Antigen escape as a shared mechanism of resistance to BCMA-directed therapies in multiple myeloma Free

Clinical outcomes for patients with relapsed/refractory multiple myeloma (MM) are poor.¹ Recently developed agents targeting B-cell maturation antigen (BCMA), including chimeric antigen receptor (CAR) T cells, bispecific antibodies (BsAb), and antibody-drug conjugates (ADCs), are remarkably effective in patients with advanced disease. Four BCMA-directed therapies for RRMM are FDA-approved in the fifth-line setting and beyond, and several agents are under clinical investigation. However, studies evaluating these therapies in patients with prior exposure to BCMA-directed treatments are limited,^2-10 and clinical guidance for the optimal sequencing of these agents is not established.

Deletions in TNFRSF17, the gene coding for BCMA, have been implicated in secondary resistance to BCMA-directed CAR T-cell therapies and BsAbs.^11-13 The primary mechanisms of resistance to these treatments remain elusive, however, with available studies focusing on patient–specific immune function, which may not represent a shared mechanism of resistance among BCMA-targeting agents.^3,14 

Additional investigation is, therefore, warranted to understand how BCMA expression affects responses to BCMA-directed therapies and how to incorporate target antigen verification in therapy selection for RRMM. This is increasingly relevant as therapies targeting G protein–coupled receptor, class C, group 5, member D (GPRC5D) and Fc receptor homolog 5 (FcRH5) become more ubiquitous.^15-17 

The study included 72 RRMM patients at Memorial Sloan Kettering Cancer Center (MSKCC) who received ≥1 dose of commercial teclistamab between 29 November 2022 and 1 November 2023. Fifty patients had a pretreatment bone marrow (BM) or plasmacytoma biopsy for BCMA-expression assessment. Of the remaining 22 patients, 5 had biopsies inadequate for analysis and 17 lacked pretreatment tissue sampling. Treatment response was determined using International Myeloma Working Group response criteria after manual review of the electronic health record by consensus of 2 investigators. Baseline patient characteristics and response data are shown in supplemental Table 1, available on the Blood website.

Formalin-fixed and paraffin-embedded BM and plasmacytoma biopsy samples were used to assess antigen expression. Hematoxylin and eosin stained tissue was used to identify areas with malignant plasma cells for immunohistochemistry (IHC)-based assays. IHC experiments were performed using a BOND-III fully automatic staining system (Leica Biosystems) following manufacturer recommendations. Staining for CD138, BCMA, and GPRC5D was performed according to manufacturer recommendations (supplemental Table 2).

Peripheral blood plasma was collected on an MSKCC institutional review board (IRB)–approved biospecimen procurement protocol (MSKCC IRB 06-107 or 12-245). Plasma was isolated and cryopreserved using institutional practice (supplemental Methods, A1). Soluble BCMA (sBCMA) concentrations were measured with the ProteinSimple SimplePlex human soluble BCMA kit (catalog no. SPCKB-PS-000768) using an Ella Automated Immunoassay System. Samples were run in quadruplets, 2 at 1:100 dilution and 2 at 1:200 dilution. All pretreatment samples were collected <14 days before the start of teclistamab therapy.

Tumor DNA for WES was collected from a residual sample used for standard-of-care tumor cytogenetic analysis. Sequencing was performed using KAPA Hyper library chemistry and xGen v2.0 (Integrated DNA Technologies, Inc) capture chemistry using a NovaSeq 6000 sequencer. Coverage averaged 344×. Because no DNA sample was available from a pre-BCMA–directed therapy time point, copy number variant analysis was conducted using the fraction and allele-specific copy number estimates from tumor sequencing (FACETS) method with 9 control normals and aggregated output from multiple runs, with data from the control normal with the greatest confidence following quality control used for analysis (supplemental Methods, A2).¹⁸ 

Pre-teclistamab plasma cell BCMA expression was available for 50 patients. Of these patients, all individuals naïve to anti-BCMA–directed therapy and most patients with anti-BCMA–directed therapy exposure (n = 47) had detectable BCMA expression in BM or plasmacytoma biopsy samples. However, the remaining 3 patients, who previously received non-teclistamab anti-BCMA–directed therapies, lacked malignant plasma cell BCMA expression, and subsequently had no response to teclistamab. BCMA expression was present in biopsy samples before receiving non-teclistamab BCMA-directed therapies (Figure 1).

