Selective Targeting of Multiple Myeloma By Bcma-Specific Central Memory CD8+ cytotoxic T Lymphocytes: A Potential Immunotherapeutic Application in Multiple Myeloma and Other Plasma Cell Disorders

Abstract Background: Multiple myeloma (MM) is a B-cell malignancy characterized by the clonal proliferation and accumulation of malignant plasma cells in the bone marrow and development of osteolytic bone lesions. Despite recent advances in treatment using novel therapeutics, MM remains incurable with high mortality rates. We have demonstrated in preclinical studies that CD8+ cytotoxic T lymphocytes (CTL) generated with immunogenic HLA-A2 or HLA-A24 peptides targeting XBP1(X-box binding protein 1), CD138 (Syndecan-1) and CS1 (SLAMF7) antigens induces robust cytotoxic activities against MM. The Phase 1/2a trials were completed or are in progress in the patients with smoldering multiple myeloma or triple negative breast cancer, respectively, using the HLA-A2 XBP1/CD138/CS1 multipeptide vaccine. The clinical data demonstrated that the multipeptide vaccine is safe and induces the XBP1/CD138/CS1-specific immune responses, evidenced by expansion of peptides-specific Tetramer+/CD45RO+memory CTL and Th-1 specific immune responses. Moreover, clinical trials combining with Lenalidomide or checkpoint inhibitor enhanced the CD8+ CTL activities induced by multipeptide vaccine, indicating the benefit of combination therapy. To expand the breadth and extent of the antigens-specific immunotherapy beyond XBP1/CD138/CS1, we have recently identified additional tumor-associated antigens (TAA) on tumor cells obtained from newly diagnosed MM patients (N=616). Here, we introduce a novel heteroclitic peptides specific to BCMA, the receptor forbinding of B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL). Due to its restricted expression pattern on MM cells and plasma cells along with its critical role in promoting MM cell growth, survival and drug resistance, we are currently in development of novel immunotherapeutic to target BCMA on MM cells with different therapeutic approaches. Objective: The aims of current study were to target BCMA as a TAA by generating the antigen-specific memory CD8+ CTL to induce effective and long-lasting immune response against MM. Findings: We report on immunogenic HLA-A2-restricted peptides derived from BCMA, which are capable of evoking antigen-specific immune responses against MM. The heteroclitic BCMA peptides displayed improved binding affinity/stability to HLA-A2 molecules from their native BCMA peptides. To define immunogenicity of the selected peptides, we generated BCMA-specific CTL (BCMA-CTL) by repeated stimulationof CD3+ T cells with respective heteroclitic peptide. The BCMA-specific engineered peptides evoked the expansion of antigen-specific CD8+ CTL and generated BCMA-CTL displaying high T cell activation (CD38, CD69) and co-stimulatory (CD40L, OX40, GITR) molecules expression. Additionally, a gradual expansion was observed in BCMA-specific memory CD8+ T cells, with a corresponding decrease in naïve CD8+ T cells. The BCMA-CTL demonstrated robust poly-functional immune responses with Th1-specific anti-MM activities [high IFN-g/IL-2/TNF-aproduction, CD8+ T cells proliferation, cytotoxicity] in antigen-specific and HLA-A2-restricted manner. The functional activities were directly correlated with the expansion of central memory CD8+ CTL in the BCMA-CTL generated from different HLA-A2+ individuals (Donor 1 BCMA-CTL: 81.0%, Donor 2 BCMA-CTL: 82.6%, Donor 3 BCMA-CTL: 67.0%). Finally, the combination with checkpoint inhibitor (anti-LAG3) or immune agonist (anti-OX40) enhanced the anti-tumor activities of BCMA-CTL, along with the induction of cytotoxic activities by central memory CD8+ T cell subset. Therefore, these studies suggest that heteroclitic BCMA peptides offer a therapeutic potential to effectively generate BCMA-specific CD8+ CTL targeting MM. Significance: Here, we introduce novel immunogenic engineered heteroclitic BCMA peptides capable of inducing antigen-specific memory CD8+ CTL with robust poly-functional immune responses against MM. These results provide the framework for therapeutic application of heteroclitic BCMA peptides in MM patients. They further support combination treatment options incorporating BCMA peptides-specific vaccine or BCMA peptides-specific adoptive T cells immunotherapy with anti-LAG3 and/or anti-OX40for patients with myeloma or other diseases expressing BCMA. Disclosures Munshi: OncoPep: Other: Board of director. Anderson:Celgene: Consultancy; Takeda Millennium: Consultancy; Bristol Myers Squibb: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncopep: Equity Ownership; C4 Therapeutics: Equity Ownership.

