Jelinek, Diane F.

Abstract B cell maturation antigen (BCMA)-targeted chimeric antigen receptor T (CART) cell therapies have emerged as a potentially curative treatment for multiple myeloma (MM). However, 50-70% of patients relapse within 3 years. The MM tumor microenvironment (TME) is highly immunosuppressive with an abundance of bone marrow (BM) cancer-associated fibroblasts (CAFs), which contribute to BCMA-CART cell failure. Therapeutic targeting of BM-CAFs has been beset by 1) an incomplete understanding of CAF interactions with CART cells and other BM cells and 2) toxicity risks due to prolonged depletion of healthy fibroblasts and inefficient drug delivery to the BM. To elucidate how BM-CAFs drive BCMA-CART resistance, we performed single cell RNA sequencing (scRNAseq) of baseline BM aspirates from responders (R, n = 6) and non-responders (NR, n = 6) to BCMA-CART cell therapy. NR BMs were enriched for inhibitory CAFs (iCAFs) that highly express fibroblast activation protein (FAP). Spatial analyses of BM cells revealed greater iCAF-iCAF and iCAF-inhibitory macrophage interactions in NR. These findings suggest that FAP+ iCAFs in NR are abundant in the BM and exhibit significant interactions with immunosuppressive macrophages. Next, we sought to validate our scRNAseq data in preclinical models. Xenograft mice were intravenously (IV) engrafted with luciferase+ (luc+) BCMA+ OPM2 cells and/or patient-derived (pd) BM-CAFs (1x106 each). Following engraftment, mice received 1x106 BCMA-CART cells (IV). BCMA-CART cells eradicated tumors in mice engrafted with OPM2 cells, but not in mice engrafted with pdBM-CAFs and OPM2 cells (p<0.01). In NSG and humanized mice (NSG mice engrafted with human CD34+ cells in the first 48 hours of life), tumor growth was significantly accelerated in the presence of pdBM-CAFs (NSG: p<0.01; humanized: p<0.001). BCMA-CART, OPM2, and pdBM-CAF co-cultures also showed significant increases in immunosuppressive cytokines including IL-1Rα, IL-4 and IL-10 and inhibited CART cell proliferation compared to co-cultures without pdBM-CAFs (IL-1Rα, IL-10: p<0.01; IL-4: p<0.001; proliferation: p<0.05). To overcome CAF-mediated CART dysfunction, we engineered BCMA-CART cells to secrete molecules to target BM FAP+ iCAFs and remodel the MM-TME (STriKEs). We designed two BCMA-CART-STriKE strategies: 1) a tri-specific composed of IL-15, anti-FAP, and anti-CD16 molecules to activate innate immune cells to kill CAFs and 2) bi-specific anti-FAP, anti-SIRPα molecules linked to an antibody Fc region (Fc fusion) targeting the CD47/SIRPα axis to induce phagocytosis of FAP+ iCAFs. We hypothesized that BCMA-CART-STriKEs would surmount CAF-mediated immunosuppression without toxicity. We generated BCMA-CART-STriKEs from healthy human donor T cells. CAR expression, STriKE secretion, and binding were confirmed via flow cytometry. Co-cultures of BCMA-CART-STriKEs, donor-matched macrophages, OPM2 cells or BCMA+ MM1S cells, and pdBM-CAFs showed significantly higher OPM2 or MM1S killing (p<0.001), immune cell activation (p<0.01), and phagocytosis (p<0.05), compared to control BCMA-CART cells secreting IL-15 or Fc fusions targeting only FAP or only SIRPα. Next, we assessed BCMA-CART-STriKE efficacy in vivo. NOD-SCID-γ-/- (NSG) mice received 1x106 pdBM-CAFs and 1x106 luc+ OPM2 or luc+ MM1S cells via IV or subcutaneous (SQ) injection. Mice were randomized after confirming tumor engraftment (via bioluminescence imaging for IV or tumor caliper for SQ) and received IV treatment with 1x106 BCMA-CART-STriKEs (or controls) and 5x105 donor-matched macrophages or CD16+ immune cells. BCMA-CART-STriKE-treated mice achieved total tumor clearance. Treatment with BCMA-CART cells secreting IL-15 led to lethal weight loss despite tumor clearance while BCMA-CART cells secreting Fc fusions targeting only FAP or SIRPα failed to eradicate tumors. Lastly, we assessed the potential toxicities of BCMA-CART-STriKEs in humanized mice engrafted with OPM2 cells and pdBM-CAFs (1x106 each). CART cells expressing BCMA and FAP CARs (BCMA-FAP CART) served as a control. BCMA-FAP CART treatment resulted in significant toxicity and weight loss while BCMA-CART-STriKE treatment did not (p<0.01). In summary, we have identified iCAF interactions with immunosuppressive immune cells as key contributors to BCMA-CART cell failure and designed novel BCMA-CART-STriKEs that overcome this failure in MM preclinical models. These findings support the translation of BCMA-CART-STriKEs to a phase I clinical trial.

