Immunotherapy In Multiple Myeloma Research Articles

Charting the Course: Sequencing Immunotherapy for Multiple Myeloma.

Multiple chimeric antigen receptor (CAR) T-cell and bispecific antibody (bsAb) therapies have been approved, demonstrating impressive clinical efficacy in relapsed/refractory multiple myeloma (MM). Currently, these treatment share overlapping approval indications in the relapsed/refractory space, highlighting the importance of optimal selection and sequencing to maximize clinical efficacy. For patients previously unexposed to T-cell-directed therapies, several factors should be weighed when both options are available. These factors include access and logistical challenges associated with CAR T-cell therapy, disease-specific factors such as tempo of disease relapse, in addition to patient-specific factors such as frailty, and distinct toxicity profiles across these agents. Sequential therapy, whether it involves CAR T-cell therapy followed by bsAb or vice versa, has demonstrated clinical efficacy. When sequencing these agents, it is crucial to consider various factors that contribute to treatment resistance with careful selection of treatments for subsequent therapy in order to achieve favorable long-term patient outcomes.

BET Inhibitor JQ1 Selectively Reduce Tregs by Upregulating STAT3 and Suppressing PD-1 Expression in Patients with Multiple Myeloma.

Multiple myeloma (MM) stands as a prevalent hematological malignancy, primarily incurable, originating from plasma cell clones. MM's progression encompasses genetic abnormalities and disruptions in the bone marrow microenvironment, leading to tumor proliferation, immune dysfunction, and compromised treatment outcomes. Emerging evidence highlights the critical role of regulatory T cells (Tregs) in MM progression, suggesting that targeting Tregs could enhance immune functionality and treatment efficacy. In this study, a notable increase in Treg proportions within MM patients' bone marrow (BM) compared to healthy individuals is observed. Additionally, it is found that the bromodomain and extraterminal domain (BET) inhibitor JQ1 selectively diminishes Treg percentages in MM patients' BM and reduces TGF-β1-induced Tregs. This reduction occurs via inhibiting cell viability and promoting apoptosis. RNA sequencing further indicates that JQ1's inhibitory impact on Tregs likely involves upregulating STAT3 and suppressing PD-1 expression. Collectively, these findings suggest JQ1's potential to modulate Tregs, bolstering the immune response in MM and introducing a promising avenue for MM immunotherapy.

Construction of CD138-targeted chimeric antigen receptor- modified T cells and their effect in multiple myeloma therapy

Objective: To construct a novel chimeric antigen receptor T (CAR-T) cell targeting CD138 and to investigate its cytotoxicity against myeloma cells. Methods: The hybridoma strain that can stably secrete the CD138 monoclonal antibody (mAb) was prepared and obtained through monoclonal antibody screening technology. The hybridoma strain cells were intraperitoneally injected into mice to produce ascites containing monoclonal antibodies, which were then collected and purified to obtain pure CD138 mAb. Further examinations were performed to assess the biological characteristics of CD138 mAb. The variable region sequence of this antibody was amplified through reverse transcription polymerase chain reaction and was used as the antigen recognition domain of CD138 CAR, which was subsequently expressed on the surface of T cells by lentiviral infection. Flow cytometry was employed to assess the phenotype of CD138 CAR-T cells. In vitro cytotoxicity and degranulation assays were performed to evaluate their antitumor effects. Results: ① We successfully prepared anti-human CD138 antibody hybridoma cell lines and screened a hybridoma cell strain, 5G2, which could persistently and stably secrete the anti-CD138 antibody. ② The purified CD138 (5G2) mAb can especially recognize CD138(+) cells with a binding affinity constant (K(D)) of 6.011×10(-9) mol/L and showed no significant binding activity with CD138(-) cells. ③The variable region sequence of the CD138 (5G2) antibody was obtained using molecular cloning technology, and CD138 (5G2) CAR was successfully constructed and expressed on T cells through lentivirus infection and, concurrently, demonstrated effective binding to recombinant human CD138 protein.④ The proliferation of T cells transduced with the CD138 (5G2) CAR was highly efficient. The phenotype analysis revealed that CD138 (5G2) CAR-T cells exhibited a greater tendency to differentiate into central memory T cells and memory stem T cells, with a reduced proportion of terminally differentiated effector memory subsets. ⑤CD138 (5G2) CAR-T cells demonstrated specific cytotoxicity against CD138(+) myeloma cell line H929, whereas CD138(-) cell line K562 remained unaffected. The percentage of residual H929 cells was (12.92±8.02) % after co-culturing with CD138 (5G2) CAR-T cells, while (54.25±15.79) % was left in the Vector-T group (E∶T=1∶2; P<0.001). ⑥Results of degranulation assays demonstrated a significant activation of CD138 (5G2) CAR-T cells after co-culture with the H929 cell line, whereas no significant activation was observed in Vector-T cells [ (25.78±3.35) % vs (6.13±1.30) %, P<0.001]. ⑦After co-culturing with CD138(+) cells, CD138 (5G2) CAR-T cells exhibited a significant increase in cytokine secretion compared to the Vector-T group [interleukin-2: (1 697.52±599.05) pg/ml vs (5.07±1.17) pg/ml, P<0.001; interferon-γ: (3 312.20±486.38) pg/ml vs (9.28±1.46) pg/ml, P<0.001; and tumor necrosis factor-α: (1 837.43±640.49) pg/ml vs (8.75±1.65) pg/ml, P<0.001]. However, no significant difference was observed in cytokine secretion levels between the two groups after co-culturing with CD138(-) cells. Conclusion: This study successfully prepared a novel monoclonal antibody against CD138, and CAR-T cells constructed with the antigen recognition domain derived from this 5G2 mAb demonstrated effective antitumor activity against myeloma cells. This can be used as a new option for the detection of the CD138 antigen and proposes a novel strategy for multiple myeloma immunotherapy.