Patient 1 received commercial belantamab mafodotin (belantamab) and achieved a complete response with minimal residual disease negativity. Following relapse from belantamab and salvage autotransplant, a restaging BM biopsy showed almost complete loss of BCMA expression on CD138-positive plasma cells (supplemental Figure 1). The patient later received CAR T-cell therapy with ciltacabtagene autoleucel (cilta-cel) and then teclistamab, with no response to either treatment.¹⁹ A BM biopsy before teclistamab administration showed a complete absence of BCMA staining (Figure 1A). A BM biopsy after teclistamab failure showed GPRC5D-expressing plasma cells (supplemental Figure 2), and the patient started talquetamab with response to therapy.¹⁵ 

Patient 2 received cilta-cel and achieved a minimal residual disease negative complete response. At cilta-cel relapse, a plasmacytoma biopsy was negative for BCMA expression (Figure 1B). The patient then had no response to teclistamab and died of disease-related complications.

Patient 3 received belantamab and achieved a partial response, with a 2-month duration of response. At relapse, a BM biopsy showed a complete absence of plasma cell BCMA expression. The patient then received teclistamab with no response and has since received cytotoxic chemotherapy with ongoing response.

Because antigen escape has never been reported as a mechanism of resistance to belantamab and was not observed in patients in either the DREAMM-1 or DREAMM-2 trials,²⁰ we performed WES on bulk tumor DNA from patient 1 after belantamab relapse and before cilta-cel administration. Copy number variant analysis showed loss of both copies of TNFRSF17 on chromosome 16 spanning from ∼12 000 kb to ∼12 220 kb (Figure 2), with a cell fraction of 60.6% for the deletion. This indicated a heterogenous tumor sample inclusive of clones with biallelic loss of TNFRSF17 and clones with intact TNFRSF17, consistent with the protein level data by IHC showing a residual small population of BCMA-expressing plasma cells (supplemental Figure 1). No mutations in members of the gamma secretase complex or in protein N-glycosylation genes that regulate BCMA expression were found.²¹ 

Because BM biopsies are not routinely performed in RRMM patients before initiating each new therapy, we assessed peripheral blood sBCMA levels as a noninvasive proxy of BCMA expression by BM plasma cells for patients 1 to 3 (Figure 3). Consistent with the known absence of BCMA-expressing BM plasma cells by IHC after prior non-teclistamab anti-BCMA–directed therapy, sBCMA levels immediately before teclistamab treatment were <10 ng/mL or undetectable, a level comparable with individuals without clonal plasma cell disorders.²² Values of sBCMA were compared with patient-specific measures of disease burden, which were kappa free light chain (KFLC) levels for patient 1, plasmacytoma biopsy plasma cell percentage for patient 2 (nonsecretory disease), and monoclonal protein levels for patient 3. In all cases, sBCMA levels were dramatically higher, relative to the metric of disease burden, before BCMA expression loss. For example, in patient 1, sBCMA levels decreased by >3.5-fold despite KFLC increasing by greater than sixfold following relapse from belantamab, when a BCMA-positive clone was still present before cilta-cel treatment (supplemental Figure 1). Subsequently, sBCMA became undetectable following cilta-cel failure despite a further KFLC increase.

To provide further context for these sBCMA results, we measured sBCMA levels in 10 patients in our cohort who retained BCMA expression on BM biopsies (supplemental Figure 3). Although there was variance in values due to differences in disease burden, the lowest measured value was 24.3 ng/mL. Thus, low or undetectable sBCMA in patients with active myeloma offers a surrogate measure for plasma cell BCMA expression, obviating the need for invasive BM testing, which was not available for 22 out of 72 patients in our cohort and reflective of the real-world practicalities of disease restaging.

