Development of ISB 1442, a CD38 and CD47 bispecific biparatopic antibody innate cell modulator for the treatment of multiple myeloma.

Antibody engineering can tailor the design and activities of therapeutic antibodies for better efficiency or other advantageous clinical properties. Here we report the development of ISB 1442, a fully human bispecific antibody designed to re-establish synthetic immunity in CD38+ hematological malignancies. ISB 1442 consists of two anti-CD38 arms targeting two distinct epitopes that preferentially drive binding to tumor cells and enable avidity-induced blocking of proximal CD47 receptors on the same cell while preventing on-target off-tumor binding on healthy cells. The Fc portion of ISB 1442 is engineered to enhance complement dependent cytotoxicity, antibody dependent cell cytotoxicity and antibody dependent cell phagocytosis. ISB 1442 thus represents a CD47-BsAb combining biparatopic targeting of a tumor associated antigen with engineered enhancement of antibody effector function to overcome potential resistance mechanisms that hamper treatment of myeloma with monospecific anti-CD38 antibodies. ISB 1442 is currently in a Phase I clinical trial in relapsed refractory multiple myeloma.

p53 dysregulation in B-cell malignancies: More than a single gene in the pathway to hell.

TP53 deletion or mutation is frequent in B-cell malignancies and is associated with a low response rate. We describe here the p53 landscape in B-cell malignancies, from B-Acute Lymphoblastic Leukemia to Plasma Cell Leukemia, by analyzing incidence of gain or loss of function of actors both upstream and within the p53 pathway, namely MYC, RAS, ARF, MDM2, ATM and TP53. Abnormalities are not equally distributed and their incidence is highly variable among malignancies. Deletion and mutation, usually associated, of ATM or TP53 are frequent in Diffuse Large B-Cell Lymphoma and Mantle Cell Lymphoma. MYC gain, absent in post-GC malignancies, is frequent in B-Prolymphocytic-Leukemia, Multiple Myeloma and Plasma Cell Leukemias. RAS mutations are rare except in MM and PCL. Multiple Factorial Analysis notes that MYC deregulation is closely related to TP53 status. Moreover, MYC gain, TP53 deletion and RAS mutations are inversely correlated with survival. Based on this landscape, we further propose targeted therapeutic approaches for the different B-cell malignancies.

Combination of lenalidomide with vitamin D3 induces apoptosis in mantle cell lymphoma via demethylation of BIK.

Mantle cell lymphoma (MCL) is a currently incurable B-cell malignancy. Lenalidomide (Len) has been demonstrated to be one of the most efficient new treatment options. Because Len and 1α,25-dihydroxyvitamin (VD3) synergize to kill breast cancer cells, we investigated whether VD3 could increase the ability of Len to induce MCL cell death. While MCL cells were weakly sensitive to Len (1 µM), the addition of VD3 at physiological dose (100 nM) strongly increased cell death, accompanied by slowdown in cell cycle progression in MCL cell lines (n=4 out of 6) and primary samples (n=5 out of 7). The Len/VD3 treatment markedly increased the expression of the BH3-only BCL2-interacting killer (Bik) without affecting the expression of other Bcl-2 molecules. Immunoprecipitation assays demonstrated that Bik was free from anti-apoptotic partners, Bcl-2 and Bcl-xL, in treated cells. Moreover, silencing of BIK prevented apoptosis induced by Len/VD3, confirming the direct involvement of Bik in cell death. Bik accumulation induced by Len/VD3 was related to an increase in BIK mRNA levels, which resulted from a demethylation of BIK CpG islands. The sensitivity of MCL cells to Len/VD3 was similar to the response to 5-azacytidine, which also induced demethylation of BIK CpG islands. These preclinical data provide the rationale to investigate the role of VD3 in vivo in the response to Len.

Autocrine insulin-like growth factor 1 and stem cell factor but not interleukin 6 support self-renewal of human myeloma cells.

