Dual Targeting of Multiple Myeloma Stem Cells and Myeloid-Derived Suppressor Cells for Treatment of Chemotherapy-Resistant Multiple Myeloma

Here we review the insights and lessons learned from early clinical trials of T-cell engaging bispecific antibodies (BsABs) as a new class of biotherapeutic drug candidates with clinical impact potential for the treatment of multiple myeloma (MM). BsABs are capable of redirecting host T-cell cytotoxicity in an MHC-independent manner to malignant MM clones as well as immunosuppressive myeloid-derived suppressor cells (MDSC). T-cell engaging BsAB targeting the BCMA antigen may help delay disease progression in MM by destroying the MM cells. T-cell engaging BsAB targeting the CD38 antigen may help delay disease progression in MM by depleting both the malignant MM clones and the MDSC in the bone marrow microenvironment (BMME). BsABs may facilitate the development of a new therapeutic paradigm for achieving improved survival in MM by altering the immunosuppressive BMME. T-cell engaging BsiABs targeting the CD123 antigen may help delay disease progression in MM by depleting the MDSC in the BMME and destroying the MM stem cells that also carry the CD123 antigen on their surface.

Several strategies are being explored to overcome the immunosuppressive cellular elements of the BMME in MM patients, including the use autologous hematopoietic stem cell transplantation (AHSCT) (4,20,(27)(28)(29) to remodel the BMME by establishing a more favorable ratio between effective MMspecific CTLs versus Tregs and other immunosuppressive cells. Treatment strategies aimed at further enhancing the anti-MM immunity can be employed as post-AHSCT interventions, including MM-or MDSC-directed monoclonal antibodies (MoAb) (20,27,28). New generation multi-parameter minimal residual leukemia (MRD) detection techniques provide a unique opportunity to evaluate the effect of new treatment modalities that are applied as part of AHSCT or post-AHSCT on the quality and length of complete remission in both newly diagnosed highrisk MM as well as RR MM (29,30).

