Non‐BCMA targeted CAR‐T cell therapies for multiple myeloma

Despite the emergence of new strategies in recent years, multiple myeloma (MM) is still an incurable disease with poor outcome. As a new treatment, chimeric antigen receptor (CAR‐) T cell therapy brought exciting news to patients with relapsed or refractory MM. B‐cell maturation antigen (BCMA) is ubiquitously expressed on the surface of myeloma cells and is considered an “ideal” target of CAR‐T cell. BCMA‐targeted CAR‐T cell therapies achieved remarkable efficacy in relapsed or refractory MM patients in several clinical trials. However, some patients had no response or relapsed after BCMA targeted CAR‐T cell therapy. Myeloma cells also express other surface markers which might be used as targets for CAR‐T cell therapy. Encouragingly, CAR‐T cells targeting these non‐BCMA markers are being tested in clinical trials or under preclinical investigation, already showing some promising results. In this review, we summarized and provided an update of these advances.


INTRODUCTION
Multiple myeloma (MM) is one of the most common hematological tumors. 1,2 The prognosis of MM patients has been improved by modern therapies such as proteasome inhibitors, lenalidomide, anti-CD38 monoclonal antibody, and autologous stem cell transplantation. [3][4][5][6] However, most patients eventually relapse with poor outcomes.
Chimeric antigen receptor (CAR-) T cell therapy is a promising immunotherapeutic approach with the potential to prolong survival of patients with malignant hematological diseases including MM, lymphomas, and leukemias. [7][8][9][10][11] The key to success of CAR-T therapy is the selection of appropriate antigen targets. Ideally, the target anti- showed encouraging results. 10,12,13 However, possibly due to the heterogeneous expression or therapy-induced down-regulation of BCMA antigen on myeloma cells, some patients did not respond to treatment or relapsed rapidly after response. 14,15 Some patients even did not express BCMA on myeloma cells, 13 Table 1).

CD138 (syndecan-1)
CD138, also known as syndecan-1, is highly expressed in MM cells and considered to be an attractive target for CAR-T cell therapy. 22 A preclinical study, CD138-directed CAR T cells can eliminate tumor cell lines and primary myeloma cells both in vitro and in vivo. 23 In a phase 1 clinical report (NCT01886976) of five patients with R/R MM who received CD138-directed CAR-T cell therapy, four patients were in stable condition longer than 3 months and one patient had disease progression. 24 In terms of safety, the patients developed cytokine release syndrome (CRS) with no epithelial cytotoxicity occurred. 24 CD138 CAR-T also has potential disadvantages. CD138 also expresses on epithelial cells, possibly resulting in skin or mucosa as the target of anti-CD138 CAR-T cells. 25,26 In the future, relevant strategies may be needed to avoid the targeted toxicity and maintain the potential anti-tumor effect of anti-CD138 CAR-T. Two clinical trials with CD138directed CAR T cells are ongoing (NCT01886976, NCT03672318).

Kappa light chain
Immunoglobulin light chain is a promising target because mature Bcell malignancies are often featured by κ light chain or λ light chain restricted expression. 27 Thus, CAR-T cells targeting light chain are dedicated to eliminate the tumor cells expressing a certain light chain with-out causing overt immunosuppression. Although plasma cells did not express cell-surface immunoglobulins, the postulated MM "stem cells" was shown to express surface immunoglobulins, 18,27 which provided a chance to target the surface immunoglobulins. Monoclonal antibody MDX-1097, which targeted cell membrane-associated κ immunoglobulin free light chains in MM, achieved encouraging efficacy and was well tolerated in clinical trials of MM patients. 28 In a phase 1 clinical study (NCT00881920), anti-κ light chain CAR-T cell was used to treat seven patients with MM, of which six patients had previously received ASCT.
The result showed that four of seven patients had responses and maintained stable condition for 6 weeks to 24 months. 27 This trial is still recruiting subjects.
However, almost all MM cells secrete immunoglobulin, but only a few cells express immunoglobulin on cell membrane. In addition, in the development of the disease, tumor cells usually downregulate the expression of surface immunoglobulin, which may weaken the target ability of anti-κ light chain CAR-T cell.