In this study, we have identified the growth factors supporting myeloma self-renewal in eight myeloma cell lines. All cell lines able to form self-colonies displayed constitutive P-AKT and P-ERK1,2 but not P-STAT3 and did not express CD45, suggesting the presence of an insulin-like growth factor 1 (IGF1) loop. We showed that a blocking anti-insulin-like growth factor 1 receptor (IGF1R) monoclonal antibody (mAb) inhibited colony formation in correlation with IGF1R expression and decreased P-AKT. Imatinib or a blocking anti-stem cell factor (SCF) mAb also inhibited colony formation of two cell lines expressing C-KIT and SCF, and decreased P-AKT. Moreover, the PI3K/AKT pathway inhibitor wortmannin inhibited colony formation. Blocking interleukin (IL)6R did not inhibit colony formation in good agreement with a lack of constitutive P-STAT3. We showed that primary cells frequently co-expressed IGF1R/IGF1 but not C-KIT/SCF or IL6R/IL6, suggesting that in vivo autonomous growth could be possible via IGF1R. Despite their similar role in clonogenic growth and shared signaling pathway, IGF1R and C-KIT had opposite prognostic values, suggesting that they were surrogate markers. Indeed, we showed that both C-KIT and IGF1R prognostic values were not independent of MMSET expression. This study highlights the autocrine role of IGF1 in myeloma cells and reinforces the interest in targeting IGF1R in IGFR1(+) CD45(+/-) patients, such as MMSET(+) patients.

Paradoxical effect of lenalidomide on cytokine/growth factor profiles in multiple myeloma.

BACKGROUND: Lenalidomide is an active immunomodulatory and antiproliferative agent in multiple myeloma. However, the molecular mechanisms driving these activities are not yet fully elucidated. Therefore, we investigated the modulation of the cytokine/growth factor patterns of myeloma cells under LEN treatment. METHODS: Lenalidomide effect on myeloma cell proliferation was investigated in a myeloma cell line collection (n=23) by (3)H-thymidine incorporation. Modulation of the cytokine/growth factor patterns of myeloma cells under LEN treatment was analysed by real-time quantitative PCR. RESULTS: Lenalidomide inhibits the proliferation of two-thirds of myeloma cell lines independently of their genetic background. We demonstrated that LEN increased TNF-α and IL-8 inflammatory cytokines and insulin-like growth factor-1 (IGF-1) growth factor in both sensitive and resistant myeloma cells to LEN. CONCLUSION: Lenalidomide favours a uniform TNF-α and IL-8 inflammatory and IGF-1 secretory profile of myeloma cells, an observation that raises important questions for therapeutic approaches incorporating the agent.

The cap-translation inhibitor 4EGI-1 induces apoptosis in multiple myeloma through Noxa induction.

BACKGROUND: Cancer cells are frequently addicted to deregulated oncogenic protein translation. The small molecule 4EG-I selectively inhibits the cap-dependent translation of mRNAs. As multiple myeloma is an incurable disease that requires new therapeutic approaches, we investigated whether targeting the translation initiation pathway could be a target for myeloma therapy. METHODS: Six myeloma cell lines and primary samples were included in this study. The 4EGI-1 effect was determined by AnnexinV staining and caspase activation. Modification of Bcl-2 protein expression was analysed, and the significance of modified proteins was analysed by knock-down experiments. RESULTS: We demonstrated that 4EGI-1 impaired the assembly of the eIF4F complex and decreased the expression of the eIF4E-regulated proteins in myeloma cells. Furthermore, we showed that 4EGI-1 induced strong apoptosis in five out of six myeloma cell lines. Apoptosis is associated with the activation of the intrinsic mitochondrial pathway. The 4EGI-1 triggered Noxa induction only in cells undergoing apoptosis through endoplasmic reticulum (ER) stress. Furthermore, Noxa silencing prevented myeloma cells from 4EGI-1-induced apoptosis. Finally, Noxa induction led to a disruption of Mcl-1/Bim complexes in parallel to the generation of 'Mcl-1-free Noxa'. CONCLUSION: Our results suggested that the use of inhibitors that directly target the translation initiation complex eIF4F could represent a potential novel approach for multiple myeloma therapy.

