toward breadth, mirroring the Env evolution pattern described for another donor who developed V2-apex targeting bnAbs. Overall, these findings suggest potential strategies for vaccine approaches based on germline-targeting and serial immunogen design.
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•Isolation of PCT64, a PGT145-like, 25-aa CDRH3 HIV Env V2 apex bnAb lineage•Env glycoform heterogeneity plays a role in the lineage precursor B cell activation•Localized diversity at key V2 epitope residues drove bnAb maturation toward breadth•Env evolution pattern is similar to CAP256, another V2 apex broad neutralizer
Understanding the molecular basis of HIV Env-specific broadly neutralizing antibodies (bnAbs) development is key for vaccine design. Landais et al. find that glycan heterogeneity played a role in the activation of V2 apex PCT64 bnAbs precursor and that viral evolution was similar to CAP256, another donor with V2 apex bnAbs.
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<0.0001) strongly supporting the involvement of pSHP1 in CD37 mediated signaling. Furthermore we demonstrated that Tyr13 is essential for the transduction of death signals since the lack of it but not Tyr274 prevented SMIP-016 induced phosphorylation of CD37, association with SHP1 and cell death. In the setting of late B-cell activation, BIM is a critical pro-apoptotic protein responsible for mitochondrial membrane stabilization and apoptosis. Interestingly, CD37 ligation of CLL cells, resulted in consistent FoxO3a dependent transcription of Bim mRNA (p<0.001) as demonstrated by ChIP, luciferase and pull down assays while level of other pro- and anti-apoptotic proteins such as BAX, BCL2 and MCL1 remain unchanged. BIM down-modulation by siRNA resulted in inhibition of depolarized mitochondria (p=0.0025) with increase in intact mitochondrial integrity (p=0.002) and in the reduction of SMIP-016-induced cytotoxicity compared to scrambled control (p<0.001) confirming the involvement of BIM in SMIP-016-mediated MMD and apoptosis. Proximal SHP1 activation by SMIP-016 directly contributes to BIM induction, since the down modulation of SHP1 by siRNA antagonized BIM up-regulation in CLL cells. Moreover lack of Tyr13 but not Tyr274 prevented SMIP-016 induced up-regulation of BIM further indicating that Tyr13 is essential for the induction of cell death. Interestingly, the deletion of Tyr274 resulted in increased SMIP-016 induced cytotoxicity (p<0.0001) indicating a negative regulatory function of this region in CD37 mediated cell death, consistent with the presence of an ITAM motif in this domain. The p85 regulatory and the p110 catalytic subunits of phosphatidylinositol 3-kinase (PI3K) have been shown to bind to phosphorylated tyrosines within the ITAM motif in other cell types. By co-immunoprecipitation studies we demonstrated that Tyr274 negatively regulates cell death by activating a PI3K dependent survival loop by recruitment of p85 and p110δ and downstream phosphorylation of GSK3β. In addition, treatment of cell lines expressing wild type CD37 or CLL cells with the PI3K inhibitor LY294002 blocked PI3K phosphorylation and resulted in increased SMIP-016 mediated cell death. In conclusion, this dual direct signaling role of a tetraspanin protein has not been previously described. The findings put forth provide rationale for therapeutic antibodies or peptides which selectively activate Tyr13. They also provide strong rationale for combination of SMIP-016 with PI3-kinase inhibitors which have been shown by our group and others promising therapeutic option for CLL.
Algate:emergent biosolutions: Employment. Stromatt:Emergent Product Development Seattle, LLC, Seattle, WA: Employment.
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