Immune Therapies in AL Amyloidosis-A Glimpse to the Future.

Light-chain (AL) amyloidosis is a rare plasma cell disorder characterized by the deposition of misfolded immunoglobulin light chains in target organs, leading to multi-organ dysfunction. Treatment approaches have historically mirrored but lagged behind those of multiple myeloma (MM). Recent advancements in MM immunotherapy are gradually being evaluated and adopted in AL amyloidosis. This review explores the current state of immunotherapeutic strategies in AL amyloidosis, including monoclonal antibodies, antibody-drug conjugates, bispecific antibodies, and chimeric antigen receptor T-cell therapy. We discuss the unique challenges and prospects of these therapies in AL amyloidosis, including the exposure of frail AL amyloidosis patients to immune-mediated toxicities such as cytokine release syndrome (CRS) and immune effector-cell-associated neurotoxicity syndrome (ICANS), as well as their efficacy in promoting rapid and deep hematologic responses. Furthermore, we highlight the need for international initiatives and compassionate programs to provide access to these promising therapies and address critical unmet needs in AL amyloidosis management. Finally, we discuss future directions, including optimizing treatment sequencing and mitigating toxicities, to improve outcomes for AL amyloidosis patients.

Role of Immune Cells and Immunotherapy in Multiple Myeloma.

The clinical signs of multiple myeloma, a plasma cell (PC) dyscrasia, include bone loss, renal damage, and paraproteinemia. It can be defined as the uncontrolled growth of malignant PCs within the bone marrow. The distinctive bone marrow milieu that regulates the progression of myeloma disease involves interactions between plasma and stromal cells, and myeloid and lymphoid cells. These cells affect the immune system independently or because of a complicated web of interconnections, which promotes disease development and immune evasion. Due to the importance of these factors in the onset of disease, various therapeutic strategies have been created that either target or improve the immunological processes that influence disease progression. The immune system has a role in the mechanism of action of multiple myeloma treatments. The main contributions of immune cells to the bone marrow microenvironment, as well as how they interact and how immune regulation might lead to therapeutic effects, are covered in this study.

Current status and challenges of immunotherapy for multiple myeloma

Multiple myeloma (MM) is the second most common hematologic malignancy and the incidence of MM in mainland China in 2016 was 1.15/100 000.With the development of China's aging society, the incidence of MM is expected to increase year by year. Immunotherapy for MM has become the fourth pillar of therapy after autologous hematopoietic stem cell transplantation, immunomodulators, and proteasome inhibitors, and is the most active area of MM treatment. Nine new drugs have been approved for multiple myeloma treatment in China, and three are expected to be approved in 2024, which will focus on immunotherapy. There are many ambiguities about the current status of research and utilization in this emerging field in China. Determining the optimal integration of these therapies into the treatment regimen for Chinese MM patients constitutes a critical challenge for clinicians. Immunotherapy for MM primarily encompasses two major categories: antibody-based drug therapy and cellular immunotherapy. Antibody-based medications primarily include monoclonal antibodies, T-cell engagers, IgG-like bispecific antibodies, and trispecific antibodies. Cellular immunotherapy mainly consists of chimeric antigen receptor T (CAR-T) cells, as well as other immune cells such as chimeric antigen receptor natural killer (CAR-NK) cells, dendritic cells, T cell receptor-engineered T cells, and peptide vaccines.This article mainly focuses on the current research status and existing issues of the aforementioned immunotherapy methods, with the aim of providing references for the treatment of MM.

Fc receptor-like 5 (FCRL5)-directed CAR-T cells exhibit antitumor activity against multiple myeloma

Multiple myeloma (MM) remains a challenging hematologic malignancy despite advancements in chimeric antigen receptor T-cell (CAR-T) therapy. Current targets of CAR-T cells used in MM immunotherapy have limitations, with a subset of patients experiencing antigen loss resulting in relapse. Therefore, novel targets for enhancing CAR-T cell therapy in MM remain needed. Fc receptor-like 5 (FCRL5) is a protein marker with considerably upregulated expression in MM and has emerged as a promising target for CAR-T cell therapeutic interventions, offering an alternative treatment for MM. To further explore this option, we designed FCRL5-directed CAR-T cells and assessed their cytotoxicity in vitro using a co-culture system and in vivo using MM cell-derived xenograft models, specifically focusing on MM with gain of chromosome 1q21. Given the challenges in CAR-T therapies arising from limited T cell persistence, our approach incorporates interleukin-15 (IL-15), which enhances the functionality of central memory T (TCM) cells, into the design of FCRL5-directed CAR-T cells, to improve cytotoxicity and reduce T-cell dysfunction, thereby promoting greater CAR-T cell survival and efficacy. Both in vitro and xenograft models displayed that FCRL5 CAR-T cells incorporating IL-15 exhibited potent antitumor efficacy, effectively inhibiting the proliferation of MM cells and leading to remarkable tumor suppression. Our results highlight the capacity of FCRL5-specific CAR-T cells with the integration of IL-15 to improve the therapeutic potency, suggesting a potential novel immunotherapeutic strategy for MM treatment.

Advances in adoptive cellular immunotherapy in multiple myeloma

Advances in adoptive cellular immunotherapy in multiple myeloma

Synergistic integration of histone deacetylase inhibitors apparently enhances the cytokine-induced killer cell efficiency in multiple myeloma via the NKG2D pathway.

