Challenges for CAR-T cell therapy in multiple myeloma: overcoming the tumor microenvironment

Abstract

Chimeric antigen receptor T (CAR-T) cell therapy has shown promising efficacy in multiple myeloma (MM) patients, leading to FDA approval of two B cell maturation antigen (BCMA)-specific CAR-T cell therapies (ide-cel and cilta-cel). Despite the remarkable response rates and response depth of MM patients to CAR-T cell therapy, patients inevitably relapse. A growing body of evidence suggests that the activity of CAR-T cells is affected by the immunosuppressive tumor microenvironment (TME). In this review we have summarized the main challenges that CAR-T cells face in the TME, including various immunosuppressive cells, structural components, hypoxia, nutrient starvation, and metabolism. Moreover, we also discussed some candidate strategies for CAR-T cell therapy to overcome immunosuppressive TME and improve the efficacy of CAR-T cell therapy in the treatment of MM.

Main article text

1. INTRODUCTION

Chimeric antigen receptor T (CAR-T) cell therapy exhibit impressive response rates in relapsed and refractory multiple myeloma (MM) patients, which led to FDA approval of two B cell maturation antigen (BCMA)-specific CAR-T cell therapies (ide-cel and cilta-cel) [1–4]. Other candidate CAR-T cell targets for MM include signaling lymphocytic activation molecule F7 (SLAMF7) [5], CD38 [6], CD138, CD19 [7], Lewis-Y, CD56, CD44v6, Kappa-light chain, and G-protein-coupled receptor class C group 5 member D (GPRC5D) [8]. The availability of these CAR-T cell products offers the hope of disease control and remission for patients lacking therapeutic options; however, the durable response has only been observed in a small group of patients [9]. Multi-functional TRUCK T cells (T-cells redirected for antigen unrestricted cytokine-initiated killing) or ‘the fourth-generation’ CAR T cells are now being evaluated in preclinical settings [10, 11]. The fourth-generation CAR-T cells incorporate cytokine-secreting vectors, such as IL-12, IL-18, or transcription factors-producing vectors, to modulate the tumor microenvironment (TME) and enhance the activation of CAR-T cells. Advances in genome editing techniques, such as CRISPR/CAS9, allows any array of genes in the genome to be incorporated or knocked out, which results in more individualized, efficient, and less toxic CAR-T cell products [12–14].

By combining the ability to recognize tumor-specific antigens in a non-major histocompatibility complex (MHC)-restricted manner with potent activation of T cells to kill the tumor cells, CAR-T cell therapy is considered one of the most promising choices for achieving a cure in MM patients; however, the efficacy of CAR-T cell therapy for hematologic malignancies is largely influenced by the TME [10, 15, 16]. The MM TME contains immunosuppressive cells, an extracellular matrix (ECM), and vasculature components that facilitate the development of tumors. The oxygen concentration and nutrient imbalance inhibit the activity of CAR-T cells. In this review, we summarized the challenges the TME poses to CAR-T cells, with a particular emphasis on MM intrinsic factors, including immunosuppressive cells, structural components, hypoxia, nutrient starvation, and metabolism. Moreover, we also discussed efforts underway to overcome these TME challenges in treating MM, taking into account the latest advances in the next-generation CAR-T cells and single-cell sequencing technology in recent years.

2. OVERCOMING IMMUNOSUPPRESSIVE CELLS

Impairment of immunosurveillance and immune effector function of the host immune system, including both adaptive and innate immunity, leads to immune escape in MM, and contributes to tumorigenesis and tumor progression [17]. Anti-tumor responses of the main effector immune cells, including natural killer (NK) cells [18] and cytotoxic T lymphocytes (CTLs) [19], are impaired in the TME. In addition, MM often induces an immunosuppressive TME, which promotes the expansion of myeloid-derived suppressor cells (MDSCs) [20], tumor-associated macrophages (TAMs) [21], and regulatory T cells (Tregs) [22]. Anti-inflammatory cytokines and chemokines secreted by these immunosuppressive cells, in turn, foster the TME and further promote immune escape and tumor growth [23, 24].

2.1 MDSCs

As a highly heterogeneous group of immature myeloid cells, MDSCs are capable of suppressing immune responses [25, 26]. There are two major subsets of MDSCs, as follows: monocytic-MDSCs (M-MDSCs), which are morphologically and phenotypically similar to monocytes; and polymorphonuclear or granulocytic-MDSCs (PMN-MDSCs), which resemble neutrophils. A subset of more immature progenitors (Lin-CD33+ cells) are a third group of MDSCs, and defined as “early-stage MDSCs (eMDSCs)” [27]. Previous studies have reported an increased number of MDSCs in MM patients compared to healthy donors [28]. In addition, an elevated level of peripheral blood (PB) monocytes correlates with poor prognosis in patients with solid tumors [29]. The humoral and cellular immunosuppressive effects of MDSCs have been elucidated in various hematologic malignancies, with T cells serving as the major targets [27]. MDSCs inhibit T cell activation by secreting reactive oxygen species (ROS) and nitric oxide (NO), and by scavenging amino acids, including arginine, cysteine, and tryptophan, which are essential for T cell proliferation [27, 28]. MDSCs also produce IL-10 and TGF-β to induce Tregs [15]. Moreover, the presence of MDSCs is correlated with patient outcome following treatment with with CAR-T cell therapy. In a phase I/IIa trial involving third-generation CAR-T cells, lymphoma and leukemia patients with lower levels of M-MDSCs had better responses than patients with higher levels of M-MDSCs [30].