Genomic sequencing of malignant plasma cells is not standard for the management of MM beyond identifying clinically relevant immunoglobulin heavy chain translocations or cytogenetic abnormalities associated with high-risk disease.²³ Other than identifying the presence of the t(11;14) translocation,²⁴ there are no current diagnostic tests for MM management that specifically direct therapy selection. Acquiring sufficient tumor material for genetic analysis in RRMM patients is often challenging, even in academic settings, as BM biopsy cell yields may be inadequate, or patients may decline an invasive test that does not impact disease management.

Here we show 3 cases of BCMA-expression loss, likely due to TNFRSF17 deletion in all cases, that can be identified by IHC or very low sBCMA levels. All 3 individuals with BCMA expression loss had primary refractoriness to teclistamab, with 1 patient additionally having primary refractoriness to cilta-cel. To our knowledge, this is also the first report of a dominant TNFRSF17 deletion occurring after belantamab treatment, including relatively rapid loss of BCMA expression after a ∼2-month partial response and possibly indicative of belantamab–mediated selective pressure leading to the expansion of a BCMA-negative clone.

Overall, these findings, combined with recently published data, indicate that all current classes of BCMA-directed therapies, including CAR T cells, BsAbs, and ADCs, can trigger BCMA-expression loss and that TNFRSF17 biallelic deletions represent a shared, albeit rare, resistance mechanism across BCMA–targeting therapeutic platforms. In contrast, TNFRSF17 monoallelic deletions coupled with TNFRSF17 extracellular domain mutations resulting in functional antigenic escape occur at a higher frequency (almost 50%) in patients progressing after anti-BCMA BsAbs.^11-13 Notably, these structural abnormalities do not lead to nearly undetectable levels of peripheral blood sBCMA or a lack of BCMA expression when assessed by IHC using antibodies that bind the cytoplasmic domain of the protein. With prospective validation, assessment of malignant plasma cell BCMA expression by IHC or sBCMA could be readily incorporated into clinical practice and allow for a guided approach to the choice of next therapy, especially for individuals refractory to prior BCMA-directed therapy. A limitation of this method is the inability to identify structural variants of BCMA, which can limit the efficacy of BCMA-directed BsAb therapy.¹³ Recent reports suggest that TNFRSF17 overexpression can limit the efficacy of BCMA-directed therapies through competition for binding, further supporting the value of assessing sBCMA levels before BCMA-directed BsAb treatment.²⁵ 

The authors acknowledge the use of the Integrated Genomics Operation Core and support from the Bioinformatics Core, both of which are funded by the National Institutes of Health (NIH), National Cancer Institute (NCI) Cancer Center support grant (CCSG, P30 CA08748), Cycle for Survival, and the Marie-Josée and Henry R. Kravis Center for Molecular Oncology. R.S.F. is supported by the Conquer Cancer Foundation and the International Myeloma Society/Paula and Rodger Riney Foundation. This work was also funded by the Khalily Myeloma Research Fund (D.J.C.) and the Guthart Myeloma Research Fund (D.J.C.). This work was also supported by NIH/NCI grant P01 CA023766. U.S. reports grants from NIH/NCI Cancer Center support grant P30 CA008748, MSK Paul Calabresi Career Development Award for Clinical Oncology K12CA184746, Paula and Rodger Riney Foundation, Parker Institute for Cancer Immunotherapy at Memorial Sloan Kettering, HealthTree Foundation, International Myeloma Society, American Society of Hematology, and Allen Foundation Inc, as well as nonfinancial support from American Society of Hematology Clinical Research Training Institute and TREC Training Workshop R25CA203650 (principal investigator: Melinda Irwin). K.H.M. reports grant support from the American Society of Hematology, the Multiple Myeloma Research Foundation, and the International Myeloma Society.

Portions of the visual abstract were created with BioRender.com.

Contribution: R.S.F. and D.J.C. conceived the project, designed all experiments, and analyzed the data; R.S.F., T.S., M.H., S.M., C.R.T., N.K., A.M.L., H.H., U.S., K.H.M., S.R., H.J.L., M.S., G.L.S., O.B.L., S.G., S.Z.U., and D.J.C. collected samples or recruited patients; R.S.F. and M.Z. analyzed immunohistochemistry data; R.S.F., W.G.Q., and K.M. performed and analyzed soluble BCMA experiments; R.S.F. and N.D.S. analyzed genomic data; R.S.F. and D.J.C. wrote the manuscript; and all authors reviewed the manuscript.