The rapid recognition of epigenetic manipulation's potential in restricting cancer cell capabilities spurred translational initiatives, including histone deacetylase inhibitors (HDACis). Clinical trials on multiple myeloma (MM) demonstrated substantial benefits of HDACis, coupled with promising outcomes from cytokine-induced killer cell (CIK) immunotherapy. Intriguingly, the unexplored synergy of HDACis and CIK cell immunotherapy in MM prompted our study. We examined clinically relevant HDACis (panobinostat/LBH589 and romidepsin) alongside CIK cells derived from peripheral blood mononuclear cells across diverse MM cell lines (U266, RPMI8226, OPM-2 and NCI-H929). Utilising various in vitro methodologies, we investigated how HDACis enhance CIK cell lysis of myeloma cells through NKG2D/NKG2D ligand interactions. The results of our analysis indicated several key findings. (1) Enhanced cytotoxicity of CIK cells in MM cells when combined with HDACis. (2) Significant increase in apoptosis, suggesting HDACis and CIK may together enhance apoptotic effects in specific MM cell lines. (3) Elevated IFN-γ secretion and alterations in granzyme Bsecretion because of the independent activity of HDACis. (4) Notably, HDACis increased the expression of MICA/B and ULBP2, crucial for inducing antitumor cytotoxicity of NKTcells. Validation through NKG2D receptor blocking in CIK cells with a purified mouse antihuman NKG2D antibody further supported our findings. Our analyses provide sufficient evidence to consider this clinically forgotten instance (HDACis-CIK cell combination) as a therapeutic priority for MM treatment. Furthermore, we suggest that NKG2D/NKG2D-ligand interactions activating NK/NKTcells may contribute to enhanced myeloma cell lysis in response to HDACis treatment by CIK cells.

Role of Natural Killer T (NKT) Cells in Myeloma Biology and Therapy.

Natural Killer T (NKT) cells are distinct innate lymphocytes that recognize lipid antigens in the context of nonpolymorphic molecule CD1d. Multiple myeloma (MM) is a hematologic malignancy wherein malignant plasma cells express CD1d and are sensitive to lysis by NKT cells. Progressive malignancy in MM is characterized by NKT cell dysfunction. Several studies have tried to harness the anti-tumor properties of NKT cells in MM to mediate tumor regression. NKT cells are also attractive targets for approaches at immune redirection in MM with chimeric-antigen receptor NKT (CAR-NKT) and bispecific antibodies. In addition to the commonly studied invariant-NKT (iNKT) cells, MM patients often also exhibit alterations in type-II NKT cells and their ligands. In patients and mouse models with Gaucher disease (GD), an inherited lipid-storage disorder with markedly increased risk for MM, distinct type-II NKT cells exhibit a T-follicular helper (NKT-TFH) phenotype and provide help to lipid-specific B cells. Chronic immune activation in this setting eventually sets the stage for malignancy, which can be targeted in both mouse models and GD patients by reducing the underlying antigen. NKT cells are thus integrally linked to MM pathogenesis and an attractive target for MM immunotherapy.

Novel therapies for multiple myeloma

B-cell maturation antigen (BCMA)-targeting therapy is the most common approach to immunotherapy and cellular therapy for multiple myeloma (MM). Three major agents, CAR-T cells, bispecific antibodies, and ADC have been developed as novel therapeutic agents. CAR-T therapy showed favorable efficacy in the treatment of relapsed and refractory MM (RR MM) and was tried in early lines of therapy. Similarly, bispecific antibodies targeting BCMA or other targets have also shown promising effects in treatment of RR MM, and have been now tested in combination with other agents. Although issues such as poor fitness or exhaustion of T cells and increased susceptibility to viral infection remain to be fully resolved, novel immunotherapies and cellular therapies should further improve the prognosis of patients with RR MM.

Impaired FADD/BID signaling mediates cross-resistance to immunotherapy in Multiple Myeloma

The treatment landscape in multiple myeloma (MM) is shifting from genotoxic drugs to immunotherapies. Monoclonal antibodies, immunoconjugates, T-cell engaging antibodies and CART cells have been incorporated into routine treatment algorithms, resulting in improved response rates. Nevertheless, patients continue to relapse and the underlying mechanisms of resistance remain poorly understood. While Impaired death receptor signaling has been reported to mediate resistance to CART in acute lymphoblastic leukemia, this mechanism yet remains to be elucidated in context of novel immunotherapies for MM. Here, we describe impaired death receptor signaling as a novel mechanism of resistance to T-cell mediated immunotherapies in MM. This resistance seems exclusive to novel immunotherapies while sensitivity to conventional anti-tumor therapies being preserved in vitro. As a proof of concept, we present a confirmatory clinical case indicating that the FADD/BID axis is required for meaningful responses to novel immunotherapies thus we report impaired death receptor signaling as a novel resistance mechanism to T-cell mediated immunotherapy in MM.