Considering the adverse effect of MDSCs on CAR-T cell cytolytic function and proliferation, it is reasonable to designate MDSCs as a treatment target along with CAR-T cell therapy. Effective strategies can target MDSCs through MDSC depletion and deactivation, as well as blocking MDSC recruitment to the TME [25, 27]. As a proof of concept, it has been observed that complete depletion of MDSCs following anti-Gr-1 antibody treatment in EL-4 tumor-bearing mice results in improved anti-tumor efficacy of CAR-T cells [31]. A high amount of arginase-1 is expressed in activated MDSCs, thus revealing that the MDSC suppressive mechanisms are reversed by an inhibitor of arginase-1 in MM [28, 32]. Furthermore, expansion of MDSCs driven by granulocyte-macrophage colony-stimulating factor (GM-CSF) and STAT3 signaling, thus GM-CSF neutralization and STAT3 inhibition may be alternative targets for limiting the immunosuppressive activity of MDSCs [33]. Furthermore, accumulation of MDSCs is significantly reduced by targeting chemokines and cytokines, such as IL-17, exosomal CD47, and S100A8/S100A9, which have important roles in the trafficking of MDSCs to the TME [34]. To better understand the immune changes in the MM TME, Zavidij et al. [19] applied single-cell RNA sequencing (scRNA-seq) to profile the MM TME. Significant up-regulation of HLA-DR was noted for CD14+ monocytes in MM patients compared to healthy controls; however, the surface expression of CD14+ was significantly lower in MM patients. Such a phenotypic change in TME T CD14+ monocytes towards MDSCs is rescued after treatment with MARCHF1/MARCH1 knockdown.

2.2 TAMs

TAMs represent one of the main immune cells in the TME, the accumulation of which in the MM TME is associated with poor prognosis [35]. Based on the activation status, TAMs can be classified into M1 and M2 macrophages [36]. A newer terminology to describe TAMs has been proposed as “M1-like” and “M2-like” TAMs; the former is associated with pro-inflammatory role and “M2-like” TAMs have an anti-inflammatory and wound-healing role in the TME. “M1-like” TAMs are induced by type-1 cytokines, such as IFN-γ and TNF-α, while “M2-like” TAMs are activated by cytokines, including IL-13 and IL-4. The transcriptomic diversity of TAMs has recently been described by two large-scale cross-tissue scRNA-seq studies [37–39]. A phenotypic plasticity spectrum from pro- to anti-inflammatory TAMs has been demonstrated, and TAMs with angiogenic and proliferative functions are also preserved in almost all TMEs [37].

TAMs sustain tumor progression, tumor cell metastasis, and invasion in multiple ways. Other features of TAMs include secretion of growth factors, such as fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF), which promote proliferation of tumor cells, a variety of proteolytic enzymes (including metalloproteinases that facilitate matrix remodeling), and ROS and NO that induce genetic instability of hematologic malignancies [15]. TAMs also express immunosuppressive cytokines, including TGF-β and IL-10, that suppress T cell immune effects, leading to metabolic starvation of T cells by secreting amino acid-depleting enzymes, including 2,3-dioxygenase (IDO) and arginase-1 [40]. By expressing multiple immune checkpoint ligands, including programmed death-ligand 1 (PD-L1), PD-L2, and B7-H4, TAMs induce immunosuppression [40]. Zheng et al. [21] first showed that TAMs activate anti-apoptosis signaling of MM via physical interaction, thereby contributing to MM cell drug resistance.

The pro-tumorigenic characteristics of TAMs make TAMs potential targets when treating MM with CAR-T cell therapy. Emerging strategies to target TAMs include blockade of monocyte/macrophage recruitment to the bone marrow (BM), direct depletion of TAMs from the TME, and re-education of M2 TAMs into M1 TAMs [36, 37]. Several preclinical studies have shown antigen-specific phagocytosis by macrophages after modification with CAR (CAR-M) [41–43]. In a recent study Morrissey et al. [41] developed a series of CAR-Ms to phagocytose Raji B cells, CD19⁺, or CD22⁺ tumor cells. It was further shown that CAR-M combined with CD47 monoclonal antibody or SIRPα enhance the phagocytic function of CAR-M in vitro [41].

2.3 Tregs

Tregs are a subset of specialized CD4+ T cells that are capable of exerting strong immunosuppressive activity and expressing the transcription factor, FOXP3 [22]. Tregs have an important role in regulating the immune system and mediating immune tolerance for homeostasis [44, 45]; however, Tregs can be differentiated to suppress cytotoxicity of CD8⁺ T cells in the TME via the following mechanisms: secretion of immunosuppressive cytokines (TGF-β or IL-10); competitive consumption of IL-2; CTLA-4-mediated inhibition of antigen-presenting cells in the form of direct contact; and secretion of granzyme and/or perforin to lytic effector T cells [40].

Sufficient evidence suggests enrichment of Tregs in the PB of MM patients, and it has also been observed that the frequency of Tregs in PB is positively associated with disease risk stage and paraprotein level [46, 47]. Furthermore, an elevated Treg level is also an adverse prognostic factor for MM patients [46]. An obvious strategy to address the immunosuppressive limitation of Tregs is to specifically eliminate Tregs from the TME [45]. The number and function of Tregs have been shown to be reduced, accompanied by restoration of anti-tumor immunity after treatment with low-dose cyclophosphamide [48]. Tregs can be transiently removed from the TME and an improved survival rate can be achieved after treatment with low-dose cyclophosphamide in a murine model of MM [49]. In addition, emerging strategies targeting Tregs include targeting immune checkpoints on Tregs and reversing Tregs towards an anti-tumor immunity phenotype [45].

