Novel Anti-B-cell Maturation Antigen Alpha-Amanitin Antibody-drug Conjugate HDP-101 Shows Superior Activity to Belantamab Mafodotin and Enhanced Efficacy in Deletion 17p Myeloma Models

B-cell maturation antigen (BCMA) plays a pathobiologic role in myeloma and is a validated target with five BCMA-specific therapeutics having been approved for relapsed/refractory disease. However, these drugs are not curative, and responses are inferior in patients with molecularly-defined high-risk disease, including those with deletion 17p (del17p) involving the tumor suppressor TP53, supporting the need for further drug development. Del17p has been associated with reduced copy number and gene expression of RNA polymerase II subunit alpha (POLR2A) in other tumor types. We therefore studied the possibility that HDP-101, an anti-BCMA antibody drug conjugate (ADC) with the POLR2A poison α-amanitin could be an attractive agent in myeloma, especially with del17p. HDP-101 reduced viability in myeloma cell lines representing different molecular disease subtypes, and overcame adhesion-mediated and both conventional and novel drug resistance. After confirming that del17p is associated with reduced POLR2A levels in publicly available myeloma patient databases, we engineered TP53 wild-type cells with a TP53 knockout (KO), POLR2A knockdown (KD), or both, the latter to mimic del17p. HDP-101 showed potent anti-myeloma activity against all tested cell lines, and exerted enhanced efficacy against POLR2A KD and dual TP53 KO/POLR2A KD cells. Mechanistic studies showed HDP-101 up-regulated the unfolded protein response, activated apoptosis, and induced immunogenic cell death. Notably, HDP-101 impacted CD138-positive but not-negative primary cells, showed potent efficacy against aldehyde dehydrogenase-positive clonogenic cells, and eradicated myeloma in an in vivo cell line-derived xenograft (CDX). Interestingly, in the CDX model, prior treatment with HDP-101 precluded subsequent engraftment on tumor cell line rechallenge in a manner that appeared to be dependent in part on natural killer cells and macrophages. Finally, HDP-101 was superior to the BCMA-targeted ADC belantamab mafodotin against cell lines and primary myeloma cells in vitro, and in an in vivo CDX. Together, the data support the rationale for translation of HDP-101 to the clinic, where it is now undergoing Phase I trials, and suggest that it could emerge as a more potent ADC for myeloma with especially interesting activity against the high-risk del17p myeloma subtype.