Secondary Genetic Events Impact the Expression of Key Immunotargets on the Surface of Multiple Myeloma Cells

Background: Multiple myeloma (MM) is characterized by complex genomic aberrations including primary translocations of the immunoglobin heavy chain (IGH) locus and secondary mutations involving key oncogenic drivers. This temporal complexity represents a constant challenge for the clinical management of MM, but at the same time may provide new avenues for immunotherapy-based approaches including CAR-T cells and T-cell engaging antibodies. In this study, we investigated the impact of secondary genetic events on the surface expression of common targets for immunotherapy in MM. Methods: Cell models harboring two hotspot activating mutations in KRAS (G12A, A146T) and TP53 (R282W) were created using the MM cell lines OPM-2 and AMO1 via CRISPR-Cas9 technology. Direct stochastic optical reconstruction microscopy ( dSTORM) was used for high-resolution receptor density estimation and epitope quantification of common immunotherapeutic targets such as CD38, BCMA and SLAMF7. Results: dSTORM imaging of cell line models carrying patient-derived secondary genetic alterations revealed a distinct pattern linked to each mutation respectively. In KRAS G12A, we observed a reduction in BCMA density by 2-fold as compared to wild type cells (5.9 ± 0.3 vs. 10.8 ± 1.2 localization clusters/µm 2, p=0005). This reduction in BCMA surface expression was accompanied by a decrease in other key immunotargets, including SLAMF7 (6.0 ± 0.8 vs 11.8 ± 0.8 localization clusters/µm 2, p &amp;lt;0,0001) and CD38 (12.2 ± 1.5 vs 17.9 ± 1.6 localization clusters/µm 2, p=0,012). In KRAS A146, CD38 density increased, whereas mean BCMA and SLAMF7 localizations were consistently reduced as compared to wild type control. To ensure that BCMA downregulation is a consistent feature in genomic high-risk disease, we next simulated a TP53 double-hit situation by introducing an inactivating hotspot mutation R282W in a TP53 deletion background. Surfaceome profiling by dSTORM revealed a minor increase in CD38 levels for TP53R282W/Del as compared to wild type control (51.5 ± 5.7 vs. 36.4 ± 4.2 localization clusters/µm 2, p=0.06). Interestingly, the expression level of BCMA, known to be comparably low on WT tumor cells, further decreased in TP53 double-hit cells (0.6 ± 0.1 vs 1.1 ± 0.1 localization clusters/µm 2, p=0.002), whereas no major differences in the cluster density of SLAMF7 were observed. This leaves room for speculation whether these effects on biallelic TP53 inactivated tumor cells may underlie reduced susceptibility towards BCMA-targeting therapy. Of note, we also observed altered cell morphology in the TP53 double-hit model, characterized by elongated stress fibers enriched with CD138, potentially interfering with receptor accessibility of surface receptors (Figure 1). Recently, secondary genetic events have been reported in BCMA and GPRC5D following targeted immunotherapy inducing acquired drug resistance. We created cell models representing homo- and heterozygous genetic alterations in BCMA and GPRC5D, studies to assess their effects on the surfaceome are ongoing and will be presented at the meeting. Summary: Our data suggest that surface epitope expression in MM cells seems to be less agnostic to secondary genomic events than expected. These findings may provide a direct link between genetic high-risk disease and inferior efficacy of anti-BCMA immunotherapies in the context of e.g. TP53 double-hit MM.

Chimeric Antigen Receptor T Cells Targeting LILRB4, an Immunoreceptor Mediating T-Cell Suppression, Are Potently Effective in Multiple Myeloma

Significant progress has been made in the treatment of multiple myeloma (MM) however, no strategy to date has proven curative and relapse invariably occurs. We recently developed an integrated pipeline to identify biologically and therapeutically relevant targets for MM immunotherapy by using Mass-Spectrometry analyses from seven MM cell lines and RNA-seq data from 900+ patients. Starting from 4,000+ candidates, ILT3 aka LILRB4, an immunoreceptor tyrosine-based inhibition motif-containing receptor emerged as a promising candidate target: it has absent/low expression in most normal tissues and high and frequent expression in many primary patient samples we used for validation, similarly to BCMA (Di Meo et al., Cell Reports Medicine 2023). We sought to examine its functional role in MM and develop Chimeric Antigen Receptor (CAR) T-cells targeting LILRB4. Interrogating RNA-seq data from MM patients, LILRB4 expression results higher in patients with high-risk cytogenetics such as t:(14;16), t:(14;20), and t:(8;14) translocations compared to patients with no genomic abnormalities. Investigating function, we find no difference between cell proliferation of U266/KMS11/OPM2 genetically depleted of LILRB4 (KO) compared to controls by measuring cell growth in vitro for 120 hours and MTS assays. Similarly, when KO and control cells are seeded in plastic chambers containing a porous membrane and serum-free medium, both KO and control cells are attracted to the lower chambers containing FBS, suggesting that LILRB4 is also not required for MM cell migration. Despite these initial observations, RNAseq data from LILRB4 KO and overexpressing (OE) U266 MM cells identify ~3,000 differentially expressed genes compared to controls. Thus, we next investigate potential extrinsic effects on T-cell proliferation by co-culturing T-cells from healthy donors with LILRB4 KO or OE U266 MM cells (T:E ratio 1:2). T cells are previously activated with anti-CD3/CD28-coated beads and IL-2. 5 days after activation, T cells and LILRB4 KO U266 MM are seeded in the same well plate. After 48h of co-culture, T-cell proliferation is decreased (-30%) in the presence of LILRB4 U266 OE MM cells, whereas it is increased (+50%) when the co-culture includes LILRB4 U266 KO cells. CD25 expression is also reduced by 20% on T cells co-cultured with LILRB4 OE cells and increased by the same extent when T cells are co-cultured with LILRB4 KO cells, suggesting a role as a negative immune receptor. This effect seems dependent on the direct cell-cell interaction given that when T and MM cells are separated by a transwell in co-culture, no effect on the T cell proliferation is observed. Finally, we develop multiple Chimeric Antigen Receptor (CAR) constructs targeting LILRB4, by using different single-chain variable fragments (scFVs) and rearranging the variable regions of the heavy (VH) and light (VL) regions. We used lentiviral LILRB4 scFV-41BB-CD3zeta vectors. LILRB4 CAR T-cells specifically kill U266, H929 and KMS11 MM cell lines in vitro with cytotoxicity ranging from 30 to 80% depending on the construct (T:E ratio 1:10). Equal efficacy is observed in U266 cells resistant to Bortezomib and BCMA KO U266cells. No effect on LILRB4 null cells is noted. These data suggest that LILRB4 is a negative immune receptor that is active on malignant plasma cells mediating T-cell suppression, similar to previously described data in leukemia (Deng et al., Nature 2018). Our work supports the rationale development of ILT3 CAR-T cell therapy as a viable therapeutic approach for MM patients especially high-risk patients. Ongoing studies are investigating the functional role of LILRB4 in mediating the cross-talk between MM ad immune cells.