Conflict-of-interest disclosure: T.S. reports receiving honoraria from Roche-Genentech. M.Z. reports receiving advisory or consulting fees from Leica Biosystems. M.H. reports research funding from Amgen, Daiichi Sankyo, GlaxoSmithKline (GSK); and has received honoraria for consultancy/participated in advisory boards for Curio Science LLC, Intellisphere LLC, Bristol Myers Squibb (BMS), and GSK. S.M. received consulting fees from Evicore, Optum, BioAscend, Janssen Oncology, BMS, AbbVie, HMP Education, and Legend Biotech; and received honoraria from OncLive, Physician Education Resource, MJH Life Sciences, and Plexus Communications. Memorial Sloan Kettering Cancer Center receives research funding from the National Cancer Institute, Janssen Oncology, BMS, Allogene Therapeutics, Fate Therapeutics, Caribou Therapeutics, and Takeda Oncology for conducting research. C.R.T. reports research funding from Janssen and Takeda and personal fees from Physician Educations Resource and MJH Life Sciences; and has participated in advisory boards for Janssen and Sanofi, outside of the submitted work. N.K. reports research funding through Amgen, Janssen, Epizyme, AbbVie; consults for Clinical Care Options, OncLive, and Intellisphere Remedy Health; and participated in advisory board for Janssen and MedImmune. A.M.L. reports receiving grants from Novartis, during the conduct of the study; grants from BMS; personal fees from Trillium Therapeutics; grants, personal fees, and nonfinancial support from Pfizer; grants and personal fees from Janssen, outside the submitted work; and also has a patent (number US20150037346A1) with royalties paid. H.H. reports grants from Celgene, Takeda, and Janssen, outside the submitted work. U.S. reports research support from Celgene/BMS and Janssen; personal fees from MashUp MD, Janssen Biotech, Sanofi, BMS, MJH Life Sciences, Intellisphere, Phillips Gilmore Oncology Communications, i3 Health, and RedMedEd. H.J.L. has served as a paid consultant for Takeda, Genzyme, Janssen, Karyopharm, Pfizer, Celgene, Caelum Biosciences, and has received research support from Takeda. M.S. served as a paid consultant for McKinsey & Company, Angiocrine Bioscience, Inc, and Omeros Corporation; received research funding from Angiocrine Bioscience, Inc, Omeros Corporation, and Amgen, Inc; served on ad hoc advisory boards for Kite, a Gilead company; and received honoraria from i3 Health, Medscape, and CancerNetwork for CME-related activity. G.L.S. receives research funding from Janssen, Amgen, BMS, Beyond Spring; serves on the data safety monitoring board (DSMB) for ArcellX; and receives research funding to the institution from Janssen, Amgen, BMS, Beyond Spring, and GPCR. O.B.L. reports serving on advisory board for MorphoSys, Kite, Daiichi Sankyo Inc, and Incyte and has served as a paid consultant for Incyte. S.G. reports personal fees and advisory role (scientific advisory board) from Actinium, Celgene, BMS, Sanofi, Amgen, Pfizer, GSK, Jazz, Janssen, Omeros, Takeda, and Kite, outside the submitted work. K.M. reports funding from Sebia, Binding site, and Siemens. S.Z.U. reports grants and personal fees from AbbVie, 404 Amgen, BMS, Celgene, GSK, Janssen, Merck, Mundipharma, Oncopeptides, 405 Pharmacyclics, Sanofi, Seattle Genetics, SkylineDX, and Takeda. D.J.C. receives research funding from Genentech. The remaining authors declare no competing financial interests.

Correspondence: Ross S. Firestone, Department of Medicine, Memorial Sloan Kettering Cancer Center, 1275 York Ave, New York, NY 10065; email: firestor@mskcc.org; Saad Z. Usmani, Myeloma Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, 530 E 74th St, New York, NY 10021; email: usmanis@mskcc.org; and David J. Chung, Adult Bone Marrow Transplant Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, 530 E 74th St, New York, NY 10021; email: chungd1@mskcc.org.