CD38
The CD38 molecule, a single chain type II transmembrane glycoprotein, is highly expressed on malignant myeloma cells, making it a suitable therapeutic target for CAR-T cell therapy. Daratumumab (CD38 monoclonal antibody) has been approved by the US Food and Drug

CD44 variant 6
CD44 variant 6 (CD44v6), an isoform of the hyaluronate receptor CD44, is overexpressed in hematological malignancies and epithelial tumors, where it contributes to the cancer stem/initiating phenotype. 38 The significance of CD44v6 is not clear. CD44v6 was shown to be expressed by 43% of advanced, high risk MM cases 36 and was associated with 13q14 chromosome deletions, a well-known risk factor in MM. 39 The minimal expression of CD44v6 on normal cells including activated T cells, monocytes and keratinocytes 38 makes However, anti-CD44v6 CAR-T still has potential targeted toxicity due to the nonspecific expression of CD44v6 on normal cells. Besides, CD44v6-targeted CAR-T cells really caused reversible monocytopenia although they did not recognize hematopoietic stem cells. 38 Additional studies are needed to further evaluate the safety of anti-CD44v6 CAR-T. Objective clinical responses to NKG2D CAR T-cell therapy alone were not seen, and CRS or CAR-T cell-related encephalopathy syndrome was not observed in these MM patients. 42 On the whole, the NKG2D-CAR approach is still in early stages of clinical development, additional studies are needed to assess the clinical efficacy of it. Indeed, a clinical study of MM patients is currently recruiting (NCT03018405).

SLAMF7/CS1
Signaling-lymphocyte-activating molecule (SLAM)F7, a cell surface glycoprotein, also known as CS1, is widely and highly expressed on the

CD56
CD56 is a specific marker of MM and strongly expressed by malignant plasma cells in 70% of myeloma patients. 47 With the progression of the disease, the loss of CD56 indicates extramedullary infiltration of MM and plasma cell leukemia. In a preclinical trial, CAR-T cells targeting CD56 in a xenograft model of myeloma effectively cleared the tumor of MM mice and showed powerful anti-tumor efficacy. 48 Therefore, CAR T-cell therapy targeting CD56 has paved the way for clinical trials in combination with another target antigen (NCT03473496, NCT03271632).
However, CD56 is also expressed on some normal cells such as central neurons and NK cells, 9,49 so the potential neurotoxicity may limit the application of CD56 CAR-T cell therapy.

Integrin β7
Because integrin β7 is highly expressed in most myeloma cells and has an active conformer, researchers have developed anti-MMG49 CAR-T cells using fragments from MMG49 monoclonal antibodies, which could specifically recognize a subset of integrinβ7. T cells transduced with MMG49-derived CAR specifically recognize and kill MM cells, exert powerful activity against MM without damaging normal hematopoietic cells. 50 Thus, active conformer of integrin β7 can serve as a promising target against MM.

CD70
CD70, a member of tumor necrosis factor receptor superfamily, has the ability to regulate the activation, proliferation, and differentiation of T

GPRC5D
The human orphan G protein-coupled receptor, class C group 5 member D (GPRC5D), highly expressed in CD138+ primary MM cells from bone marrow samples, is another feasible target antigen of MM. 15,55 In a preclinical study, GPRC5D-targeted CAR-T cells were shown to exhibit significant anti-tumor effects on MM cell lines and myeloma cell murine xenograft model. 15 In particular, GPRC5D-targeted CAR-T cells still showed activity in a murine model of BCMA escape mediated relapse. 15 Overall, these results suggest that GPRC5D could play an important role in CAR-T therapy of MM patients.

CD229
The surface antigen CD229, also known as SLAMF3, is not only commonly and strongly overexpressed on the malignant plasma cells but also highly expressed on pre-plasma cells carrying the phenotype of myeloma-propagating and chemotherapy-resistant cells in RRMM patients. 56 CD229 CAR-T cells have been reported in a preclinical study with dramatic ability eradicating MM cells including MMpropagating cells without fratricide, 57 indicating that targeting CD229 maybe a promising target for patients with MM.

TACI
Transmembrane activator and CAML interactor (TACI), a member of the tumor necrosis factor receptor (TNFR) superfamily, was found to be expressed on MM cells, making it an alternative strategy for CAR-T therapy.
It has been showed that a proliferation-induced ligand (APRIL) -

MULTI-TARGETS STRATEGIES FOR MM
Tumor recurrence caused by antigen loss and escape have become

CONFLICTS OF INTEREST
All the authors declare no financial competing interests.