Download Full-text

Targeting LAG3/GAL-3 to overcome immunosuppression and enhance anti-tumor immune responses in multiple myeloma

Leukemia ◽

10.1038/s41375-021-01301-6 ◽

2021 ◽

Author(s):

Jooeun Bae ◽

Fabrizio Accardi ◽

Teru Hideshima ◽

Yu-Tzu Tai ◽

Rao Prabhala ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

Immune Responses ◽

Checkpoint Inhibitor ◽

Immune Checkpoints ◽

Specific Memory ◽

Immune Profiling ◽

Improve Patient ◽

Inhibitor Treatment

AbstractImmune profiling in patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and multiple myeloma (MM) provides the framework for developing novel immunotherapeutic strategies. Here, we demonstrate decreased CD4+ Th cells, increased Treg and G-type MDSC, and upregulation of immune checkpoints on effector/regulatory and CD138+ cells in MM patients, compared MGUS/SMM patients or healthy individuals. Among the checkpoints profiled, LAG3 was most highly expressed on proliferating CD4+ Th and CD8+ Tc cells in MM patients BMMC and PBMC. Treatment with antibody targeting LAG3 significantly enhanced T cells proliferation and activities against MM. XBP1/CD138/CS1-specific CTL generated in vitro displayed anti-MM activity, which was further enhanced following anti-LAG3 treatment, within the antigen-specific memory T cells. Treg and G-type MDSC weakly express LAG3 and were minimally impacted by anti-LAG3. CD138+ MM cells express GAL-3, a ligand for LAG3, and anti-GAL-3 treatment increased MM-specific responses, as observed for anti-LAG3. Finally, we demonstrate checkpoint inhibitor treatment evokes non-targeted checkpoints as a cause of resistance and propose combination therapeutic strategies to overcome this resistance. These studies identify and validate blockade of LAG3/GAL-3, alone or in combination with immune strategies including XBP1/CD138/CS1 multipeptide vaccination, to enhance anti-tumor responses and improve patient outcome in MM.

Download Full-text

Bcma Heteroclitic Peptide Encapsulated Nanoparticle Enhances Antigen Stimulatory Capacity and Tumor-Specific CD8+ cytotoxic T Lymphocytes Against Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-117292 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3195-3195 ◽

Cited By ~ 1

Author(s):

Jooeun Bae ◽

Parayath Neha ◽

Mansoor Amiji ◽

Nikhil Munshi ◽

Kenneth Anderson

Keyword(s):