Optimal, Off-the-Shelf, CAR-iNKT Cell Platform-Based Immunotherapy for Multiple Myeloma

Autologous CAR-T cell immunotherapy targeting BCMA confers significantly improved progression free survival as compared to standard-of-care in relapsed/refractory multiple myeloma. However, despite &amp;gt;50% complete remission rates, patients continue to relapse. Of note, in current licenced BCMA CAR-T products, 4-1BB comprises the co-stimulatory domain of a 2 nd generation CAR. Expression of CD1d by myeloma plasma cells (PC) lends itself to immunotherapy with the CD1d-restricted, glycolipid-reactive iNKT cells which can be deployed ‘off-the-shelf’ without causing aGVHD. We hypothesised that ‘off-the-shelf’ anti-BCMA CAR-iNKT would be an alternative platform for the treatment of MM. We sought to identify the CAR design that would function optimally in conjunction with iNKT cells. To this end, we designed several different second (28z, 4-1BBz, OX40z) and third (28z-4-1BBz, 28z-OX40z) generation BCMA CARs and tested their anti-myeloma activity. Lentiviral transduction efficiency of iNKT cells was comparable for all five CARs and all five CAR-iNKT were able to kill two BCMA-expressing but not BCMA knock out myeloma cell lines in short- and long-term cytotoxicity assays. The level of cytotoxicity varied with donor, timing of the assay and cell lines, with the only consistent pattern seen with the 3 rdgeneration CD28-4-1BB CAR. This showed the least reactivity at low E:T ratios in both 4 and 16hrs assays for both donors tested. Testing cytotoxicity against primary myeloma PC (n=3) showed that the 3 rd gen CARs were less active than the three 2 nd gen counterparts. Short term proliferation assays using Incucyte Zoom demonstrated a higher proliferative potential of CD28z CAR-iNKT cells with CD28z-4-1BBz CAR showing the least proliferation. In a longer term 35-day assay, following stimulation with the iNKT cell ligand aGalCer, we found that CD28z CAR-iNKT cells showed the highest expansion. To further probe functional differences between the five CARs, we tested the corresponding CAR-iNKT cells in acoustic force avidity assays. These highlighted CD28z CAR-iNKT cells as having the highest avidity amongst CAR-iNKT cells with untransduced iNKT cells the lowest (Fig. 1A ). Using confocal microscopy, we explored how these differences in avidity impacted the ability of CAR-iNKT cells to shape the immune synapse (IS) and engage the cytolytic apparatus. We found that while conjugates of untransduced iNKT cells with myeloma cells all fell in the distal centrosome group, of the five CARs, CD28z and 4-1BBz CAR-iNKT conjugates recorded the highest and the lowest respectively proximal and docked conjugates, i.e., those signifying imminent or active discharge of the cytolytic granules. To delve further into the mechanism(s) of higher avidity and ability to established IS by CD28z CAR-iNKT, we compared the transcriptomes of CD28z vs 4-1BBz CAR-iNKT from three donors. We found 64 genes differentially expressed (14 up- and 50 down-regulated in CD28z CAR-iNKT, padj&amp;lt;0.05). Amongst the upregulated genes we identified, currently investigating genes that encode proteins that were previously implicated in the strength and duration of IS. To investigate the in vivo correlates of these functional assays, we deployed a xenograft myeloma model using MM1.S luciferase-expressing cells (Fig. 1B ). All five CAR iNKT cells were i.v transferred to myeloma-bearing mice at the limiting dose of 10 6 CAR+ cells/mouse and disease burden was monitored by bioluminescence. In line with the functional assays, we found that mice receiving CD28z CAR-iNKT cells demonstrated the lowest disease burden and survived the longest (p&amp;lt;0.01). While disease burden and survival of animals receiving CD28z-4-1BBz CAR-iNKT was the same as controls, 4-1BBz CAR-iNKT-treated mice showed significantly higher disease burden and shortened survival as compared to CD28z CAR-iNKT. Further disease burden assessment by flow-cytometry at sacrifice showed that CD28z CAR-iNKT-treated animals had the lowest (p&amp;lt;0.01) frequency of myeloma cells in their peripheral blood, bone marrow and spleen. Finally, in ongoing xenograft assays, we find that compared to 10 6 28z BCMA CAR-iNKT and 5x10 6 untransduced iNKT, 5x10 6 28z BCMA CAR-iNKT cells maintain better disease control (p=0.008, day7). We conclude that CD28z by enhancing effector-target avidity and strength of IS constitutes the optimal CAR design for clinical development of CAR-iNKT therapy of multiple myeloma.

Immune checkpoint inhibitors for multiple myeloma immunotherapy.