DUAL TARGETING OF MM CELLS AND MDSCS
Dual targeting of MM cells and MDSCs using biotherapeutic agents has emerged as a very promising new therapeutic platform with a particularly high clinical impact potential. For example CD38 antigen is present on MM cells as well as MDSCs (4,20,(31)(32)(33)(34). Daratumumab, a complement-activating anti-CD38 MoAb capable of causing antibody-dependent cellular cytotoxicity (ADCC) and apoptosis in MM cells, showed significant single agent activity in relapsed MM patients and improved the survival outcome when used in combination with other active anti-MM agents such as bortezomib and dexamethasone or lenalidomide and dexamethasone (4,20,(31)(32)(33)(34). Similar results were obtained using alternative anti-CD38 MoAbs, such as Isatuximab (4,20). Daratumumab has been shown to expand the immunoreactive CTL populations via depletion of CD38 + immunosuppressive cellular elements of the BMME (33). The immunomodulatory effects of Daratumumab improved the clinical responses of previously resistant MM patients to standard combination therapy (4,20). Unfortunately, increased expression levels of complement inhibitors CD55 and CD59 as well as decreased cell surface expression levels of CD38 on MM cells may decrease the clinical activity of anti-CD38 antibodies (4,35).
Bispecific CD3xCD38 antibodies have also been developed (50, 51) and entered clinical trials in patients with relapsed or refractory MM, such as AMG424 (NCT0344566) (51) and GBR1342 (NCT0330911). A CD38-reactive tri-specific antibody targeting CD3 and CD28 co-receptors on T-cells has also been developed to achieve augmented and sustained T-cell activation via CD28 engagement (52). A bispecific T-cell engaging CD3xCD307 antibody, named BFCR4350A, was developed targeting the FcRH5/CD307 antigen (53) on MM cells and it is currently being evaluated in a Phase 1 clinical trial (NCT03275103) (54).
Talquetamab is a GPRC5DxCD3 BsAB targeting the orphan G protein-coupled receptor GPRC5D that is abundantly expressed on MM cells. In a Phase 1 study in R/R MM patients testing both IV and SC administration schedules (NCT03399799), the RP2D was identified as 405 mcg/kg administered SC on a weekly basis (55). The RP2D level was well tolerated and exhibited promising activity with an overall response rate of 63%. CRS (79%), neutropenia (64%), anemia (57%) and dysgeusia (57%) were the most common treatment-emergent AEs. Furthermore, 7% of patients developed neurotoxicity and 32% developed infections. The overall response rate at the RP2D was 63% (55).
Targeting the BMME in MM with SL-401 has been shown to reduce the viability of PDCs and inhibit PDC-induced MM cell growth, impair the viability of CD123 + MM stem cells, and prevent osteoclastogenesis in preclinical model systems (60). SL-401 is being assessed in combination with standard of care in a clinical study (NCT02661022) in relapsed/refractory MM patients with promising early evidence of clinical activity (61,62). Seroproteomics analysis of MM patient serum samples reportedly showed a reduction of PDCderived soluble proteins in SL-401 treated patients (63).
CD123-targeting, CD3-engaging BsAB, such as Flotetuzumab (59) and APVO436 (64) bring cytotoxic T-cells (CTLs) within close vicinity of target CD123 + cells to create "cytolytic synapses" as a short bridge between target cells and CTLs, triggering CTL activation and destruction of targeted cells (Figures 2, 3). These dual-function anti-MM drug candidates are currently in clinical trials for treatment of CD123-expressing hematologic malignancies with early clinical proof of concept for their ability to destroy CD123 + malignant clones, including CRs in relapsed or refractory AML patients (NCT02152956, NCT03647800). However, the clinical potential of a CD123xCD3 bispecific antibody in MM therapy may be limited as the bulk population of MM cells lack CD123 and depletion of CD123 + MM stem cells alone is unlikely to be an effective strategy for monotherapy. Therefore, clinical feasibility and efficacy studies of combinations of CD123 targeting BsAB with active anti-MM drugs such as pomalidomide that appeared to have augmented activity in the presence of the anti-CD123 fusion toxin tagraxofusp (3,4,8,60), biotherapeutic agents, such as CD3xBCMA BsABs, daratumumab, elotuzumab (4)(5)(6)(7)20), and CAR-T cells (4,65) are needed to gain insights into the clinical impact potential of CD123xCD3 BsABs. Notably, MDSCs have been shown to significantly suppress the CTL engaging activity of a BCMAxCD3 BsAB, but the MDSCsuppressed CTL activity could be restored by addition of a hypomethylating agent (HMA) capable of epigenetically altering the MDSC transcriptome via reversal of the aberrant DNA methylation (66). Therefore, MDSC-targeting BsAB could be potentiated by HMAs. It is noteworthy that a combination of the CD123xCD3 BsAB APVO436 with azacitidine is being evaluated in one of the cohorts in the ongoing expansion phase of a Phase 1B AML study (NCT03647800).