T Cells ◽

Immune Responses ◽

Cancer Vaccine ◽

Cd8 T Cells ◽

Release Kinetics ◽

Memory Cd8 T Cells ◽

Functional Activities ◽

Free Peptide ◽

Specific Memory ◽

Equity Ownership

Abstract Background: B-cell Maturation Antigen (BCMA), a member of the tumor necrosis factor (TNF) receptor superfamily and the receptor for binding of B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL), is a promising therapeutic target for MM. Due to its restricted expression pattern on MM cells and plasma cells along with its role in promoting MM cells growth, survival, and drug resistance, BCMA is being targeted by several immunotherapeutic strategies including antibodies, immunotoxins, bispecific T-cell engagers, and CAR-T cells. Recently, we have identified nanomedicine-based therapeutics targeting BCMA as a promising area of translational research to effectively evoke and augment anti-tumor responses in MM patients. Several nanomedicines are available and more advanced nanoparticle constructs are under development for antigen encapsulation. To this end, we have designed a heteroclitic BCMA peptide encapsulated nanoparticle-based cancer vaccine to overcome the limitations of free peptide vaccines including poor peptide stability, susceptibility to enzyme degradation, and low antigen uptake and delivery. Furthermore, the nanotechnology-based cancer vaccine was developed to induce more robust BCMA-specific CD8+ cytotoxic T lymphocytes (CTL) activities in MM patients, with more sustained antigen release and increased bioavailability and presentation of the immunogenic peptide. Here, we examine the potential of a novel nanomedicine-based therapeutic delivery system specific to BCMA antigen to treat the patients with MM. Objective: The purpose of this study was to design the optimal nanoparticle encapsulated BCMA antigen constructs to efficiently evoke and develop the BCMA-specific CD8+ CTL with functional anti-myeloma activities. Findings: Nanoparticles [liposome or poly(D,L-lactide-co-glycolide) (PLGA)] with different antigen-release kinetics demonstrated their capacity to effectively deliver heteroclitic BCMA peptideto antigen-presenting cells and evoke BCMA antigen-specific CTL with anti-MM activities. The heteroclitic BCMA peptide encapsulated nanoparticles demonstrated a higher uptake by human dendritic cells than free peptide, with the highest uptake mediated with liposome-based nanoparticles. In contrast, BCMA-specific CTL induced with PLGA-based nanoparticle demonstrated the highest functional activities and specific immune responses against MM cells. The PLGA/BCMA peptide nanoparticle induced BCMA-specific CTL displayed the highest increases in CD107a degranulation, the antigen-specific CD8+ T cells proliferation and Th-1 type cytokines (IFN-g, IL-2, TNF-a) production to MM patients' tumor cells and MM cell lines compared to BCMA-CTL generated with free BCMA peptide or liposome/BCMA peptide nanoparticle. These observations were aligned with the highest level of CD28 costimulatory molecules upregulation, Tetramer+ CTL generation and peptide-specific responses within the BCMA-CTL generated by PLGA/BCMA nanoparticles. Furthermore, the PLGA/BCMA nanoparticles triggered a more robust induction of antigen-specific memory CD8+ T cells, which demonstrated significantly higher anti-tumor activities, evidenced by CD107a degranulation and IFN-g production, compared to non-memory CD8+ T cells within the BCMA-CTL. Especially, the increased central memory CTL development and their anti-tumor activities evoked by PLGA/BCMA peptide were associated with the optimal peptide release kinetics and enhanced immunogenicity of the antigen via this nanotechnology. Thus, these results demonstrate that the heteroclitic BCMA peptide encapsulated nanoparticle strategy supports the peptide delivery into dendritic cells and then subsequently to T cells, resulting in effective induction of BCMA-specific central memory CTL with poly-functional activities against MM. Significance: These results demonstrate the utility of nanotechnology using encapsulated heteroclitic BCMA peptide to enhance the immunogenicity of BCMA peptide-specific therapeutics against MM. Importantly, our observations provide the framework for therapeutic application of PLGA-based heteroclitic BCMA peptide delivery to enhance the BCMA-specific memory T cell immune responses, overcome the limitations of current peptide-based cancer vaccine, and improve the patient outcome in MM. Disclosures Munshi: OncoPep: Other: Board of director. Anderson:Bristol Myers Squibb: Consultancy; Celgene: Consultancy; Millennium Takeda: Consultancy; C4 Therapeutics: Equity Ownership, Other: Scientific founder; OncoPep: Equity Ownership, Other: Scientific founder; Gilead: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Dominant Expression of PVRIG and TIGIT Inhibitory Pathways in Bone Marrow of Multiple Myeloma Patients