2.4 Other cellular compartments

Mesenchymal stem/stromal cells (MSCs) are a subset of multipotent stem cells that give rise to chondrocytes, adipocytes, osteocytes, and other lineages [50]. MSCs from BM are CD90-, CD73-, and CD105-positive, and negative for CD45, CD34, CD14, and HLA-DR [50, 51]. MSCs represent an integral component of the MM TME, and the presence of MSCs is essential for MM development, progression, and metastasis [52]. By using scRNA-seq, Jong et al. [53] identified two MSC clusters that are strongly enriched in MM BM, and differentially expressed genes of these MM-specific MSC clusters show significant enrichment for an inflammation-related pathway. A longitudinal analysis, however, has revealed that successful anti-tumor induction therapy does not revert BM inflammation, indicating the essential role of MSC-centric TME inflammation in MM persistence [53].

Osteoclasts are the critical mediator of bone absorption and are responsible for bone destruction in > 80% of MM patients [54, 55]. Osteoclasts in the MM TME are large multi-nucleated cells, which are differentiated from CD14⁺ myeloid cells and induced by OC-activating cytokines and receptor activators of nuclear factor-κB (RANKL) by BM supporting cells and MM cells [56]. Traditionally, osteoclasts are thought to be essential for regulation of bone metabolism [57]; however, accumulating evidence has shown that osteoclasts are important immunomodulatory compartments and are involved in the formation of an immunosuppressive MM TME [23, 56]. The expression of a variety of immunosuppressive molecules, including PD-L1, IDO, CD200, and Galectin-9, are upregulated during osteoclastogenesis. The proliferation of CD4+ and CD8+ T cells is significantly inhibited by osteoclasts, thus resulting in attenuated T-cell-mediated anti-myeloma immunity. Furthermore, the immunosuppressive effects of osteoclasts are alleviated, and IDO expression is decreased after CD38 monoclonal antibody is applied [23].

3. OVERCOMING STRUCTURAL COMPONENTS

The TME, apart from immunosuppressive cells, also includes structural components, including ECM and blood vessels. MM cells and cancer-associated fibroblasts synthesize and alter the ECM, while stimulating the growth of host blood vessels. In this way, the nature of TME is altered, which in turn promotes the growth and metastasis of MM.

3.1 ECM

As a major component of the MM TME, ECM is composed of different macromolecules, including proteoglycans, glycosaminoglycans, laminins, collagens, and their receptors in the TME [15]. It is essential for normal tissue homeostasis to maintain the integrity of the ECM network because the network provides structural support for the normal tissue and regulates cell morphology, function, and cell-matrix interactions [58]. MM cells modify the composition of the ECM via enzymatic and non-enzymatic processes, thereby promoting drug resistance, tumor cell survival, and proliferation. Eckhardt et al. [59] reported that transcription of ECM-related components is dysregulated during tumor progression through gene-expression profiling. In addition, TME and ECM are dynamic regulatory reservoirs for various effectors, including cytokines, growth factors, enzymes, and chemokines, which bind to glycosaminoglycans mainly via heparan sulfate side chains [58]. MM cells release ECM-bound effectors by secreting heparanase to cleaves heparan sulfate, which converts these effectors to bioactive mediators and regulates the composition and organization of the ECM [60]. In addition, MM cells interact directly with the ECM by binding syndecan-1 or very late antigen-4, thus leading to cell cycle dysregulation and activation of anti-apoptotic pathways within MM cells [61].

In addition to modification of the ECM, a permissive TME can form for MM cells. Of increasing interest is the formation of the pre-metastatic niche by MM cells by altering the ECM in the monoclonal gammopathy of undetermined significance (MGUS) stage [15]. Such an alteration arises from the deposition of ECM fragments, as well as new macromolecules by the enzymatic activity of malignant plasma cells [62]. Multiple ECM receptors, ECM proteins, and ECM-modulating enzymes, including laminin α4, integrin α5β5, and matrix metalloproteinase-2, have been reported to be upregulated in a step-by-step fashion from MGUS to MM by proteome profiling [63]. It is unclear, however, how the presence of the ECM affects the efficacy of CAR-T cell therapy. Peri-tumoral ECM collagen fibers have been reported to be a barrier that limit the access of T cells to the TME [64]. Therefore, infiltration of CAR-T cells into the TME can be promoted by matrix degradation, which in turn enhances the efficacy of CAR-T cell therapy.

3.2 Tumor vasculature

Under physiologic conditions, angiogenesis in the BM is regulated by numerous pro- and anti-angiogenic modulators, including cells, cytokines, and growth factors. The dynamic balance of angiogenesis is disrupted in the TME, and MM cells promote the growth of blood vessels by upregulated expression of pro-angiogenic modulators and downregulated expression of anti-angiogenic modulators, thereby leading to increased angiogenesis and tumor progression [65]. The level of pro-angiogenic factors or degree of angiogenesis in the MM TME is associated with the stage of disease, prognosis, and depth of response [66]. Furthermore, MM patients with active disease have a higher microvascular density compared to MGUS or inactive MM patients. A stepwise elevation in TME angiogenesis has been reported among patients with MGUS to smoldering MM, symptomatic MM, and relapsed MM [67]. Such elevated levels of angiogenesis in MM patients may be associated with the accumulation of pro-angiogenic cytokines, including VEGF, syndecan-1, and bFGF [67].

Aberrant angiogenesis is critical for the survival, proliferation, progression, and metastasis of tumor cells. Furthermore, aberrant angiogenesis provides a barrier that limits access and infiltration of CAR-T cells into the TME. To overcome the tumor vasculature, CAR-T cells can be designed to target pro-angiogenic modulators, including VEGF receptor (VEGFR)-1, VEGFR-2, prostate-specific membrane antigen (PSMA), or tumor endothelial marker 8 [40]. It has been shown that treatment with anti-VEGF antibodies results in increased recruitment of effector T cells and improved responses in metastatic renal cell carcinoma [68]. Corollary studies have demonstrated a reduced frequency of MDSCs in the TME after treatment with VEGFR-2-specific CAR-T cell therapy combined with IL-12, and showed promising efficacy in murine models with multiple vascularized tumors [69]. Additional studies are needed to target pro-angiogenic modulators in the TME and modify next-generation CAR-T cells to further enhance the efficacy of CAR-T cell therapies.