Introduction
Multiple myeloma is a clonal plasma cell malignancy characterized clinically in symptomatic patients by hypercalcemia, renal insu ciency, anemia, painful bony lesions, and an increased risk of infectious complications.Several new drug classes have been introduced over the last two decades, including proteasome inhibitors (PIs), immunomodulatory drugs (IMiDs), and monoclonal antibodies (mAbs) to cell surface targets such as CD38 and Signaling lymphocytic activation molecule family member 7 (1).These have added to the effectiveness of older therapeutics such as corticosteroids and alkylating agents, and use of these drugs in rational combination regimens has substantially improved long-term outcomes.Encouragingly, this trend seems likely to continue with the development of novel immunotherapeutics that work through various mechanisms of action, including antibody drug conjugates (ADCs) (2), T-cell engaging bispeci c antibodies (3), and chimeric antigen receptor (CAR)-guided T-cells (4).B-cell maturation antigen (BCMA) has been an especially popular target for these drugs since it is commonly and fairly speci cally expressed on plasma cells, and plays an important role in plasma cell differentiation (5).Moreover, binding of the BCMA ligands A proliferation-inducing ligand (APRIL) and Bcell-activating factor (BAFF) promotes myeloma cell proliferation and survival, as well as immune suppression in the tumor microenvironment (5), further contributing to disease pathobiology.However, not all patients bene t equally from BCMA-targeted agents, and this is especially true for those with molecularly or clinically de ned high-risk disease (6, 7).For example, lower response rates were noted among high-risk patients in the pivotal trials of the ADC belantamab mafodotin (8), the bispeci c teclistamab (9), and the CAR T-cell product idecabtagene vicleucel (10), which were the rst drugs in each mechanistic category to achieve regulatory approvals.Thus, novel strategies and drugs to overcome the adverse prognosis of patients with high-risk features are still needed.
The high-risk disease category encompasses several unique molecular subtypes of myeloma (6, 7), and patients with deletion of 17p (del17p) consistently represent one of these that have inferior long-term outcomes (11,12,13,14,15,16).While the proportion of myeloma patients with del17p is approximately 10% when the disease is newly diagnosed, it then increases substantially in the relapsed/refractory setting, especially with more advanced and aggressive variants such as plasma cell leukemia (17,18,19,20).TP53 loss is felt to be the pathognomonic lesion in del17p myeloma patients, but neighboring genes may also be co-deleted.One example is RNA polymerase II subunit A (POLR2A), which is located about 200 kilobases from TP53, and has been described to be co-deleted in a large proportion of colorectal cancer cases (21).Decreased POLR2A expression through such haploinsu ciency may enhance sensitivity to α-amanitin (21,22), a cyclic peptide that targets POLR2A at low concentrations (23).This provided us with the rationale to further evaluate HDP-101 (24,25,26), a novel ADC with a structural derivative of α-amanitin optimized for stability conjugated to an engineered cysteine at position 265, and linked to a BCMA-targeted antibody through a Cathepsin B-cleavable linker.
In the current line of research, we developed pre-clinical models of TP53-wild type and deleted myeloma, both with and without POLR2A suppression, and found that HDP-101 showed potent anti-proliferative and pro-apoptotic activity especially in the former.HDP-101 overcame adhesion-mediated and novel drug resistance, and was equally effective against clonotypic cells de ned by expression of Aldehyde dehydrogenase (ALDH).Mechanistic studies showed HDP-101 up-regulated the unfolded protein response (UPR) gene program, while proteomic studies con rmed induction of multiple cellular UPR arms, activation of apoptosis, and induction of immunogenic cell death (ICD).In cell line-derived xenograft models (CDXs) in immune-de cient mice, a single HDP-101 dose was su cient to cure myeloma, and also to prevent tumor re-engraftment on subsequent tumor cell rechallenge, with evidence of a dependence on natural killer (NK) cells and macrophages.Finally, HDP-101 was superior to the previously approved BCMA-targeted ADC belantamab mafodotin against cell lines in vitro and in an in vivo del17p model.Together, the data further validate the e cacy of this new BCMA ADC, which is undergoing Phase I trials, and suggest it could be equipotent, or perhaps even more effective against myeloma with the high-risk del17p abnormality.

Primary patient samples and myeloma cell lines
Primary cells were collected under a protocol approved by the MD Anderson Institutional Review Board after informed consent was obtained in compliance with the Declaration of Helsinki.Human myeloma and the HS-5 (RRID:CVCL_3720) human stromal cell lines were maintained in RPMI 1640 medium (Corning Cellgro; Manassas, VA) supplemented with L-glutamine, 5% fetal bovine serum and 1% penicillin/streptomycin, as well as interleukin (IL)-6 in the case of ANBL-6 (RRID:CVCL_5425) and KAS-6/1 (RRID:CVCL_9544) cells.Drug-resistant myeloma lines were developed as described previously (28).
Please refer to the Supplementary Methods section for additional details on this and other aspects of the techniques employed.

Co-culture, viability, and apoptosis assays
Please consult the Supplementary Methods section for details on these assays, which were typically analyzed by ow cytometry (30).

Western blotting, gene expression pro ling, and real time polymerase chain reaction
Cell lysates were prepared, processed, and analyzed as detailed in the Supplementary Methods section, and Table S1 provides a list of the antibodies used.Please also consult this section for details on gene expression pro ling analyses and real time PCR (qPCR).

Xenograft modeling
These studies were performed following protocols approved through the Institutional Animal Care and Use Committee.Six-to 8-week-old female non-obese diabetic, severe combined immunode ciency (NOD-SCID) mice, or NOD.Cg-Prkdc scid IL2rγ tm1Wjl /SzJ (NSG) mice (The Jackson Laboratory; Bar Harbor, ME), were injected via tail vein with 1×10 6 luciferase-labeled myeloma cells.Once weekly bioluminescent imaging was performed after administration of 10 µl/g D-luciferin substrate (Thermo Fisher Scienti c) using the IVIS Spectrum In Vivo Imaging System (PerkinElmer; Waltham, MA).Treatment was initiated as detailed in the text, weekly imaging was performed and, if needed, mice were euthanized by CO 2 inhalation per institutional guidelines.