Blood ◽

10.1182/blood-2021-148052 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4717-4717

Author(s):

Masha Frenkel ◽

Zoya Alteber ◽

Ning Xu ◽

Mingjie Li ◽

Haiming Chen ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Plasma Cells ◽

Myeloma Cell ◽

Inhibitory Receptor ◽

Preclinical Models ◽

Cell Numbers

Abstract Introduction Blocking inhibitory immune checkpoints holds promise to treat multiple myeloma (MM) patients. However, currently available checkpoint inhibitors have not shown significant clinical benefits for MM patients, warranting the need for alternative checkpoint blockers. The immune checkpoint TIGIT was recently shown to be the most upregulated immune inhibitory receptor on CD8+ T cells in MM patients' bone marrow (BM), compared to other checkpoints (Guillerey C., Blood. 2018). Preclinical models demonstrated the dominant effects of TIGIT blockade or depletion, by significantly improving mice survival, reducing myeloma cell numbers and exhausted T cell hallmarks (Minnie S., Blood. 2018). As a result, several clinical trials using anti-TIGIT monoclonal antibodies have been recently initiated in MM patients. The DNAM-1 family, in addition to TIGIT, also includes the inhibitory receptor PVRIG, that competes with the co-activating receptor DNAM-1 for the binding to the shared ligand PVRL2, similarly to the TIGIT/PVR/DNAM-1 interaction. Accordingly, TIGIT and PVRIG co-blockade were shown to synergize in enhancing T cell activity and anti-tumor activity in preclinical models (Whelan S., Cancer Immunol. Res. 2019). PVRL2 together with PVR (ligand of TIGIT) were shown to be highly expressed on plasma cells and on CD14+ cells in BM of MM patients (Lozano E., Clin. Cancer Res. 2020). This study aimed at evaluating DNAM-1 axis receptors expression in MM patients' BM. Methods Fresh BM aspirates were collected from 21 MM patients with progressive disease (PD) or in complete response (CR) after obtaining IRB approval. BM mononuclear cells were isolated and single cell suspensions were obtained followed by staining with anti-human antibodies to evaluate DNAM-1 axis members and PD-1 expression. BM biopsies from 6 MM patients (each patient had 4 core on the Tissue Micro-Array T291 USBiomax) were stained for PVRL2 expression by immuno-histochemistry (IHC). Results Flow cytometry analysis of PD-1 and DNAM-1 axis receptors revealed a significant lower fraction of PD1+ cells among cell populations examined compared with other receptors. TIGIT expression was the highest on NK, CD8+ and NKT cells compared to CD4+ T cells, which is in line with previous published data (Lozano E. Clin. Cancer Res. 2020). In contrast, DNAM-1 was expressed on CD8+ T, NK and NKT cells with prominent high expression on CD4+ T cells (Fig 1A). The highest expression among the receptors was of PVRIG on all lymphoid populations, except CD4+ where DNAM-1 was more highly expressed. Importantly, 50% of CD8+ T cells co-expressed TIGIT and PVRIG, supporting a combinatorial therapeutic approach (Fig. 1B). Additionally, the expression of the PVRL2 ligand on MM plasma and endothelial cells was demonstrated by IHC. FACS analysis further supported PVRL2 expression on plasma cells in MM BM (Fig 2). A higher expression of PVRIG, TIGIT and PD-1 was present on DNAM-1 negative CD8+ T cells (Fig 3A, B), suggesting accumulation of exhausted cells in MM tumor microenvironment (TME) as previously described (Minnie S., Blood. 2018). PVRIG had significantly higher expression on DNAM+ cells, compared to PD-1 and TIGIT (Fig 3C), suggesting the potential of its blockade to enhance DNAM-1 activation and subsequent proliferation of earlier differentiated memory cells in MM TME. Finally, CR patients had a trend for higher DNAM-1 expression on CD8+ T cells compared to those with PD (Fig 3D). This is consistent with other reports in mice showing that the expression of DNAM-1 negatively correlates with BM myeloma cell numbers (Minnie S., Blood. 2018). Conclusions DNAM-1 axis receptors are dominantly expressed on lymphocytes in BM of MM patients, with PVRIG exhibiting the most prominent expression. The reduced expression of DNAM-1 in PD patients' TME, compared to CR patients, suggests a link between DNAM-1 axis and clinical outcomes. Recent data suggest TIGIT is an attractive target for blockade in MM. Our new findings highlight for the first time the dominant expression of PVRIG, as well as TIGIT, and suggest that combined blockade of TIGIT with PVRIG may potentially benefit MM patients, placing the DNAM-1 axis as a dominant pathway in MM therapy. Figure 1 Figure 1. Disclosures Frenkel: Compugen Ltd.: Current Employment, Other: in the event of frontal participation, I will be reimbursed for my travel expenses by Compugen Ltd.. Alteber: Compugen Ltd.: Current Employment. Cojocaru: Compugen Ltd.: Current Employment. Ophir: Compugen Ltd.: Current Employment.