Multiple myeloma (MM) is related to immune disorders, recent studys have revealed that immunotherapy can greatly benefit MM patients. Immune checkpoints can negatively modulate the immune system and are closely associated with immune escape. Immune checkpoint-related therapy has attracted much attention and research in MM. However, the efficacy of those therapies need further improvements. There need more thoughts about the immune checkpoint to translate their use in clinical work. In our review, we aggregated the currently known immune checkpoints and their corresponding ligands, further more we propose various ways of potential translation applying treatment based on immune checkpoints for MM patients.

Lipid Handling Induces Ferroptosis and Conveys Sensitivity to Anti-BCMA Immunotherapy in Multiple Myeloma

Lipid metabolic changes in cancer are attracting increased attention. BAFF/APRIL axis is a multifaceted immune regulator of adipose tissue function and an emerging target for anti-BCMA therapy in multiple myeloma (MM). However, lipid analyses are scarce in MM onset and response to treatment. First, we measured metabolites and proteins in sera obtained from matched bone marrow and peripheral blood of 17 newly diagnosed and 9 relapsed/refractory MM patients, by LC-MS/MS. String analysis identified crucial pathways including fibrinogen regulation, inflammatory response, structural integrity of lipoproteins and lipid transport, with a significant increase of apoliproteins APO-A1 (key component of HDL particle and cofactor for lecithin-cholesterol acyltransferase (LCAT) which is responsible for the formation of most plasma cholesteryl esters), A2,C2 (which hydrolyzes triglycerides to provide free fatty acids for cells), C3, components of HDL and LDL, confirming a deranged lipid handling in RR-MM patients. Second, we studied the lipid metabolic changes upon anti-BCMA exposure with Elranatamab (EL), an investigational, off-the-shelf, humanized BCMA-CD3-directed bispecific antibody engineered to elicit potent T-cell-mediated anti-MM activity. Combining in vitro assays, FACS, qRT-PCR, immunofluorescence, Western Blot and RNA-seq we evaluated the response to EL treatment in 3 human myeloma cell lines U266, NCI-H929 and OPM2 (3 cell lines chosen for their different basal bioenergetics profile). MM cells were co-incubated with various concentrations of EL in presence of CD3+T cells isolated from healthy donors in 96- well U-bottom plates for 4 h and 24 h at an E:T ratio of 1:5 and 1:10, then stained with anti-CD138-PE antibody . CD138 + cells were detected by flow cytometry and counted as surviving MM cells. BODIPY™ 581/591 C11 (Lipid Peroxidation Sensor) and BODIPY 493/503 for Neutral Lipid Droplets Staining for FACS and immunofluorescence were used to quantify lipid peroxidation and cytoplasmatic LDs on surviving CD138 + cells. Despite the BCMA target was equally expressed on U266, NCI-H929 and OPM2, the sensitivity to EL was different, inducing cell cycle arrest in the G2/M phase in U266 and NCI-H929 cell lines but not in OPM2, in E:T ratio independent-manner. The expression of the activation markers CD69 and CD25 on T cells and the release of cytokines by T cells were target-dependent and occurred only in the presence of BCMA-positive cells. Oil-Red-O staining revealed an increased lipid content after EL exposure in all tested MM cell lines, with OPM2 the richest in basal lipids content. To dissect the contribution of lipophagy versus lipolysis we performed an autophagy flux analysis by western blot and RNA-seq essay, showing that lipolysis was the emerging response to EL treatment. In EL-sensitive U266 we found an increase of lipid peroxides, hallmark of ferroptosis, a form of cell death due to missed control of membrane lipid peroxidation, a phenomenon negligible in EL-resistant OPM2. Lipidomics demonstrated higher levels of monounsaturated fatty acids (MUFA) than polyunsaturated fatty acids (PUFA) in OPM2, associated to an increased expression of Stearoyl CoA desaturase 1 (SCD1), the enzyme that catalyzes the rate-limiting step in the production of MUFA. While the PUFA oxidation is pro-ferroptotic, MUFA activation promotes a ferroptosis-resistant cell state. Indeed, a ferroptosis-primed resistant phenotype in EL-sensitive U266 was associated to downregulation of lipid peroxides scavengers GPX4 and SLC7A11 and up-regulation of pro-ferroptotic acyl-CoA synthetase long-chain family member 4 (ACSL4). Exogenous MUFA (via supplementation with palmitic acid 125 µM conjugated to BSA 1:6) alleviated ferroptosis induction in U266 making them resistant to EL, a phenotype associated to overexpression of CD36, the main free fatty acids (FFA) transporter. Conversely, in OPM2, inducing iron-dependent lipid peroxidation through RSL3 (3 µM) treatment rescued EL resistance, resulting in a time-dependent increase in cytosolic lipid peroxidation beginning at 6 hours, and overt cell death via ferroptosis at 18 hours. Taken together, our findings suggest that MUFA/PUFA ratio drives ferroptosis and mediates EL sensitivity, addressing lipid handling as a novel means to tailor immunotherapy in RRMM.

Single Cell Profiling Unravels Plasma Cell Gene Expression Signatures Predictive of Response to Daratumumab Based Therapy, and Prognosis in Multiple Myeloma