Statistical analyses
Data are represented as the mean plus either standard deviation (SD; for triplicate data from the same experiment) or standard errors of the mean (SEM; for multiple independent experiments).The signi cance of drug-effect relationships was determined by one-tailed unpaired t-tests or ANOVA using Graph-Pad Prism, and p < 0.05 was considered statistically signi cant.

HDP-101 potently reduces viability of myeloma cells
To evaluate the activity of HDP-101, we studied a panel of BCMA-expressing myeloma cell lines (Fig. 1A) representing common molecular subtypes, including translocation (t)(11;14), t(4;14), t(14;16), gain and ampli cation of 1q, and different TP53 status and IL-6 dependency.HDP-101 potently reduced viability in all of these lines in a dose-dependent manner (Fig. 1B), as evidenced by median inhibitory concentrations (IC 50 ) ranging from 40 pM to 38.6 nM (Table S2).As the IC 50 values across the cell lines did not correlate with surface BCMA expression (R 2 = 0.094), we examined the impact of the GSI RO4929097 (27), which increased surface BCMA levels (Fig. S1A).When the GSI was added to HDP-101, the combination enhanced the reduction in viability compared with either agent alone (Fig. S1B).One prominent feature of myeloma clinically is the bene t that neoplastic cells receive from their interactions with the marrow microenvironment, including adhesion-mediated drug resistance (31).To model this, we co-cultured myeloma cells with Green uorescent protein (GFP)-labeled HS-5 stromal cells, exposed these co-cultures to HDP-101 and control compounds, and evaluated them for Annexin V and TO-PRO-3 staining by gating on GFP-positive and-negative cells.HS-5 cells were essentially unaffected by HDP-101 as the viable (Annexin V − /TO-PRO-3 − ) cell fraction was stable under all tested conditions (Fig. 1C).In contrast, HDP-101 reduced the proportion of viable MM1.S myeloma cells to 36.7% from the baseline of 74.0%, while this fraction was 37.1% in the presence of HS-5 cells (Fig. 1C).Notably, free α-amanitin, the unconjugated BCMA antibody, and an anti-digoxigenin/α-amanitin control ADC did not impact viability.Qualitatively similar ndings were noted when this same experiment was performed with H929 cells (Fig. S2).One especially relevant product of the microenvironment is APRIL, since it binds BCMA and contributes to multiple aspects of the transformed phenotype (5).Importantly, when excess APRIL or soluble BCMA were added, while there was slight interference with the activity of HDP-101 at low ADC concentrations (Fig. S3A-B), at higher, physiologically relevant levels, HDP-101 overcame these soluble factors.Finally, acquired drug resistance is another prominent aspect of myeloma, and agents that overcome resistance hold promise for relapsed/refractory disease.There was no difference between the e cacy of HDP-101 in MM1.S versus MM1.R (RRID:CVCL_8794) dexamethasone-resistant cells (Fig. 1D, left upper panel), which showed greater potency in these studies compared to Fig. 1B since cells were incubated for 96 hours, demonstrating also a time-dependent effect.Similarly, the sensitivity of RPMI 8226 cells (RRID:CVCL_0014) resistant to melphalan (RRID:CVCL_J434) or doxorubicin (RRID:CVCL_J431) was similar to (Fig. 1D), or even slightly higher than their WT counterparts (Table S3).Moving to models of novel drug resistance, HDP-101 activity was not reduced in bortezomib-resistant RPMI 8226 or KAS-6/1, or in car lzomib-resistant RPMI 8226, U266 (RRID:CVCL_0566), or KAS-6/1 cells (Fig. 1D, Table S3).Finally, resistance to the cell-autonomous effects of lenalidomide was studied, and no reduction in e cacy was seen (Fig. 1D, Table S3).
Del17p reduces POLR2A expression, increasing HDP-101 sensitivity Del17p producing TP53 loss has been linked to POLR2A co-deletion and haploinsu ciency in colorectal and prostate carcinoma (21,22).Therefore, we evaluated the Multiple Myeloma Research Foundation's CoMMpass SM database, and rst con rmed that POLR2A copy number was reduced in del17p patients compared with those without del17p (mean 0.0373 vs. -0.5688,respectively, p < 0.0001; Fig. 2A).This was associated with a signi cant reduction in POLR2A mRNA levels by RNASeq (16.72978FPKM (fragments per kilobase of exon model per million reads mapped) vs. 12.88536 FPKM, respectively, p < 0.0001; Fig. 2B).Notably, no change was seen in BCMA copy number and mRNA expression with del17p (Fig. S4).To model this in vitro, we started with TP53 WT H929, MM1.S, and MOLP-8 cells and used genome editing to knockout (KO) TP53 (Fig. S5A).Next, POLR2A was subjected to knockdown (KD) using shRNAs (Fig. S5B), and we exposed these to HDP-101.Compared to WT cells, TP53 KO alone showed no impact on HDP-101 sensitivity in H929 and MM1.S cells, while there was a small degree of sensitization in MOLP-8 cells (Fig. 2C).In contrast, POLR2A KD alone had a more signi cant sensitizing effect and was comparable to that seen in the dual TP53/POLR2A KO/KD cells (Fig. 2C).Similarly, by Annexin V staining, HDP-101 induced evidence of decreased cell viability and increased apoptosis with increasing surface exposure of phosphatidyl-serine in the POLR2A KD and dual TP53/POLR2A KO/KD models (Fig. 2D).Finally, we also evaluated mitochondrial membrane potential changes, and the TP53 WT/POLR2A KD and TP53 KO/POLR2A KD cells both had substantially decreased potentials (Fig. 2E).In addition, cell cycle analysis was performed on the four H929-derived cell lines, and HDP-101 increased the apoptotic, sub-G0/G1 cell population to a greater extent in the two POLR2A KD models (Table S4).Together, the data support the statement that HDP-101 shows enhanced e cacy driven by reduced POLR2A levels, including in models of del17p myeloma, where sensitivity is increased compared to WT 17p controls.