Download Full-text

Immunotherapy with Long-Lived Anti-CD38 × Anti-CD3 Bispecific Antibodies Stimulates Potent T Cell-Mediated Killing of Human Myeloma Cell Lines and CD38+ Cells in Monkeys: A Potential Therapy for Multiple Myeloma

Blood ◽

10.1182/blood.v124.21.4727.4727 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4727-4727 ◽

Cited By ~ 6

Author(s):

Seung Y. Chu ◽

Yvonne Miranda ◽

Sheryl Phung ◽

Hsing Chen ◽

Rumana Rashid ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

Plasma Cells ◽

Bispecific Antibodies ◽

Cytotoxic T Cell ◽

Antigen Specificity ◽

Employment Equity ◽

Activation Markers ◽

Equity Ownership

Abstract CD38, being highly expressed on malignant plasma cells, is an attractive target of new therapies for multiple myeloma (MM). Several anti-CD38 antibodies including daratumumab are in clinical development; however, a limitation of such monospecific antibodies is their inability to stimulate cytotoxic T cell killing of myeloma cells. To exploit the potent mechanism of T cell immunotherapy yet preserve the favorable drug and dosing properties of therapeutic antibodies, we designed bispecific antibodies that recruit T cells to CD38+ MM cells. Such bispecifics act via a "redirected T cell-cytotoxicity" (RTCC) mechanism because they stimulate targeted T cell-mediated killing regardless of T cell receptor antigen specificity. Unlike other bispecific formats, these antibodies possess a full Fc domain and spontaneously form stable heterodimers that are readily manufactured. Their Fc domain was also engineered to abolish binding to Fcγ receptors (to reduce the potential for nonselective T cell activation), yet preserve binding to human FcRn (to maintain long serum half-life). We first generated a library of humanized and affinity-optimized anti-CD38 × anti-CD3 antibodies and measured their potency using RTCC assays in which antibodies stimulated killing of the human MM cell line RPMI8226 by human T cells. From this screen, we selected two candidates for further assessment. XmAb13243 and XmAb13551 have 21 and 0.2 nM affinities, respectively, for human CD38, and have identical T cell-engaging domains with 8 nM affinity for human CD3. XmAb13243 stimulated RTCC with an EC50 of 2.5 ng/ml (20 pM) after 24 hr, while XmAb13551 had an EC50 of ~100 pg/ml (~1 pM). In contrast to bispecific formats lacking an Fc domain, XmAb13243 and XmAb13551 had long half-lives in mice of ~7.6 and 8.3 days, respectively. Because these bispecifics were optimized for human CD38 and CD3 binding and do not crossreact with mouse antigens, we next evaluated efficacy in immunodeficient SCID mice engrafted with human PBMCs. In this model, engrafted human B cells differentiate into CD38+ plasma cells, which produce high levels of human Ig. Bispecific antibodies