BH3-only protein Bik is involved in both apoptosis induction and sensitivity to oxidative stress in multiple myeloma.

BACKGROUND: although gene expression profile of multiple myeloma (MM) patients shows a wide range of Bik/Nbk expression, varying from absent to high, its regulation and function in myeloma cells is poorly understood. Thus, we addressed these questions in MM. METHODS: human myeloma cell lines (HMCLs) and primary purified myeloma cells were studied for Bcl-2 family protein expression by western blot and further correlation analysis was performed. Correlative study between Bik and thyrotroph embryonic factor (TEF) transcription factor expression was analysed by PCR. Stress oxidative response was analysed by flow cytometry. RESULTS: a strong expression of Bik protein was found only in one out of three of HMCL and correlated to Bcl-2 expression (P=0.0006). We demonstrated that Bik could be regulated at the protein level by Bcl-2 and at the transcriptional level by TEF. Bik overexpression sensitises myeloma cells to oxidative stress whereas Bik silencing increases resistance to H(2)O(2) oxidative stress. Furthermore, Bik ectopic expression disrupts Bim/Bcl-2 and Bim/Bcl-xL endogenous complexes triggering Bim release that could induce Bax and Bak activation. CONCLUSIONS: ours results suggest that Bik has a role in both, apoptosis induction and sensitivity to oxidative stress in myeloma cells. Small BH3 mimetic molecules should be considered for further apoptosis-based therapy in myeloma cells expressing endogenous Bik/Bcl-2 complexes.

Pathogen-associated molecular patterns are growth and survival factors for human myeloma cells through Toll-like receptors.

Multiple myeloma (MM) patients are strongly vulnerable to infections, which remain a major cause of death. During infection, human immune cells sense the presence of invading pathogens through the Toll-like receptor family (TLR), which recognizes pathogen-associated molecular patterns (PAMP). We hypothesized that MM cells also could sense the presence of microorganisms, thus promoting myeloma disease progression. Here, we report that human myeloma cell lines (HMCL) and primary myeloma cells express a broad range of TLR, and are sensitive to the corresponding PAMP. Toll-like receptor 1, 7 and 9 are most frequently expressed by HMCL. The expression pattern of TLR does not correlate with the one of B cells, as TLR2 and 10 are lost while TLR3, 4 and 8 are acquired by some HMCL. Culture with TLR7- and TLR9-ligands saves HMCL from serum-deprivation or dexamethasone-induced apoptosis. Similarly, both ligands increase myeloma cell growth. These effects are mediated by an autocrine secretion of interleukin-6 (IL-6) since the neutralization of IL-6 blocks the growth and survival of HMCL. Thus, TLR expression and function are not restricted to the cells of the immune system and could be of advantage for cancer cells. In MM, recurrent infections could promote tumor growth and favor escape from standard therapies.

Normal and malignant human plasma cells: proliferation, differentiation, and expansions in relation to CD45 expression.

In this study, we have evaluated the proliferation and the phenotype of human plasma cells of different origins, i.e., from tonsil, peripheral blood, bone marrow as well as plasma cells generated in vitro from memory B cells. We have demonstrated that plasma cells from tonsil, peripheral blood, as well as those generated in vitro, were highly proliferating and presented a homogeneous CD45bright phenotype. In contrast, bone marrow plasma cells were heterogeneous for CD45 expression but their proliferation was restricted to the CD45bright compartment. Subsequently, their CD45 expression decreased with proliferation arrest and final maturation. We also studied the proliferation of abnormal plasma cells, i.e., peripheral blood reactive plasmacytoses and multiple myeloma (MM). All reactive plasmacytoses turned out to be homogeneous expansions of CD45bright plasma cells with unusually high labeling index. In contrast, CD45 expression was heterogeneous in MM as in normal bone marrow. However, a minor CD45bright population was also always the most proliferating one as opposed to a major population of less or non-proliferating myeloma cells characterized by a weaker or a lack of CD45 expression. In conclusion, proliferation is linked to plasma-cell generation and a CD45bright phenotype is the hallmark of the most proliferating normal, reactive as well as malignant plasma cells.