Hematopoietic and stromal cells within the bone marrow (BM) provide membrane-bound and/or soluble factors that are vital for the survival of plasma cells (PCs). Recent reports in murine BM demonstrated the dynamic formation and dispersion of PC clusters. To date, PC clustering in normal human BM has yet to be thoroughly examined. The goal of this study was to determine whether PC clusters are present in human BM and whether clustering changes as a function of age. Quantification of PCs and clustering in BM sections across six different age groups revealed that fewer PCs and PC clusters were observed in the youngest and oldest age groups. PC clustering increased with age until the sixth decade and then began to decrease. A positive correlation between the number of PCs and PC clusters was observed across all age groups. PC clusters were typically heterogeneous for immunoglobulin heavy- and light-chain expression. Taken together, these data demonstrate that PC clusters are present in human BM and that PC clustering increases until middle adulthood and then begins to diminish. These results suggest the spatial distribution of BM PC-supportive stromal cells changes with age:

Abstract The ability of IFN-α to induce an anti-viral state in a wide variety of cell types as well as to inhibit cellular growth has long been appreciated. It is less clear, however, whether both these effects lie downstream of a common signaling pathway. In this study we have taken advantage of an atypical human myeloma cell line (KAS-6/1) displaying a dramatic proliferative response to IFN-α in an effort to resolve the signaling requirements for IFN-α-induced anti-viral and growth regulatory effects. Thus, we have analyzed the ability of IFN-α to induce a number of known receptor-initiated events in this cell line and have compared these responses with those exhibited by a cell lineage- and maturation stage-matched myeloma cell line (ANBL-6) that displays typical IFN-α responsiveness. Despite the widely contrasting effects of IFN-α on cellular proliferation, IFN-α was shown to be comparable in its ability to induce the expression of early response genes as well as induce resistance to viral infection in both cell lines. By contrast, the effects of IFN-α on the activation of mitogen-activated protein kinase (MAPK) were strikingly distinct. Finally, although inhibition of MEK and MAPK activation had no effect on the induction of the anti-viral response, it completely blocked IFN-α-stimulated proliferation of the KAS-6/1 cells. In summary, our analysis of the role of the MAPK and anti-viral signaling pathways using these two cell lines suggests that the anti-viral and growth regulatory effects of IFN-α display a differential requirement for activation of the MAPK pathway.

Abstract A variety of signals, mediated via either the B cell Ag receptor (BCR), or non-BCR molecules such as CD40 or cytokine receptors, have been shown to be crucial for the regulation of B cell survival, growth, and differentiation. Although it is clear that a variety of signaling pathways can be activated in B cells in a stimulus-dependent manner, it remains unknown whether differential activation of these signaling pathways is the underlying mechanism controlling B cell fate, i.e., growth vs differentiation. Initial studies reported here indicated that stimulation of highly purified peripheral blood B cells with the polyclonal B cell activators Staphylococcus aureus and CD40 ligand (CD40L) resulted in the rapid induction of phosphatidylinositol 3-kinase (PI 3-kinase) activity. Moreover, pretreatment of B cells with wortmannin, a specific inhibitor of PI 3-kinase, resulted in a complete block in induction of PI 3-kinase activity. The effects of wortmannin, as well as a second PI 3-kinase inhibitor, LY294002, on the induction of both B cell growth and differentiation were therefore investigated. Although these PI 3-kinase inhibitors variably inhibited B cell DNA synthesis in a stimulus-dependent manner, both drugs effected a near-complete block of the ability of each of these stimuli to induce Ig production. Furthermore, separation of B cells into naive IgD+ and postswitch IgD- B cells failed to reveal differential sensitivity of these populations to wortmannin. These results suggest that activation of PI 3-kinase, or other wortmannin- and LY294002-sensitive targets, is a crucial event that occurs during the differentiation of normal human B lymphocytes. The differential sensitivity of B cell responses to inhibitors of PI 3-kinase supports the notion that distinct signal transduction pathways are involved in differentiation vs proliferation of normal human B lymphocytes.

Abstract The role of IL-12 in human peripheral blood B cell responsiveness was examined. To analyze the ability of IL-12 to directly mediate B cell growth and/or differentiation, FACS-purified (> 99% pure) B cells were studied and a polyclonal B cell-activating system utilizing Cowan I Staphylococcus aureus was used. Whereas IL-2 is highly effective in this system in promoting both B cell growth and differentiation, IL-12 was observed only to augment modestly B cell growth and to be ineffective by itself as a B cell differentiation factor for S. aureus-stimulated B cells. However, IL-12 markedly enhanced Ig secretion when added in the presence of IL-2. Moreover, when the ability of IL-12 to augment IL-2-dependent B cell Ig secretion was compared with the ability of several known auxiliary B cell differentiation factors, IL-12 was observed to be the most potent cytokine that could costimulate with IL-2. Analysis of IL-12-stimulated B cell cultures failed to reveal outgrowth of T cells and NK cells. In addition, assessment of IFN-gamma levels in IL-12-driven B cell culture supernatants and analysis of IFN-gamma effects on B cell responses added additional support to the conclusion that IL-12 directly modulates B cell function. Finally, these results suggest that IL-12 is a potent constimulus of B cell differentiation and that the signals conveyed by IL-12 seem to be qualitatively distinct from the differentiative signals delivered by other cytokines such as IL-2.