dosed at 0.2, 1, and 5 mg/kg, 7 and 15 days after engraftment, suppressed human IgG2, IgM, and IgE to below detectable levels by Day 14 (> 50-fold for IgG2, > 1,000-fold for IgM, and > 80-fold for IgE). Daratumumab at 5 mg/kg was markedly less potent than bispecifics, reducing IgG2 by 2-fold, IgM by 6-fold, and IgE by 3-fold. The control bispecific anti-RSV × anti-CD3 (which binds to T cells but not to CD38+ cells) had no effect on IgG2, IgM, or IgE levels. To investigate activity against an immune response requiring production of new human plasma cells, mice were vaccinated with tetanus toxoid 8 days after engraftment. Anti-CD38 × anti-CD3 bispecifics suppressed human anti-tetanus antibody titers to baseline (> 100-fold), while daratumumab suppressed titers by only 2-fold. We next assessed efficacy in cynomolgus monkeys. Unlike daratumumab, which does not crossreact with monkey CD38, XmAb13243 and XmAb13551 bind to both CD38 and CD3 in monkeys (23 and 0.3 nM, respectively, to CD38, and 6 nM to CD3 for both). We treated monkeys with a single dose of XmAb13243 or XmAb13551 at 2, 5, and 20 μg/kg. T cells were activated within 1 hr, as measured by dramatic increases in CD25 and CD69 activation markers. Within 8 hr, T cells depleted circulating CD38+ cells by > 95% at the 20 μg/kg dose. Our results demonstrate that XmAb13243 and XmAb13551 effectively recruit T cells to kill CD38+ cells in vivo. Our preclinical data in monkeys and humanized mice provide a rationale for clinical testing of anti-CD38 × anti-CD3 bispecific antibodies in patients with multiple myeloma and other CD38+ malignancies. Disclosures Chu: Xencor: Employment, Equity Ownership. Miranda:Xencor, Inc.: Employment, Equity Ownership. Phung:Xencor, Inc.: Employment, Equity Ownership. Chen:Xencor, Inc.: Employment, Equity Ownership. Rashid:Xencor, Inc.: Employment, Equity Ownership. Endo:Xencor, Inc.: Employment, Equity Ownership. Chan:Xencor, Inc.: Employment, Equity Ownership. Pong:Xencor, Inc.: Employment, Equity Ownership. Bonzon:Xencor, Inc.: Employment, Equity Ownership. Muchhal:Xencor, Inc.: Employment, Equity Ownership. Leung:Xencor, Inc.: Employment, Equity Ownership. Bernett:Xencor, Inc.: Employment, Equity Ownership. Moore:Xencor, Inc.: Employment, Equity Ownership. Szymkowski:Xencor, Inc.: Employment, Equity Ownership. Desjarlais:Xencor, Inc.: Employment, Equity Ownership.

Download Full-text

CXCR3 Is Required for the Efficient Recruitment of Bone Marrow-Resident Memory T Cells to Inflamed Tissues,

Blood ◽

10.1182/blood.v118.21.3242.3242 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3242-3242

Author(s):

Robbert van der Voort ◽

Claudia Brandao ◽

Thomas J. Volman ◽

Viviènne Verweij ◽

Klaas van Gisbergen ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Immune Responses ◽