4. OVERCOMING HYPOXIA, NUTRIENT STARVATION, AND METABOLISM

Hypoxia and nutrient starvation are the main features of the MM TME. Hypoxia of the TME comes from the reduced oxygen concentration due to inadequate blood supply, increased oxygen consumption by MM cells, and hydrogen peroxide production [11, 15]. It has been shown that the oxygen concentration in the BM of MM patients or murine models is lower compared with healthy controls, which results from rapid cell division and aberrant angiogenesis [70]. Hypoxia can significantly increase the expression of CXCR4 in the TME, which ultimately promotes the homing and metastasis of circulating malignant plasma cells to other parts of the BM [71]. In addition, intra-tumoral hypoxia promotes epithelial-mesenchymal transition-like features of MM cells, and decreases E-cadherin expression and BM adhesion of MM cells, thereby enhancing tumor proliferation and migration [71]. To overcome the hypoxic TME, Juillerat et al. [72] designed a new type of hypoxia-induced factor (HIF) CAR, which fuses CD19-specific scFv and HIF1α sub-domains to γ and β chains of the IgE receptor. The function of such oxygen-sensitive CAR-T cells can be modulated by oxygen levels in the TME, paving the way for creating more “self-decision-making” CAR-T cells in the future.

Essential amino acids for T cell metabolism, such as such as tryptophan, are reduced in MM TME [73]. MM cells, MDSCs, and osteoblasts degrade tryptophan by overexpressing IDO, leading to suppressed proliferation of T cells. Furthermore, it has been shown that the expression of IDO in the TME also inhibits the immune effector activity of CAR-T cells, which results in failure of anti-CD19⁺ CAR-T cells to control the progression of CD19⁺ IDO-expressing tumors [74]. Therefore, the combination of inhibitors targeting IDO to modify the microenvironment while killing MM cells can be considered to maximize the efficacy of CAR-T cells.

The metabolism, differentiation, and function of T cells can also be modulated by metabolic stress from the TME. CAR-T cells with different co-stimulatory domains rely on different metabolic pathways. Choi et al. [75] showed that the 4-1BB co-stimulatory domain enhances the mitochondrial respiratory chain and catabolism in CAR-T cells. In contrast, the CD28 co-stimulatory domain favors the glycolysis pathway and lactate production [76].

5. CONCLUSION

Despite the significant efficacy achieved by CAR-T cell therapy, relapse following therapy has become one of the major problems for such a treatment paradigm. In addition to tumor progression and problems related to CAR-T cell products, TME is a determinant of the efficacy of CAR-T cell therapy in MM [77, 78]. In this review we summarized the main challenges for CAR-T cells in the TME, including immunosuppressive cells, structural components, hypoxia, nutrient starvation, and metabolism. We also proposed strategies to improve the efficacy of CAR-T cell therapy by modifying the TME.

CAR-T cell therapy, as ‘the living drug,’ is different from other immunotherapies because CAR-T cell therapy is more susceptible to the immunosuppressive TME that limits its efficacy. There is ample evidence that soluble factors, including IDO, IL-10, TGF-β [79], and immune checkpoints, such as PD-1 and CTLA-4 [80], cause T cell dysfunction. Furthermore, trafficking of CAR-T cells into the TME, aberrant tumor vasculature, and suppression of CAR-T cells due to immunosuppressive cells are some key challenges that the TME poses to CAR-T cells. In this review we summarized strategies for CAR-T cells to overcome the MM TME. These include depleting immunosuppressive cells, blocking immune checkpoint pathways, and modifying CARs to target the immunosuppressive cells and structural components in the TME. In addition, there are several other strategies under preclinical investigation, including alternative manufacturing processes [81], dual-target CAR-T cells, such as dual BCMA/CD19 CAR-T cells [82], and allogeneic CAR-T cells [83]. The ideal CAR-T cells for treatment in MM should meet the following characteristics: (1) target different MM cell sites to prevent antigen loss; (2) utilize the “self-decision-making” function to overcome the TME, while reducing toxicity and side effects; and (3) retain the naïve/memory phenotype to increase persistence. With the advances in gene editing technology and vector designs, better individualized and multi-functional CAR-T cells will be designed.

ACKNOWLEDGEMENTS

This investigation was supported by the International Cooperation Projects of National Natural Science Foundation (grant no. 81920108006 to L. Qiu), CAMS Innovation Fund for Medical Sciences [CIFMS] (grant no. 2022-I2M-1-022 to L. Qiu), and the National Natural Science Foundation (grant no. 81630007 to L. Qiu and grant nos. 82270218 and 81670202 to G. An).

CONFLICTS OF INTEREST

No disclosures were reported by the authors.

REFERENCES

1. Demel I, Bago JR, Hajek R, Jelinek T. Focus on monoclonal antibodies targeting B-cell maturation antigen (BCMA) in multiple myeloma: update 2021. Br J Haematol. 2021. Vol. 193:705–22. 3321697210.1111/bjh.17235

2. Shah N, Chari A, Scott E, Mezzi K, Usmani SZ. B-cell maturation antigen (BCMA) in multiple myeloma: rationale for targeting and current therapeutic approaches. Leukemia. 2020. Vol. 34:985–1005. 3205500010.1038/s41375-020-0734-z

3. Cohen YC, Cohen AD, Delforge M, Hillengass J, Goldschmidt H, et al.. Efficacy and safety of ciltacabtagene autoleucel (cilta-cel), a B-cell maturation antigen (BCMA)-directed chimeric antigen receptor (CAR) T-cell therapy, in lenalidomide-refractory patients with progressive multiple myeloma after 1-3 prior lines of therapy: updated results from CARTITUDE-2. Blood. 2021. Vol. 138:3866 10.1182/blood-2021-146072