Background: Multiple Myeloma (MM) is the second most common hematologic malignancy and remains incurable. Daratumumab (dara) is a potent anti-CD38 monoclonal antibody used for MM treatment, but responses are heterogeneous, and resistance is inevitable. There is hence an urgent need for biomarkers to predict the response to dara. We hypothesized that gene expression profiles (GEP) of tumor subpopulations could be used to predict the response to dara-based therapy. Methodology: We used the Enhanced Single-Cell Analysis with Protein Expression (ESCAPE) RNA-Seq platform (Singleron Biotechnologies) to simultaneously profile the gene and protein expression of plasma cells from the bone marrow of 15 MM patients prior to treatment with dara-based regimens. These data were combined with publicly available single-cell data from Cohen et al. 2021, resulting in a total of 32 samples. Response was defined based on the international myeloma working group (IMWG) criteria, considering patients achieving a very good partial response (VGPR) or better as “responders,” while those achieving a partial response (PR) or worse were deemed “non-responders”. The data were split into 23 training samples (15 responders and 8 non-responders) and 9 validation samples (3 responders and 6 non-responders), each set containing a combination of cases from the two sources of single-cell data. An interpretable machine-learning model was then built to predict the response of each individual cell. The median response score for each sample was used to predict the patient-level response. The Shapley Additive Explanations (SHAP) values from the dara response prediction model were then used as a starting point for creating a general prognostic model. The 200 most influential genes from the dara response prediction model were used to create a linear response model based on the TCGA (MMRF-Compass) bulk RNAseq dataset. The bulk RNAseq dataset was split 80:20 into training and test datasets. Results from the test dataset are presented here. Results: We analyzed single-cell data from a total of 13,094 plasma cells from 32 BM samples. These included 17 cases of relapsed MM described by Cohen et al., who were treated with dara, carfilzomib, lenalidomide, and dexamethasone. The patients treated at our centre comprised 7 cases of relapsed MM treated with dara, thalidomide, and dexamethasone, and 8 samples from patients with newly diagnosed MM treated with dara, bortezomib, and dexamethasone on clinical trials. All 9 patients in the test dataset had their responses correctly predicted (Fig 1). A separate cohort of non-dara-treated patient samples was also tested using the same model, and the response prediction was not accurate (AUC=0.67), suggesting that this model has specificity for predicting response to dara and is not a general response classifier (data not shown). Based on the SHAP values from the dara response prediction model, we developed a prognostic model from bulk RNAseq data consisting of four genes. A combination of this four-gene signature plus the international staging system (ISS) score resulted in a superior prediction of overall survival compared to the ISS alone with a hazard ratio of 13.5 compared to 2.5 for the ISS alone in the same dataset (Fig 2). Conclusions: Combining single-cell RNA sequencing with machine-learning methods may have value in predicting response to immunotherapy in MM. We propose that gene signatures derived from single-cell data may augment clinical decision-making for determining treatment allocation. We also show that these signatures may be combined with existing prognostic scores, resulting in improved delineation of risk groups. Furthermore, the applicability of our gene signature to a bulk gene expression dataset enhances its clinical relevance. Our study demonstrates the power of single-cell omics to identify novel predictors of response to therapy and prognosis in MM, which may be translated into clinical use.

CD155 on Multiple Myeloma Cells Is Epigenetically Regulated, Reduce Patients Survival and Compromises the Action of Novel Immunotherapies

Epigenetics, especially DNA methylation, is a key molecular regulator of gene expression. This regulation is important for cytotoxic cell activation (i.e., T-cells and NK cells) and is crucial for the clearance of malignant cells. Immune checkpoint (IC) events mediate cytotoxicity, one IC receptor involved in the process is CD155. Such receptor interacts primarily with TIGIT on cytotoxic cells and induces the inhibition of cytotoxicity. Multiple myeloma (MM) represents the 10% of all hematological malignancies and it has been described that MM cells hinder cytotoxic cell activation by inhibition and exhaustion. Furthermore, novel immunotherapeutic strategies such as Bispecific T-cell Engagers (BiTE) and Chimeric Antigen Receptor (CAR) therapies are being evaluated in MM treatment, with promising results. Nevertheless, they will confront a hostile environment. This is a very active field with frequent descriptions of new targets and therapeutic approaches being studied each day. We investigated whether CD155 is epigenetically regulated in MM and its importance in inducing inhibition of anti-tumor response in the context of novel immunotherapies through the CD155/TIGIT axis. To achieve our objectives, we focused on the promoter region of CD155 (the region up to 1500 base pairs upstream of the TSS) and characterized it at DNA, RNA and protein levels through bisulfite sequencing, quantitative PCR, and flow cytometry. We confirmed CD155 as an epigenetically regulated gene, thus demonstrating an existing correlation between CD155 promoter methylation and gene expression. In vitro treatment with azacytidine was performed to assess the active repression of CD155 expression by DNA methylation. Six different MM cell lines were characterized (two not expressing: AMO-1, KMS-12-BM, four expressing: RPMI-8226, MM.1S, EJM and JJN-3). From there, we created CD155 depletion models on expressing cell lines by short hairpin RNA interference and developed an in vitro coculture system with T-cells (from healthy donors). We observed a loss of expression between the 60% to 90% (when compared with controls). Depletion of CD155 did not significantly impact cell growth, apoptosis, or cell cycle. On the coculture with healthy T-cells, more cytotoxicity was detected against CD155-depleted cells (in RPMI-8226 Mann-Whitney P=0.020; JJN-3 and EJM P&amp;lt;0.001). To incorporate novel immunotherapeutic strategies, we carried out the same cocultures with 100pg/mL of anti-BCMA/CD3 BiTE, against anti-BCMA CAR-T or anti-BCMA CAR-NK cells (for RPMI-8226 and JJN-3 Mann-Whitney P=0.008). Coculture combinations were also performed in the presence of 10μg/ml of neutralizing αTIGIT (RPMI-8226, Mann-Whitney P =0.005; JJN-3 P=0.004) and/or αPD1 antibody. When novel immunotherapy approaches were included in the system, we observed how the depletion of CD155 was related to a cytotoxicity increase in all cases. In the presence of neutralizing αTIGIT, cytotoxicity was increased against CD155-expressing cells, while we observed no difference against CD155-depleted cells. In the presence of αPD1, we saw a significant restoration of T-cell cytotoxicity against both models and the antibody combination showed a synergic effect in CD155 expressing cells. Finally, we analyzed the existence of a correlation between CD155 gene expression and survival in newly diagnosed MM samples from CoMMpass project public data (N= 793). These data supported our results as the newly diagnosed MM patients with higher expression of CD155 had shorter overall survival than lower expressing ones (Logrank test P&amp;lt;0.001). In summary, in the context of MM, the expression of CD155 is regulated by the methylation status of its promoter region. Expression of CD155 effectively reduces cytotoxic cell response, which is mediated by the interaction of CD155 and TIGIT. The addition of αTIGIT and αPD1 validated that the CD155 cytotoxic cell inhibition is mediated in part by TIGIT interaction, independently of PD1/PD-L1 axis. This interaction seems to impact patients treated with current therapies, since their outcome correlates with CD155 expression. Based on our results, novel immunotherapies focused on enhancing immune cytotoxicity will benefit from CD155/TIGIT axis blockade. These results warrant further investigation on CD155 as a biomarker and target for novel immunotherapies for MM and other malignancies.