Cd8 T Cells ◽

Memory T Cells ◽

Central Memory ◽

T Cell Subsets ◽

Effector Memory ◽

Memory T Cell

Abstract Abstract 3242 Although the importance of the bone marrow (BM) in hematopoiesis is well known, its function in adaptive immune responses has only recently been acknowledged. Currently it is known that the BM contains fully functional CD4+ and CD8+ T cells that can engage in both primary and secondary immune responses. Interestingly, most of these T cells belong to the memory T cell lineage, identifying the BM as one of the largest memory T cell reservoirs in the body. Since not much is known about the trafficking of BM T cells, we compared the homing phenotype and function of T cell subsets in the BM, blood, spleen and peripheral lymph nodes (pLN). In addition, we determined the expression of chemokine mRNA and protein levels in the BM and other lymphoid organs. We confirmed that at least 80% of the CD4+ and 60% of the CD8+ BM T cells have a memory phenotype, and that most CD4+ T cells belong to the effector memory lineage, while the CD8+ population predominantly consists of central memory T cells. Most BM T cells expressed the chemokine receptor CXCR3, the adhesion molecules P-selectin glycoprotein ligand 1 and VLA-4, and increased levels of CD44 and LFA-1, as compared to T cells from the spleen. In addition, L-selectin was absent from most CD4+ BM T cells, but present on virtually all CD8+ T cells. Notably, the percentage of CXCR3+ T cells within the effector memory and central memory subsets from BM was higher than within the same subsets from pLN. Furthermore, BM contained significant mRNA levels of the CXCR3 ligands CXCL9, CXCL10 and CXCL11. An in vivo migration assay using a mixture of fluorescent-labeled T cells from CXCR3-deficient mice and control mice indicated however that during homeostasis CXCR3 does not play a major role in BM entry or retention. These data suggest that CXCR3 expressed by memory T cells is rather involved in BM exit, than in BM entry. Indeed, we observed that, as compared to control mice, CXCR3−/− mice contained significantly more CD4+ and CD8+ T cells in their BM. Additional in vitro assays demonstrated that CD4+ and CD8+ BM T cells migrated vigorously in response to CXCL9 and CXCL10, generally released in high concentrations during inflammation. Finally, we demonstrate that CXCR3−/− effector/effector memory T cells, but not wild type T cells, accumulate in the BM of mice infected with lymphocytic choriomeningitis virus. Altogether, these data demonstrate that the BM is a major reservoir of memory T cells that employ CXCR3 to quickly respond to chemotactic signals from inflamed tissues. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Tigit, CD226 and PD-L1/PD-1 Are Highly Expressed By Marrow-Infiltrating T Cells in Patients with Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.2102.2102 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2102-2102 ◽

Cited By ~ 5

Author(s):

Mahesh Yadav ◽

Cherie Green ◽

Connie Ma ◽

Alberto Robert ◽

Andrew Glibicky ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Nk Cells ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Plasma Cells ◽