HDP-101 induces ER stress and ICD while reducing antiapoptotic modulators
To gain mechanistic insights into the impact of HDP-101, gene expression analysis was performed on TP53 WT H929 and MM1.S cells (Fig. 3A), and also on their TP53 KO counterparts (Fig. S6).Pairwise analysis (Fig. S7) revealed that HDP-101 down-regulated more genes than were up-regulated, consistent with its mechanism as a POLR2A inhibitor.Enriched genes with a false discovery rate of < 5% are presented in Table 1 as hallmark gene sets (32) identi ed from the Molecular Signatures Database, with a focus on gene sets up-regulated in all four models.Notably, the UPR, a target of several therapeutics effective against myeloma (33), was induced by HDP-101.As this gene set contained Activating transcription factors (ATF)-5 and ATF6, as well as X-box protein (XBP)-1, we performed Western blotting to determine if there was evidence of UPR activation at the protein level.Both ATF6, which represents one arm of the ER UPR, and ATF5, which regulates the mitochondrial UPR, were induced by HDP-101 (Fig. 3B), and this tended to occurr to a greater extent in the POLR2A KD cell models.Similarly, Inositol-requiring enzyme 1 (IRE1) was induced, which led to enhanced downstream expression of the short isoform of XBP1 (Fig. 3B).On the down-regulated side, and consistent with the known mechanism of action of αamanitin (34), RNA polymerase II and III levels were decreased (Fig. 3C).Notably, HDP-101 also decreased expression levels of several anti-apoptotic proteins, including Myeloma cell leukemia 1 (MCL1) and BCLx L , as well as the X-linked inhibitor of apoptosis (Fig. 3C).These occurred in association with increased markers of apoptosis, including of cleaved poly-(ADP-ribose) polymerase (PARP1) and cleaved Caspases 3 and 7 (Fig. 3B,C).Compared to the isotype negative control, HDP-101 enhanced cell surface levels of both CRT and HMGB1 (Fig. 4A), consistent with induction of ICD as seen with bortezomib as a positive control.Next, we expanded our panel to include H929 and MM1.S cells that were TP53 WT, TP53 KO, POLR2A KD, or dual KO/KD, and looked at CRT and HMGB1, as well as externalization of Heat shock protein 70 (HSP70) and HSP90.Once again compared to the isotype negative control, HDP-101 induced externalization of all four markers of ICD in both cell lines, with a trend seen towards increasing ICD in the H929 model system (Fig. 4B).Finally, we further broadened our analysis to look at our larger panel of myeloma cell lines representing multiple molecular subtypes of myeloma.Compared to the isotype control, HDP-101 enhanced cell surface levels of Calreticulin, HMGB1, and HSP70 in all of the cell lines (Fig. S8), suggesting consistent activation of the UPR and ICD.