Farnesyl transferase inhibitor R115777 induces apoptosis of human myeloma cells.

R115777, a nonpeptidomimetic farnesyl transferase inhibitor has recently demonstrated a significant antileukemic activity in vivo in acute myeloid leukemia. Multiple myeloma (MM) is a fatal hematological malignancy characterized by an accumulation of long-lived plasma cells within the bone marrow. In the present study, we have investigated the effect of the R115777 on growth and survival of myeloma cells. We have found that R115777 induced (1) a significant and dose-dependent growth inhibition of the three myeloma cell lines tested; and (2) a significant and time-dependent apoptosis. R115777 also induced apoptosis in the bone marrow mononuclear cell population of four MM patients, being almost restricted to the malignant plasma cells. Finally, we have investigated the effect of the R115777 in the Ras/MAPK and JAK/STAT pathways which are implicated in survival and/or proliferation in MM. The phosphorylation of both STAT3 and ERK1/2 induced by IL-6 was totally blocked at 15 microM of R115777 and partially blocked when R115777 was used at 10 and 5 microM. The induction of apoptosis by R115777 in myeloma cells and its implication in the regulation of JAK/STAT signalling suggest that R115777 might be an interesting therapeutical approach in MM.

Interleukin-6 is a growth factor for nonmalignant human plasmablasts.

Interleukin-6 (IL-6), although often regarded as a B-cell differentiation factor, was recently described as the essential survival factor for human plasmablasts in vivo in reactive plasmacytosis. The present study reinvestigated the roles of IL-6 and IL-2 in the generation of plasma cells from human memory B cells in vitro. The cells involved in this differentiation process were identified as preplasmablasts (CD20+/-CD38+/-CD138-), plasmablasts (CD20-CD38++CD138-), and early plasma cells (CD20-CD38+++CD138+++). IL-2 or IL-10 induced a strong generation of plasmablasts and early plasma cells (PCs). Compared to IL-2 or IL-10, IL-6 alone was inefficient at PC generation. However, when combined with IL-2 or IL-10, IL-6 enhanced generation of early PCs. Moreover, anti-IL-6 monoclonal antibody markedly reduced IL-2-induced generation of early plasma cells, but not of plasmablasts. These roles of IL-2 and IL-6 were consistent with the difference in the expression of their respective receptors (R). CD25 (IL-2Ralpha) was increased 72 +/- 10-fold on activated B cells, but decreased and then disappeared on plasmablasts. Conversely, CD126 (IL-6Ralpha) was barely expressed on activated B cells, but increased 18 +/- 2-fold on preplasmablasts. Finally, IL-6 enhanced the proliferation (2-fold increase) of IL-2-generated plasmablasts. In conclusion, the data indicate that IL-6 is a growth factor for nonmalignant human plasmablasts.

Reactive plasmacytoses in multiple myeloma during hematopoietic recovery with G- or GM-CSF.

We report on three cases of reactive plasmacytoses (RP) in the course of multiple myeloma (MM). The three patients achieved complete remission following high dose melphalan and peripheral blood stem cell transplantation. These transient plasmacytoses had all the characteristics of RP, i.e. expansion of highly proliferative polyclonal plasma cells (PC) with a normal phenotype and genotype and corresponding to expansion of both PC progenitors (plasmablasts) and PC precursors (early plasma cells). These cells were easily distinguished from malignant PC of the corresponding patients evaluated at diagnosis, especially by their phenotypic and genotypic features.

The cancer germ-line genes MAGE-1, MAGE-3 and PRAME are commonly expressed by human myeloma cells.