Abstract Insulin-like growth factors (IGF-I, IGF-II) have long been recognized as important mitogens in many types of malignancies. Because the role of IGFs in growth control of myeloma cells has not been extensively examined, we have used a panel of IL-6-responsive myeloma cell lines to address this issue. Initial studies demonstrated that IGF-I and IGF-II significantly enhanced DNA synthesis by each of the four cell lines, even when assayed in the absence of IL-6. The specificity of the IGF response was confirmed using an IGF-I receptor Ab, and additional studies demonstrated that IGF responsiveness did not result from induction of autocrine IL-6 expression. When IL-6 responsiveness was assayed, three of four cell lines synthesized DNA in response to IL-6 alone; however, the magnitude of responsiveness was greatly enhanced by addition of IGFs. Similar results were obtained when proliferation and cell cycle progression were analyzed. By contrast, the KP-6 cell line was responsive to IL-6 only when IGF was present. Finally, we analyzed the effects of IGF-I on normal B lymphocytes. IGF, however, did not stimulate B cell DNA synthesis, suggesting that IGF responsiveness may represent a key difference between normal and malignant B cells. In summary, these studies suggest that IGFs may play an important role in multiple myeloma by virtue of their ability to directly stimulate tumor cell growth as well as modulate the magnitude of IL-6-driven growth.

Abstract Evidence supporting the importance of the 3-phosphoinositide signaling pathway in lymphocyte activation is rapidly accumulating. In our study, we assessed the effects of two PI 3-kinase inhibitors, wortmannin and LY294002, on T cells as a means to analyze the role of the PI 3-kinase-signaling pathway in the generation of T cell help for B cell growth and differentiation. For these studies, B cells were cocultured with CD3-activated mitomycin C-treated T cells to induce B cell responsiveness. Of interest, wortmannin or LY294002 pretreatment of the T cell population significantly inhibited T cell-dependent induction of B cell proliferation and differentiation. The failure of wortmannin-treated CD3-activated mitomycin C-treated T cells to provide help in driving the differentiation of B cells to Ig-secreting cells could not be corrected by the addition of exogenous IL-2. Further studies designed to elucidate the mechanism by which wortmannin-treated T cells failed to provide B cell help indicated that wortmannin and LY294002 significantly inhibited the induction of CD40 ligand and, to a lesser extent, intercellular adhesion molecule-1 expression. These results suggest that the PI 3-kinase-signaling pathway, or other wortmannin- and LY294002-sensitive pathways, may be important for the induction of expression of crucial interaction molecules, such as CD40 ligand, on T cells and thus indicates that D-3 phosphoinositides play a pivotal role in regulating T cell-dependent B cell activation.

Abstract Expression of autocrine growth factors by myeloma cells is an important mechanism that may contribute to tumor expansion. IL-6 is one of several cytokines that uses the signal transducer gp130 as a receptor component. Of these cytokines, those that have been shown to be paracrine growth factors for some myeloma cells include IL-6, IL-11, ciliary neurotrophic factor, leukemia inhibitory factor, and oncostatin M (OSM). Only IL-6, however, has been identified as an autocrine growth factor for myeloma cells. In this study we used a panel of three IL-6-responsive myeloma cell lines to investigate the expression of other autocrine growth factor loops. Initial studies employing neutralizing mAbs to IL-6 or gp130 revealed that the growth of the DP-6 and KP-6 cell lines was inhibited by both mAbs, whereas the growth of the KAS-6/1 cell line was inhibited only by the anti-gp130 mAb. Anti-OSM neutralizing mAb also inhibited KAS-6/1 cell growth. Autocrine OSM production by the KAS-6/1 cells was confirmed using a sensitive ELISA. Although the anti-OSM mAb had no significant effects on KP-6 and DP-6 cell growth, OSM was detected in DP-6 supernatants. These results suggest that OSM production and responsiveness by myeloma cells are distinct phenotypes and not necessarily related in all myeloma cells. Finally, we analyzed the significance of OSM-mediated myeloma cell growth by assessing the effects of OSM on normal, in vitro-generated plasmablasts. OSM markedly enhanced plasmablast Ig secretion but did not affect growth. Thus, the nature of the response elicited by OSM in myeloma cells is distinct from its effects on normal B lineage cells. Moreover, because gp130-mediated signaling results in myeloma cell growth, autocrine expression of any gp130-utilizing cytokine has the potential to significantly augment tumor expansion.