Activity of HDP-101 in vivo may engage NK cells and macrophages
We next tested the ability of HDP-101 to exert effects against primary samples derived from patients, and separated these initially into fractions enriched for CD138-positive tumor cells and CD138-negative tumor microenvironment (TME) cells.HDP-101 showed minimal effects on the proportion of TME cells that were in the early apoptotic (Annexin V + /TO-PRO-3 − ), late apoptotic (Annexin V + /TO-PRO-3 + ), or necrotic (Annexin V − /TO-PRO-3 + ) stages of cell death (Fig. 5A) compared to the isotype control.In contrast, HDP-101 induced a substantial decrease in the viable cell fraction (Annexin V − /TO-PRO-3 − ) and an increase predominantly in the late apoptotic fraction under the condition tested (Fig. 5A).To examine the activity of HDP-101 in vivo, we developed a systemic xenograft using luciferase (luc)-labeled MM.1S WT TP53 cells in NOD-SCID mice.Once engrafted, mice were randomized to produce three groups with equivalent disease burden to receive a single dose of vehicle, HDP-101 at 4 mg/kg, or the anti-digoxigenin/αamanitin ADC control at 4 mg/kg.Compared to vehicle, the negative control/non-targeted α-amanitin-ADC did not substantially reduce disease burden (Fig. 5B,C).In contrast, HDP-101 rst slowed disease progression, and this was followed by complete disease regression by day 32 (Fig. 5C).Similar patterns were seen in the MM1.S TP53 KO, POLR2A KD, or dual KO/KD models, with regression of disease and no evidence of relapse even after a single 2 mg/kg dose of HDP-101 (Fig. 5D).Amanitins are able to impact quiescent cells (24,25,26) that could be equivalent to myeloma-initiating cells, which have been hypothesized as potentially contributing to drug resistance and disease relapse after other anti-myeloma therapies (36, 37).Therefore, we considered whether elimination of such cells could have contributed to the lack of relapse after HDP-101, and used staining for Aldehyde dehydrogenase (ALDH) to identify them (36).ALDH-positive cell fractions of H929 and MM.1S cells proved to be more clonogenic than their ALDH-negative counterparts (Fig. S9A,B).Notably, HDP-101 showed similar activity against the two, with no signi cant difference in the IC 50 (Fig. S9C).
As disease relapse was not see in NOD-SCID mice xenografted with MM1.S cells and treated with HDP-101 at 100 days, we rechallenged a pilot cohort of ve of these mice with luc-labeled MM1.S cells.
Whereas engraftment was virtually 100% at baseline, none of these mice developed detectable tumor after another 100 day follow-up period (Fig. 6A).While these mice are immune de cient, recent reevaluation of this model has indicated that they retain aspects of the innate immune system, including NK cells and macrophages (38).To determine if these could be contributing to the lack of re-engraftment, we repeated this experiment except that, just prior to disease re-challenge, we randomized mice to receive a dose of either control rabbit antiserum or a rabbit anti-Asialo-GM1 antibody.While the control group showed a range of uorescence values after the initial MM1.S re-injection (Fig. 6B) consistent with some baseline circulating tumor cells, this signal rapidly disappeared (Fig. 6C).In contrast, treatment with anti-Asialo-GM1 to deplete NK cells and macrophages promoted tumor engraftment and led to a signi cantly higher disease burden (Fig. 6B,C).