Intracytoplasmatic BCMA Point Mutation and Teclistamab Resistance: Genomic Assessment of a Multidrug-Resistant Multiple Myeloma Patient

Introduction Proteasome inhibitors (PIs) are standard drugs for the treatment of Multiple Myeloma (MM), bispecific antibodies (BsAbs) and CART cells are rapidly and successfully being integrated into treatment regimens. Still, several current studies have shown that drug resistance remains one of the biggest clinical challenges. For instance, deletions in BCMA (alias TNFRSF17) were shown to confer a target loss and therefore resistance to BCMA CARTs (Da Via et al. Nat Med 2021, Samur et al. Nat Commun 2021) as well as to a CD3xBCMA bispecific T cell-engager (Truger et al. Blood Adv 2021). Besides the extracellular ligand-binding domain, BCMA further consist of a membrane spanning and a cytoplasmatic region that activates cell functions. To date not much is known in regard to alterations affecting the latter and their role in resistance. Here we present genetic profiling of a multi-refractory MM patient who relapsed to PIs, anti- BCMA (Teclistamab) and anti- GPRC5D (Talquetamab) BsAbs. Methods We performed FISH and a targeted capture-hybridization DNA sequencing panel (tchDNA-Seq) as previously described in Rosa-Rosa et al. Cancers 2022 in two sequential samples: first a bone marrow biopsy at diagnosis and second, 43 months later, a FFPE biopsy of the retroperitoneal mass of a progression plasmacytoma, after PI but prior Teclistamab treatment. The MM-specific tchDNA-Seq panel includes over 100 genes involved in MM progression, drug resistance, immunotherapy and canonical loci to cover IG rearrangements and frequent translocations. In order to predict the impact of the observed gene mutations on the protein structure, we generated 3D projections using the PyMOL molecular visualization system and references from the Protein Data Bank. The KinasePhos3 program was used to predict the most probable kinase to phosphorylate new, by the mutation generated, sites. Results Via FISH, the patient was stratified as a high-risk patient as 13% of the tumor cells showed a del17p at diagnosis, 97% a t(4;14) translocation, and 88% chromosomal gains for 1q21 and 11q13. The patient initially responded well to VRd+HCT. After 3.6 years he relapsed and developed an abdominal plasmacytoma. He was non-responsive to the following Isa-Kd treatment and relapsed again, two months after Teclistamab induction. To the subsequent PomCyDex and Talquetamab treatments he was primary refractory. In the plasmacytoma, four SNVs were detected: PSMC5 p.G152V with a VAF of 42%, BCMA p.C128Y (3%) and the two B2M mutations B2M p.S14F (47%) and B2M p.L107F (11%). At diagnosis, the VAF was 1% for PSMC5 p.G152V and B2M p.S14F, the other two SNVs were absent. In B2M, mutations in residues 13-16 have been associated with impaired protein expression and complex formation (Challa-Malladi et al. Cancer Cell 2011). The PSMC5 SNV was topologically identified within a glycine-rich loop in the ATP binding pocket of the proteasome in direct interaction with the ATP amine group, therefore likely impacting the proteolytic capacity increasing the PI tolerance. B CMA p.C128Y is located in the C-terminal cytoplasmatic tail within a highly conserved 25-aa protein segment (position 119 to 143) that has been shown to be necessary for TRAF association and NF-κB, Elk-1, and JNK activation (Hatzoglou et al. J Immunol 2000). A cysteine is mutated to a tyrosine. With high probability, this new site will be phosphorylated by kinases of the c-Src family (score 0.92), e.g. Fyn (0.71) or Lyn (0.66). Moreover, this new phosphotyrosine would become a binding site for the kinase SH2 domain (Diop et al. Int J Mol Sci 2022). Conclusions Here we report emerging mutations in PSMC5 and BCMA, that are likely causal for PI and Teclistamab resistance. PSMC5 p.G152 represents a mutational hotspot as four more PI-resistant patients with substitutions were recently reported in Haertle et al. Cancers 2023. The intracytoplasmatic BCMA SNV creates a new kinase phosphorylation and binding site resulting in aberrant downstream signaling. B2M forms part of the MHC1 molecules, responsible for antigen presentation to CD8+ T cells and the B2M serum level is a prognostic indicator in MM. However, the role of B2M alterations in disease progression and drug resistance needs to be elucidated. Predicting the biological impact based on 3D structural models remains speculative, thus, functional confirmation introducing the BCMA and B2M alterations in cell line models by genetic engineering is pending.