CYTOKINE RELEASE SYNDROME (CRS)
CD3-engaging BsABs act as agonists and activate T-cells in the presence of tumor cells expressing the target tumor-associated antigen, which can lead to excessive T-cell activation with release of inflammatory cytokines and development of the potentially lifethreatening systemic inflammation, known as cytokine release syndrome (CRS) (67)(68)(69)(70)(71). For example, BsAB AMG330 binds CD33 antigen on AML cells as well as MDSCs cells and CD3ɛ on T-cells. In an open-label Phase 1 study (Clinicaltrial.gov identifier: NCT#02520427), AMG330 was given at doses ranging from 0.5-720 mg/d in the manner of continuous IV infusion among 55 patients with R/R AML (NCT02520427). AMG 330-related AEs included CRS (67%; Grade ≥3 in 13%) as the most frequent AEs (72). Similarly, CRS was observed in 63% of AML patients treated with AMG673, a new version of AMG330 (Grade ≥3 in 18%; Clinicaltrial.gov identifier: NCT03224819) (72). Flotetuzumab (MGD006) is a bispecific, dual-affinity re-targeting (DART) antibody reactive with both CD3 antigen on T-cells and CD123 antigen on AML cells and MDSCs. This CD3 engaging bispecific antibody exhibited promising single agent activity in therapyrefractory AML patients with primary induction failure as well as patients with an early first relapse. CRS was observed in all AML patients treated with Flotetuzumab (73) and 58% of AML patients treated with Vibecotamab (XmAb14045), another CD3xCD123 BsAB (74). By comparison, only 10 of 46 patients (21.7%) treated with the CD3xCD123 BsAB APVO436 developed CRS (64).
IL-6 is one of the driving pro-inflammatory cytokines that contribute CRS and its pulmonary, cardiovascular, renal, and neurologic complications (60,(64)(65)(66)(67)(68)(69)(70)(71). Cytokine profiling in patients who developed CRS after APVO436 infusion indicates that the predominant cytokine in this inflammatory cytokine response is IL-6, which agrees with our current knowledge regarding CRS that occurs in the context of BsAB therapy (20,60,(64)(65)(66)(67)(68)(69)(70)(71)75). Within 1-2 days following the first dose of APVO436, the mean serum IL-6 concentration in these patients who developed CRS was elevated 145-fold over baseline (755 vs 5.2) and at the end of one week it was still elevated 83-fold over baseline. In most cases, CRS events were transient and medically manageable with standard of care including the use of dexamethasone and anti-IL-6:IL-6R antibody Tocilizumab or anti-IL-6 antibody Siltuximab (antibody against IL-6). However, CRS can be life-threatening even with the use of Tocilizumab or Siltuximab (60,(64)(65)(66)(67)(68)(69)(70)(71). Therefore, development of consistently effective prevention and treatment regimens against CRS remains an urgent and unmet medical need. Identification of such regimens would further advance the field of immunotherapy. We recently reported the robust anti-inflammatory activity of RJX in animal models of CRS (76). RJX has been shown to block the production of IL-6, TNF-a, as well as TGF-b and reverse inflammation-induced tissue injury and multi-organ damage in mouse models of sepsis and CRS (76). RJX is currently being evaluated for its ability to prevent COVID-19 associated CRS in a double-blind randomized clinical study (NCT04708340). Because of its safety and easy use, RJX may emerge as an attractive adjunct to BsAB platforms to mitigate the risk of severe CRS.

NEUROTOXICITY
Neurotoxicity is a treatment-emergent adverse event (AE) for BsABs, and it is often associated with CRS (77). The signs and symptoms vary from patient to patient and include headache, tremor, confusion, expressive and nominal dysphasia, impaired attention, apraxia, and lethargy occurring as early and common manifestations (77)(78)(79). Consensus grading criteria were developed by the ASTCT based on the use of the Immune Effector Cell-Associated Encephalopathy (ICE) screening tool (78). The CD19xCD3 BsAB blinatumumab has been reported to cause neurotoxicity in 70% of patients with B-lineage non-Hodgkin's lymphomas (NHL). By comparison, it is less common with CD20xCD3 or CD123xCD3 BsABs (80). In a recent  (64). A single case of Grade 3 confusion was encountered on the first day of treatment and resolved within a day. Neurotoxicity did not show any dose-dependence. Gender, race, age, absolute lymphocyte count or percentage of lymphocytes in peripheral blood did not predict neurotoxicity. Neurotoxicity occurred in 3 patients who also experienced CRS and in 2 patients who did not develop CRS (75). Conversely, of 10 patients who developed CRS, 7 did not experience any neurotoxicity (75).

CONCLUSION
Recombinant T-cell engaging humanized BsABs redirect host T-cell cytotoxicity in an target antigen-expressing cells in patients with hematologic malignancies. They can be used both for targeting drugresistant MM clones as well as the immune-suppressive cell populations in the BMME (Figure 3). Dual targeting of drugresistant MM clones and immunosuppressive MDSC has the potential to change the therapeutic landscape for MM and improve the survival outcomes of high-risk as well as relapsed/refractory MM patients. The definition of optimal strategies for overcoming the  immunosuppressive BMME in MM may require randomized clinical studies with parallel cohorts and adaptive trial designs. CD3xCD123 BsAB have clinical impact potential in MM as they may help treatment outcomes by blocking immune evasion via depletion of CD123 + MDSC and by reducing the drug-resistant tumor load via CTL-mediated MHC-independent destruction of MM stem cells.

AUTHOR CONTRIBUTIONS
FMU conceived the review, analyzed the contents of relevant publications, wrote the original draft of the manuscript revised the manuscript, provided final approval for submission of the final version. No medical writer or editor was involved.