In this study, we have investigated the mRNA expression of the cancer germ-line genes MAGE, BAGE, GAGE, RAGE and the tumor-overexpressed gene PRAME by human myeloma cell lines and malignant plasma cells from patients with multiple myeloma (MM). By reverse transcription-PCR, we show that all myeloma cell lines (n = 16) express at least one of these genes, except RAGE-1 that was never expressed. We show that malignant plasma cells from the majority of MM patients (n = 21) expressed MAGE-1, MAGE-3 and PRAME. On the contrary, polyclonal reactive plasma cells did not express any of these genes. By flow cytometry, we show that mage-1 protein is expressed within myeloma cells and cell lines and that anti-mage-1.HLA-A1 cytotoxic T lymphocytes efficiently killed MAGE-1+HLA-A1+ MDN myeloma cells. Taken together, our data show that mage-1 and mage-3 could constitute specific targets for tumor immunotherapy of MM patients.

Reactive plasmacytoses are expansions of plasmablasts retaining the capacity to differentiate into plasma cells.

Circulating plasma cells in 10 cases of reactive plasmacytosis had a shared phenotype with early plasma cell (CD19(+) CD38(+) CD138(+) CD40(+) CD45(+) CD11a+ CD49e- CD56(-)). In most cases, a minor subpopulation of CD28(+) plasma cells was also detected. Reactive plasma cells were highly proliferative, suggesting the presence of circulating progenitors (plasmablasts). After CD138(+) plasma cell removal, highly proliferative CD138(-) plasmablasts differentiated into CD138(+) plasma cells within a few days. This differentiation, which was associated with increased CD38 and decreased HLA-DR expression, was further confirmed by a large increase in intracellular Ig content (associated with Ig secretion) and was concomitant with extensive secretion of interleukin-6 (IL-6). The addition of neutralizing anti-IL-6 and anti-CD126 (IL-6 receptor) monoclonal antibodies totally prevented Ig secretion and cell differentiation by inducing apoptosis of plasmablasts, which indicates that IL-6 is an essential survival factor for plasmablasts. This report provides the first characterization of normal plasmablasts and shows that their phenotype is not exactly that of multiple myeloma cells.

Isolation of human lymphocyte antigens class I-restricted cytotoxic T lymphocytes against autologous myeloma cells.

Peripheral blood T cells from a patient with multiple myeloma in complete remission were selected in vitro against an autologous myeloma cell line (SBN-1), using a protocol designed for the selection of relatively rare precursor cytotoxic T cells (pCTL). Delayed addition (2 weeks) of interleukin 2 induced T-cell proliferation, and a bulk culture (T-cell line) was obtained 2 days later. This T-cell line displayed cytotoxicity against SBN-1. A CD8+ CD4- cytotoxic T-cell clone (CT5) was then obtained that recognized SBN-1 but not autologous EBV+ B-lymphoblastoid cells, autologous T PHA-blasts, or Daudi, Raji, K562, and 11 allogeneic myeloma cell lines. Moreover, CT5 cytotoxic activity against SBN-1 was blocked by monoclonal antibodies recognizing human lymphocyte antigen class I molecules. This seems to be the first demonstration of myeloma-specific pCTL in peripheral blood T cells of patients with multiple myeloma.

Differential expression of Bcl-2 in human plasma cell disorders according to proliferation status and malignancy.

Multiple myeloma (MM) is a malignancy characterized by a very slow proliferation of malignant plasma cells leading to their accumulation within the bone marrow. This suggests that resistance to apoptosis may play a critical role both in the pathogenesis and resistance to treatment of MM. Bcl-2 is a key protein for the regulation of apoptosis. However, it has been shown that this protein also regulates the state of proliferation. In the current study, we show that malignant plasma cells from both the bone marrow and peripheral blood express high levels of Bcl-2 and are slowly proliferating cells. In contrast, myeloma cells from extramedullary sites (ie pleural effusion, ascitis, mammary and gastric plasmacytoma) express Bcl-2 weakly while being highly proliferative. Normal non-dividing bone marrow plasma cells express high levels of Bcl-2 protein. In contrast, four highly proliferative reactive plasmacytosis express weak levels of Bcl-2. We conclude that there is an inverse correlation between Bcl-2 expression and the proliferation rate of both normal and malignant plasma cells. These data may be explained by the double function of Bcl-2, ie its well known function as an anti-apoptotic molecule and its intriguing function as an inhibitory molecule of cell proliferation.