HDP-101 is more potent ADC than belantamab mafodotin
Belantamab mafodotin (BelaMaf), a BCMA-targeted ADC linked to monomethyl-auristatin F, was the rst drug in its class to achieve a regulatory approval for myeloma.However, more recently, it was withdrawn as a randomized study of this agent did not show su cient superiority compared to a standard of care.
Given the encouraging activity of HDP-101, we compared it directly to BelaMaf in both our in vitro and in vivo model systems.In the former, HDP-101, an ADC with a drug:antibody ratio (DAR) of 2 ( 24), more potently reduced viability of all of the myeloma cell lines we tested at 48 hours than was the case for BelaMaf (Table S2), which has a DAR of 4. At 96 hours, HDP-101 showed both greater overall e cacy than BelaMaf in our H929-and MM1.S-based WT, TP53 KO, POLR2A KD, and dual KO/KD models, as well as preferential activity in the del17p dual knockout cells (Table 2).Next, we tested primary patient-derived samples, and found that HDP-101 induced a greater loss of viability than did BelaMaf at two different concentrations (Fig. 7A).Finally, we prepared a xenograft based on the del17p MM1.S dual TP53 KO/POLR2A KD model, and treated these with a single dose of isotype control, or either 0.1 or 0.5 mg/kg of BelaMaf or HDP-101.This dose was selected to produce sub-total myeloma cell killing by HDP-101 to allow for better comparisons between the activity of the two ADCs.At day 60 (Fig. 7B), control mice had substantial disease burden that was reduced only modestly by BelaMaf at either of the two doses (Fig. 7C).In contrast, a statistically signi cant tumor growth inhibition was seen at both doses of HDP-101 (Fig. 7C) which, as expected, was greater at the higher dose, where some mice had no measurable tumor (Fig. 7B).