4. Lin Y, Raje NS, Berdeja JG, Siegel DS, Jagannath S, et al.. Idecabtagene vicleucel (ide-cel, bb2121), a BCMA-directed CAR T cell therapy, in patients with relapsed and refractory multiple myeloma: updated results from phase 1 CRB-401 study. Blood. 2020. Vol. 136:26–7. 10.1182/blood-2020-134324

5. Gogishvili T, Danhof S, Prommersberger S, Rydzek J, Schreder M, et al.. SLAMF7-CAR T cells eliminate myeloma and confer selective fratricide of SLAMF7+ normal lymphocytes. Blood. 2017. Vol. 130:2838–47. 2908931110.1182/blood-2017-04-778423

6. Li C, Mei H, Hu Y, Guo T, Liu L, et al.. A bispecific CAR-T cell therapy targeting BCMA and CD38 for relapsed/refractory multiple myeloma: updated results from a phase 1 dose-climbing trial. Blood. 2019. Vol. 134:930 10.1182/blood-2019-130340

7. Ormhøj M, Bedoya F, Frigault MJ, Maus MV. CARs in the lead against multiple myeloma. Curr Hematol Malig Rep. 2017. Vol. 12:119–25. 2823315110.1007/s11899-017-0373-2

8. Teoh PJ, Chng WJ. CAR T-cell therapy in multiple myeloma: more room for improvement. Blood Cancer J. 2021. Vol. 11:84 3392719210.1038/s41408-021-00469-5

9. Manier S, Ingegnere T, Escure G, Prodhomme C, Nudel M, et al.. Current state and next-generation CAR-T cells in multiple myeloma. Blood Rev. 2022. Vol. 54:100929. 3513113910.1016/j.blre.2022.100929

10. Lindo L, Wilkinson LH, Hay KA. Befriending the hostile tumor microenvironment in CAR T-cell therapy. Front Immunol. 2020. Vol. 11:618387. 3364329910.3389/fimmu.2020.618387

11. Gowrishankar K, Birtwistle L, Micklethwaite K. Manipulating the tumor microenvironment by adoptive cell transfer of CAR T-cells. Mamm Genome. 2018. Vol. 29:739–56. 2998740610.1007/s00335-018-9756-5

12. Jo S, Das S, Williams A, Chretien A, Pagliardini T, et al.. Endowing universal CAR T-cell with immune-evasive properties using TALEN-gene editing. Nat Commun. 2022. Vol. 13:3453 3577327310.1038/s41467-022-30896-2

13. Liu X, Zhang Y, Cheng C, Cheng AW, Zhang X, et al.. CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells. Cell Res. 2017. Vol. 27:154–7. 2791085110.1038/cr.2016.142

14. Ren J, Liu X, Fang C, Jiang S, June CH, et al.. Multiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition. Clin Cancer Res. 2017. Vol. 23:2255–66. 2781535510.1158/1078-0432.CCR-16-1300

15. Kawano Y, Moschetta M, Manier S, Glavey S, Görgün GT, et al.. Targeting the bone marrow microenvironment in multiple myeloma. Immunol Rev. 2015. Vol. 263:160–72. 2551027610.1111/imr.12233

16. Epperly R, Gottschalk S, Velasquez MP. A bump in the road: how the hostile AML microenvironment affects CAR T cell therapy. Front Oncol. 2020. Vol. 10:262. 3218513210.3389/fonc.2020.00262

17. Tai Y, Lin L, Xing L, Cho S, Yu T, et al.. APRIL signaling via TACI mediates immunosuppression by T regulatory cells in multiple myeloma: therapeutic implications. Leukemia. 2019. Vol. 33:426–38. 3013546510.1038/s41375-018-0242-6

18. Seymour F, Cavenagh JD, Mathews J, Gribben JG. NK cells CD56^bright and CD56^dim subset cytokine loss and exhaustion is associated with impaired survival in myeloma. Blood Adv. 2022. Vol. 6:5152–9. 10.1182/bloodadvances.2022007905

19. Zavidij O, Haradhvala NJ, Mouhieddine TH, Sklavenitis-Pistofidis R, Cai S, et al.. Single-cell RNA sequencing reveals compromised immune microenvironment in precursor stages of multiple myeloma. Nat Cancer. 2020. Vol. 1:493–506. 3340950110.1038/s43018-020-0053-3

20. Malek E, de Lima M, Letterio JJ, Kim B, Finke JH, et al.. Myeloid-derived suppressor cells: The green light for myeloma immune escape. Blood Rev. 2016. Vol. 30:341–8. 2713211610.1016/j.blre.2016.04.002

21. Zheng Y, Cai Z, Wang S, Zhang X, Qian J, et al.. Macrophages are an abundant component of myeloma microenvironment and protect myeloma cells from chemotherapy drug-induced apoptosis. Blood. 2009. Vol. 114:3625–28. 1971050310.1182/blood-2009-05-220285

22. Feng X, Zhang L, Acharya C, An G, Wen K, et al.. Targeting CD38 suppresses induction and function of T regulatory cells to mitigate immunosuppression in multiple myeloma. Clin Cancer Res. 2017. Vol. 23:4290–300. 2824989410.1158/1078-0432.CCR-16-3192

23. An G, Acharya C, Feng X, Wen K, Zhong M, et al.. Osteoclasts promote immune suppressive microenvironment in multiple myeloma: therapeutic implication. Blood. 2016. Vol. 128:1590–603. 2741864410.1182/blood-2016-03-707547

24. Zou Z, Guo T, Cui J, Zhang L, Pan L. Onset of regulatory B cells occurs at initial stage of B cell dysfunction in multiple myeloma. Blood. 2019. Vol. 134:1780 10.1182/blood-2019-128703

25. Veglia F, Sanseviero E, Gabrilovich DI. Myeloid-derived suppressor cells in the era of increasing myeloid cell diversity. Nat Rev Immunol. 2021. Vol. 21:485–98. 3352692010.1038/s41577-020-00490-y

26. Federico C, Alhallak K, Sun J, Duncan K, Azab F, et al.. Tumor microenvironment-targeted nanoparticles loaded with bortezomib and ROCK inhibitor improve efficacy in multiple myeloma. Nat Commun. 2020. Vol. 11:6037 3324715810.1038/s41467-020-19932-1

27. Lv M, Wang K, Huang XJ. Myeloid-derived suppressor cells in hematological malignancies: friends or foes. J Hematol Oncol. 2019. Vol. 12:105 3164076410.1186/s13045-019-0797-3

28. Görgün GT, Whitehill G, Anderson JL, Hideshima T, Maguire C, et al.. Tumor-promoting immune-suppressive myeloid-derived suppressor cells in the multiple myeloma microenvironment in humans. Blood. 2013. Vol. 121:2975–87. 2332125610.1182/blood-2012-08-448548

29. Zhang X, Fu X, Li T, Yan H. The prognostic value of myeloid derived suppressor cell level in hepatocellular carcinoma: a systematic review and meta-analysis. PLoS One. 2019. Vol. 14:e0225327. 3179043710.1371/journal.pone.0225327

30. Enblad G, Karlsson H, Gammelgård G, Wenthe J, Lövgren T, et al.. A phase I/IIa trial using CD19-targeted third-generation CAR T cells for lymphoma and leukemia. Clin Cancer Res. 2018. Vol. 24:6185–94. 3009743310.1158/1078-0432.CCR-18-0426

31. Ma C, Kapanadze T, Gamrekelashvili J, Manns MP, Korangy F, et al.. Anti-Gr-1 antibody depletion fails to eliminate hepatic myeloid-derived suppressor cells in tumor-bearing mice. J Leukoc Biol. 2012. Vol. 92:1199–206. 2307724710.1189/jlb.0212059

32. Van Valckenborgh E, Schouppe E, Movahedi K, De Bruyne E, Menu E, et al.. Multiple myeloma induces the immunosuppressive capacity of distinct myeloid-derived suppressor cell subpopulations in the bone marrow. Leukemia. 2012. Vol. 26:2424–8. 2252278910.1038/leu.2012.113

33. Guha P, Gardell J, Darpolor J, Cunetta M, Lima M, et al.. STAT3 inhibition induces Bax-dependent apoptosis in liver tumor myeloid-derived suppressor cells. Oncogene. 2019. Vol. 38:533–48. 3015867310.1038/s41388-018-0449-z

34. Zhou Q, Bucher C, Munger ME, Highfill SL, Tolar J, et al.. Depletion of endogenous tumor-associated regulatory T cells improves the efficacy of adoptive cytotoxic T-cell immunotherapy in murine acute myeloid leukemia. Blood. 2009. Vol. 114:3793–802. 1972405910.1182/blood-2009-03-208181

35. Panchabhai S, Kelemen K, Ahmann G, Sebastian S, Mantei J, et al.. Tumor-associated macrophages and extracellular matrix metalloproteinase inducer in prognosis of multiple myeloma. Leukemia. 2016. Vol. 30:951–4. 2620292610.1038/leu.2015.191

36. Xiang X, Wang J, Lu D, Xu X. Targeting tumor-associated macrophages to synergize tumor immunotherapy. Signal Transduct Target Ther. 2021. Vol. 6:75 3361925910.1038/s41392-021-00484-9

37. Ma RY, Black A, Qian BZ. Macrophage diversity in cancer revisited in the era of single-cell omics. Trends Immunol. 2022. 3569052110.1016/j.it.2022.04.008

38. Mulder K, Patel AA, Kong WT, Piot C, Halitzki E, et al.. Cross-tissue single-cell landscape of human monocytes and macrophages in health and disease. Immunity. 2021. Vol. 54:1883–900.e5. 3433187410.1016/j.immuni.2021.07.007

39. Cheng S, Li Z, Gao R, Xing B, Gao Y, et al.. A pan-cancer single-cell transcriptional atlas of tumor infiltrating myeloid cells. Cell. 2021. Vol. 184:792–809.e23. 3354503510.1016/j.cell.2021.01.010

40. Rodriguez-Garcia A, Palazon A, Noguera-Ortega E, Powell DJ Jr, Guedan S. CAR-T cells hit the tumor microenvironment: strategies to overcome tumor escape. Front Immunol. 2020. Vol. 11:1109. 3262520410.3389/fimmu.2020.01109

41. Morrissey MA, Williamson AP, Steinbach AM, Roberts EW, Kern N, et al.. Chimeric antigen receptors that trigger phagocytosis. Elife. 2018. Vol. 7:e36688. 2986296610.7554/eLife.36688

42. Kang M, Lee SH, Kwon M, Byun J, Kim D, et al.. Nanocomplex-mediated in vivo programming to chimeric antigen receptor-M1 macrophages for cancer therapy. Adv Mater. 2021. Vol. 33:2103258. 3451055910.1002/adma.202103258

43. Zhang W, Liu L, Su H, Liu Q, Shen J, et al.. Chimeric antigen receptor macrophage therapy for breast tumours mediated by targeting the tumour extracellular matrix. Br J Cancer. 2019. Vol. 121:837–45. 3157075310.1038/s41416-019-0578-3

44. Togashi Y, Shitara K, Nishikawa H. Regulatory T cells in cancer immunosuppression - implications for anticancer therapy. Nat Rev Clin Oncol. 2019. Vol. 16:356–71. 3070543910.1038/s41571-019-0175-7