Discussion
Outcomes for myeloma have signi cantly improved through the development of several now widely used drug classes and, as a result, the median overall survival of most patients treated with modern induction regimens is up to 10-15 years or more from diagnosis (39,40,41).However, there remains a high-risk group who still have substantially shorter remission durations despite the use of these therapies (7).Patients whose disease harbors del17p have consistently been identi ed as one of these groups, and one study noted that this may especially be the case in those with biallelic TP53 inactivation, or so-called "double hit" myeloma (16).More recently, a second study con rmed this nding in patients treated with intensive therapy among whom "double hit" patients had a median survival of 36 months versus 152.2 months for controls (42), and even a single del17p conferred an inferior prognosis, with a median survival of 52.8 months.It is likely that these numbers will be improved by more novel therapies that induce deeper levels of remission, including those that target BCMA, but advances that leverage an understanding of the unique biology of each high-risk molecular subtype could be especially of bene t.
In light of the above, we studied HDP-101, a BCMA ADC conjugated to an optimized α-amanitin, with the rationale that del17p could produce haploinsu ciency of POLR2A, which is located nearby TP53, and heighten sensitivity to α-amanitin.Consistent with this possibility, del17p was associated in primary samples with reduced mRNA and protein levels of POLR2A, and HDP-101 showed enhanced e cacy against del17p models with biallelic TP53 loss and POLR2A knockdown (Fig. 2).Mechanistically, HDP-101 reduced mitochondrial transmembrane potentials and induced apoptosis in association with activation of the ER and mitochondrial UPRs and induction of ICD (Figs. 2-4).Moreover, HDP-101 worked in concert with gamma-secretase inhibitors (Fig. S7) and overcame adhesion-mediated drug resistance (Figs. 1C, S2), con rming earlier studies (25,26).Importantly, HDP-101 retained activity in models of acquired conventional and novel drug resistance (Table S3), indicating promise for activity in the relapsed/refractory setting where there is an unmet medical need.A single, well-tolerated dose of HDP-101 rapidly reduced disease burden to undetectable levels in vivo (Fig. 5), and relapse was not noted over a 100-day follow-up period even in the del17p models, which showed more aggressive growth without treatment.Importantly, HDP-101 showed enhanced e cacy compared with BelaMaf (Table 2, Fig. 7), the only other BCMA-targeted ADC to have achieved a regulatory approval endpoint.Notably, BelaMaf was recently withdrawn from the market after a randomized study showed a lack of su cient superiority to a control treatment in the setting of relapsed/refractory myeloma, supporting the possibility that more potent agents like HDP-101 are needed.
ADCs with microtubule-targeting agents such as BelaMaf's auristatins work well against proliferating cells (2) while amanitins can target quiescent cells (25), and may thus have an advantage against hypoproliferative tumors like myeloma, as well as against myeloma-initiating or stem-like cells.The latter was supported by our studies of ALDH-positive clonogenic cells (Fig. S9), which were equally sensitive to HDP-101 compared with ALDH-negative cells.This ability to impact both committed plasma cells and their precursors may in part underlie the ability of HDP-101 to eradicate myeloma in in vivo models, including those with del17p, and to show greater e cacy than BelaMaf pre-clinically.It is interesting in this regard to note that one recent study described TP53 loss as contributing to the tumor-initiating and drug resistance potential of clonogenic myeloma cells through activation of the Notch signaling pathway and upregulation of inhibitor of DNA binding (ID1/ID2) genes (43).
Another interesting aspect of HDP-101's mechanism of action was its strong ability to induce immunogenic cell death, which was notable in vitro (Fig. 4).Moreover, rechallenge of previously treated mice at day 100 with new aliquots of tumor cells was not successful in producing tumor re-engraftment (Fig. 6).While this could theoretically be due to the presence of persistent levels of HDP-101 that retain anti-tumor activity, this seems unlikely since the clearance of ADCs is remarkably rapid in NSG mice based on prior studies showing a half-life of 1.4 days for an anti-CD30 construct (44).Furthermore, engraftment was enhanced by depletion of NK cells and macrophages using an anti-Asialo-GM1 antibody while this was not the case for control serum, undermining the possibility that this was due to remaining drug.These ndings, which suggest the involvement of an anti-tumor effect from residual innate immune system cells in these mice, deserve follow-up in human models, since this agent is currently undergoing Phase I testing (45), to determine the immunologic effects of this drug in patients.

Conclusions
The novel BCMA-targeted ADC HDP-101 showed robust anti-myeloma e cacy against both in vitro and in vivo models of multiple myeloma, overcame conventional and novel drug resistance, and induced both direct pro-apoptotic and immunogenic cell death mechanisms.Importantly, through its amanitin-based warhead, HDP-101 showed a uniquely enhanced e cacy against models of del17p myeloma which harbor haplo-insu ciency of its target, POLR2A.Since del17p represents a high-risk myeloma subtype that typically has an inferior bene t from other therapeutics, HDP-101 could represent an important advance in this unmet medical need population, and Phase I testing is currently underway.As no engraftment was seen, this experiment was repeated and, at 100 days, mice were rst given a dose of either control rabbit serum (n=17) or rabbit anti-Asilao-GM1 (n=19), followed by injection with luc-MM1.S TP53 WT cells and monitored for disease burden.Whole animal imaging is shown for the entire cohorts at weeks 3 and 4 (B), and median uorescent ux is shown for the entire cohorts in weeks 1 through 4 (C).

Figures
Figure 7

Table 1
Hallmark gene sets identi ed from the Molecular Signatures Database using gene expression data from myeloma cells exposed to HDPhave the ability to induce both direct cytotoxic effects as well as ICD as part of their mechanisms of action (2) and, given the link between UPR activation and ICD(35), we next sought to determine if HDP-101 could so as well.Starting with the MM1.S cell model, isotype antibody or HDP-101treated cells were analyzed to detect externalization of Calreticulin (CRT) or High mobility group box 1 (HMGB1) by immuno uorescence.

Figure 2 E
Figure 2

Figure 5 E
Figure 5

Figure 6 Prior
Figure 6

Table 2
Median inhibitory concentrations (pM) for H929 and MM1.S-based myeloma cell line models treated with belantamab mafodotin or HDP-101 for 96 hours*