45. Li C, Jiang P, Wei S, Xu X, Wang J. Regulatory T cells in tumor microenvironment: new mechanisms, potential therapeutic strategies and future prospects. Mol Cancer. 2020. Vol. 19:116 3268051110.1186/s12943-020-01234-1

46. Feyler S, von Lilienfeld-Toal M, Jarmin S, Marles L, Rawstron A, et al.. CD4(+)CD25(+)FoxP3(+) regulatory T cells are increased whilst CD3(+)CD4(-)CD8(-)alphabetaTCR(+) double negative T cells are decreased in the peripheral blood of patients with multiple myeloma which correlates with disease burden. Br J Haematol. 2009. Vol. 144:686–95. 1913397810.1111/j.1365-2141.2008.07530.x

47. Beyer M, Kochanek M, Giese T, Endl E, Weihrauch MR, et al.. In vivo peripheral expansion of naive CD4+CD25highFoxP3+ regulatory T cells in patients with multiple myeloma. Blood. 2006. Vol. 107:3940–9. 1641044510.1182/blood-2005-09-3671

48. Swan D, Gurney M, Krawczyk J, Ryan AE, O’Dwyer M. Beyond DNA damage: exploring the immunomodulatory effects of cyclophosphamide in multiple myeloma. Hemasphere. 2020. Vol. 4:e350. 3230978710.1097/HS9.0000000000000350

49. Sharabi A, Laronne-Bar-On A, Meshorer A, Haran-Ghera N. Chemoimmunotherapy reduces the progression of multiple myeloma in a mouse model. Cancer Prev Res (Phila). 2010. Vol. 3:1265–76. 2071990310.1158/1940-6207.CAPR-10-0138

50. Lan T, Luo M, Wei X. Mesenchymal stem/stromal cells in cancer therapy. J Hematol Oncol. 2021. Vol. 14:195 3478931510.1186/s13045-021-01208-w

51. Krampera M, Le Blanc K. Mesenchymal stromal cells: putative microenvironmental modulators become cell therapy. Cell Stem Cell. 2021. Vol. 28:1708–25. 3462423210.1016/j.stem.2021.09.006

52. Schinke C, Qu P, Mehdi SJ, Hoering A, Epstein J, et al.. The pattern of mesenchymal stem cell expression is an independent marker of outcome in multiple myeloma. Clin Cancer Res. 2018. Vol. 24:2913–9. 2956313610.1158/1078-0432.CCR-17-2627

53. de Jong M, Kellermayer Z, Papazian N, Tahri S, Hofste Op Bruinink D, et al.. The multiple myeloma microenvironment is defined by an inflammatory stromal cell landscape. Nat Immunol. 2021. Vol. 22:769–80. 3401712210.1038/s41590-021-00931-3

54. Rajkumar SV, Dimopoulos MA, Palumbo A, Blade J, Merlini G, et al.. International Myeloma Working Group updated criteria for the diagnosis of multiple myeloma. Lancet Oncol. 2014. Vol. 15:e538–48. 2543969610.1016/S1470-2045(14)70442-5

55. Terpos E, Ntanasis-Stathopoulos I, Dimopoulos MA. Myeloma bone disease: from biology findings to treatment approaches. Blood. 2019. Vol. 133:1534–9. 3076045410.1182/blood-2018-11-852459

56. Tai Y, Cho S, Anderson KC. Osteoclast immunosuppressive effects in multiple myeloma: role of programmed cell death ligand 1. Front Immunol. 2018. Vol. 9:1822. 3014769110.3389/fimmu.2018.01822

57. Jacome-Galarza CE, Percin GI, Muller JT, Mass E, Lazarov T, et al.. Developmental origin, functional maintenance and genetic rescue of osteoclasts. Nature. 2019. Vol. 568:541–5. 3097182010.1038/s41586-019-1105-7

58. Karamanos NK, Piperigkou Z, Passi A, Götte M, Rousselle P, et al.. Extracellular matrix-based cancer targeting. Trends Mol Med. 2021. Vol. 27:1000–13. 3438924010.1016/j.molmed.2021.07.009

59. Eckhardt BL, Parker BS, van Laar RK, Restall CM, Natoli AL, et al.. Genomic analysis of a spontaneous model of breast cancer metastasis to bone reveals a role for the extracellular matrix. Mol Cancer Res. 2005. Vol. 3:1–13. 15671244

60. de Boer C, Armstrong Z, Lit VAJ, Barash U, Ruijgrok G, et al.. Mechanism-based heparanase inhibitors reduce cancer metastasis in vivo. Proc Natl Acad Sci U S A. 2022. Vol. 119:e2203167119. 3588178610.1073/pnas.2203167119

61. Hideshima T, Bergsagel PL, Kuehl WM, Anderson KC. Advances in biology of multiple myeloma: clinical applications. Blood. 2004. Vol. 104:607–18. 1509044810.1182/blood-2004-01-0037

62. Peinado H, Zhang H, Matei IR, Costa-Silva B, Hoshino A, Rodrigues G, et al.. Pre-metastatic niches: organ-specific homes for metastases. Nat Rev Cancer. 2017. Vol. 17:302–17. 2830390510.1038/nrc.2017.6

63. Slany A, Haudek-Prinz V, Meshcheryakova A, Bileck A, Lamm W, et al.. Extracellular matrix remodeling by bone marrow fibroblast-like cells correlates with disease progression in multiple myeloma. J Proteome Res. 2014. Vol. 13:844–54. 2425656610.1021/pr400881p

64. Kuczek DE, Larsen AMH, Thorseth M, Carretta M, Kalvisa A, et al.. Collagen density regulates the activity of tumor-infiltrating T cells. J Immunother Cancer. 2019. Vol. 7:68 3086705110.1186/s40425-019-0556-6

65. Ribatti D, Vacca A. New insights in anti-angiogenesis in multiple myeloma. Int J Mol Sci. 2018. Vol. 19:2031. 3000234910.3390/ijms19072031

66. Jakob C, Sterz J, Zavrski I, Heider U, Kleeberg L, et al.. Angiogenesis in multiple myeloma. Eur J Cancer. 2006. Vol. 42:1581–90. 1679796510.1016/j.ejca.2006.02.017

67. Rajkumar SV, Mesa RA, Fonseca R, Schroeder G, Plevak MF, et al.. Bone marrow angiogenesis in 400 patients with monoclonal gammopathy of undetermined significance, multiple myeloma, and primary amyloidosis. Clin Cancer Res. 2002. Vol. 8:2210–16. 12114422

68. Dumond A, Montemagno C, Vial V, Grépin R, Pagès G. Anti-vascular endothelial growth factor C antibodies efficiently inhibit the growth of experimental clear cell renal cell carcinomas. Cells. 2021. Vol. 10:1222. 3406767110.3390/cells10051222

69. Chinnasamy D, Yu Z, Kerkar SP, Zhang L, Morgan RA, et al.. Local delivery of interleukin-12 using T cells targeting VEGF receptor-2 eradicates multiple vascularized tumors in mice. Clin Cancer Res. 2012. Vol. 18:1672–83. 2229113610.1158/1078-0432.CCR-11-3050

70. Martin SK, Diamond P, Gronthos S, Peet DJ, Zannettino ACW. The emerging role of hypoxia, HIF-1 and HIF-2 in multiple myeloma. Leukemia. 2011. Vol. 25:1533–42. 2163728510.1038/leu.2011.122

71. Azab AK, Hu J, Quang P, Azab F, Pitsillides C, et al.. Hypoxia promotes dissemination of multiple myeloma through acquisition of epithelial to mesenchymal transition-like features. Blood. 2012. Vol. 119:5782–94. 2239460010.1182/blood-2011-09-380410

72. Juillerat A, Marechal A, Filhol JM, Valogne Y, Valton J, et al.. An oxygen sensitive self-decision making engineered CAR T-cell. Sci Rep. 2017. Vol. 7:39833. 2810605010.1038/srep39833

73. Ray A, Song Y, Du T, Tai Y, Chauhan D, Anderson KC. Targeting tryptophan catabolic kynurenine pathway enhances antitumor immunity and cytotoxicity in multiple myeloma. Leukemia. 2020. Vol. 34:567–77. 3146273710.1038/s41375-019-0558-x

74. Heyman B, Yang Y. Chimeric antigen receptor T cell therapy for solid tumors: current status, obstacles and future strategies. Cancers (Basel). 2019. Vol. 11:191. 3073635510.3390/cancers11020191

75. Choi BK, Lee DY, Lee DG, Kim YH, Kim S, et al.. 4-1BB signaling activates glucose and fatty acid metabolism to enhance CD8+ T cell proliferation. Cell Mol Immunol. 2017. Vol. 14:748–57. 2697277010.1038/cmi.2016.02

76. Kawalekar OU, O’Connor RS, Fraietta JA, Guo L, McGettigan SE, et al.. Distinct signaling of coreceptors regulates specific metabolism pathways and impacts memory development in CAR T cells. Immunity. 2016. Vol. 44:380–90. 2688586010.1016/j.immuni.2016.01.021

77. Usmani SZ, Berdeja JG, Truppel-Hartmann A, Fei Y, Wortman-Vayn H, et al.. KarMMa-4: idecabtagene vicleucel (ide-cel, bb2121), a BCMA-directed CAR T-cell therapy in high-risk newly diagnosed multiple myeloma. J Clin Oncol. 2021. Vol. 39:TPS8053. 10.1200/JCO.2021.39.15_suppl.TPS8053

78. Shi X, Yan L, Shang J, Kang L, Yan Z, et al.. Anti-CD19 and anti-BCMA CAR T cell therapy followed by lenalidomide maintenance after autologous stem-cell transplantation for high-risk newly diagnosed multiple myeloma. Ame J Hematol. 2022. Vol. 97:537–47. 3511402210.1002/ajh.26486

79. Tang L, Zhang Y, Hu Y, Mei H. T cell exhaustion and CAR-T immunotherapy in hematological malignancies. BioMed Res Int. 2021. Vol. 2021:6616391. 10.1155/2021/6616391

80. Simula L, Ollivier E, Icard P, Donnadieu E. Immune checkpoint proteins, metabolism and adhesion molecules: overlooked determinants of CAR T-cell migration. Cells. 2022. Vol. 11:1854. 10.3390/cells11111854

81. Costello CL, Cohen AD, Patel KK, Ali SS, Berdeja JG, et al.. Phase 1/2 study of the safety and response of P-BCMA-101 CAR-T cells in patients with relapsed/refractory (r/r) multiple myeloma (MM) (PRIME) with novel therapeutic strategies. Blood. 2020. Vol. 136:29–30. 10.1182/blood-2020-142695

82. Jiang H, Dong B, Gao L, Liu L, Ge J, et al.. Clinical results of a multicenter study of the first-in-human dual BCMA and CD19 targeted novel platform fast CAR-T cell therapy for patients with relapsed/refractory multiple myeloma. Blood. 2020. Vol. 136:25–6. 10.1182/blood-2020-138614

83. Mailankody S, Liedtke M, Sidana S, Matous JV, Chhabra S, et al.. Universal updated phase 1 data validates the feasibility of allogeneic anti-BCMA ALLO-715 therapy for relapsed/refractory multiple myeloma. Blood. 2021. Vol. 138:651 10.1182/blood-2021-145572