Color Flow

Abstract Introduction:TIGIT (T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif [ITIM] domain) is an inhibitory immunoreceptor expressed by T and natural killer (NK) cells that is an important regulator of anti-tumor and anti-viral immunity. TIGIT shares its high-affinity ligand PVR (CD155) with the activating receptor CD226 (DNAM-1). We have recently shown that TIGIT blockade, together with PD-L1/PD-1 blockade, provides robust efficacy in syngeneic tumor and chronic viral infection models. Importantly, CD226 blockade abrogates the benefit of TIGIT blockade, suggesting additional benefit of TIGIT blockade through elaboration of CD226-mediated anti-tumor immunity, analogous to CTLA-4/CD28 regulation of T-cell immunity. Whether TIGIT and CD226 are expressed in patients with multiple myeloma (MM) and how TIGIT expression relates to PD-L1/PD-1 expression is unknown. Here we evaluate expression of TIGIT, CD226, PD-1 and PD-L1 in patients with MM to inform novel immunotherapy combinations. Methods:We performed multi-color flow cytometry (n = 25 patients), and multiplex qRT-PCR (n = 7) on bone marrow specimens from patients with MM to assess expression of TIGIT, CD226, PD-1, and PD-L1 on tumor and immune cells. Cells were stained with fluorescently conjugated monoclonal antibodies to label T cells (CD3, CD4, CD8), NK cells (CD56, CD3), plasma cells (CD38, CD45, CD319, CD56), inhibitory/activating receptors (PD-1, TIGIT, PD-L1, CD226), and an amine-reactive viability dye (7-AAD). Stained and fixed cells were analyzed by flow cytometry using BD FACSCanto™ and BD LSRFortessa™. Results:TIGIT, CD226 and PD-L1/PD-1 were detectable by flow cytometry in all patients with MM who were tested, with some overlapping and distinct expression patterns. TIGIT was commonly expressed by marrow-infiltrating CD8+ T cells (median, 65% of cells), CD4+ T cells (median, 12%) and NK cells. In contrast, CD226 was more commonly expressed by marrow-infiltrating CD4+ T cells (median, 74%) compared with CD8+ T cells (median, 38%). PD-1 was expressed by marrow-infiltrating CD8+ T cells (median 38%) and CD4+ T cells (median, 16%). TIGIT was co-expressed with PD-1 on CD8+ T cells (67%-97% TIGIT+ among PD-1+), although many PD-1-negative CD8+ T cells also expressed TIGIT (39%-78% of PD-1-negative). PD-L1 was also expressed by CD8+ (median, 23%) and CD4+ (median, 8%) T cells in addition to MM plasma cells (median, 95%), albeit with significantly lower intensity on T cells compared with plasma cells. The expression of TIGIT and PD-L1 mRNA was highly correlated (R2 = 0.80). Analysis of PVR expression will also be presented. Conclusions: TIGIT, CD226, PD-1, and PD-L1 were commonly expressed in MM bone marrow, but with different patterns. Among CD8+ T cells, the frequency of TIGIT+ T cells was almost twice that of PD-1+ T cells, whereas the majority of CD4+ T cells expressed CD226. TIGIT blockade may complement anti-PD-L1/PD-1 immunotherapy by activating distinct T-cell/NK-cell subsets with synergistic clinical benefit. These results provide new insight into the immune microenvironment of MM and rationale for targeting both the PD-L1/PD-1 interaction and TIGIT in MM. Disclosures Yadav: Genentech, Inc.: Employment. Green:Genentech, Inc.: Employment. Ma:Genentech, Inc.: Employment. Robert:Genentech, Inc.: Employment. Glibicky:Makro Technologies Inc.: Employment; Genentech, Inc.: Consultancy. Nakamura:Genentech, Inc.: Employment. Sumiyoshi:Genentech, Inc.: Employment. Meng:Genentech, Inc.: Employment, Equity Ownership. Chu:Genentech Inc.: Employment. Wu:Genentech: Employment. Byon:Genentech, Inc.: Employment. Woodard:Genentech, Inc.: Employment. Adamkewicz:Genentech, Inc.: Employment. Grogan:Genentech, Inc.: Employment. Venstrom:Roche-Genentech: Employment.

Download Full-text

Alterations in the antigen processing-presenting machinery of transformed plasma cells are associated with reduced recognition by CD8+ T cells and characterize the progression of MGUS to multiple myeloma

Blood ◽

10.1182/blood-2009-06-228676 ◽

2010 ◽

Vol 115 (6) ◽

pp. 1185-1193 ◽

Cited By ~ 33

Author(s):

Vito Racanelli ◽

Patrizia Leone ◽

Maria Antonia Frassanito ◽

Claudia Brunetti ◽

Federico Perosa ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

Cd8 T Cells ◽

Antigen Processing ◽

Plasma Cells ◽

Disease Status ◽

Transcriptional Level ◽

Proteasome Subunits ◽

Polymerase Chain ◽

Lower Expression

Abstract We hypothesized that progression of monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) reflects the escape of transformed plasma cells from T-cell recognition because of impaired antigen processing-presenting machinery (APM). We studied plasma cells and CD8+ T cells from bone marrow of 20 MGUS patients, 20 MM patients, and 10 control patients. Immunofluorescence and flow cytometry revealed significantly different patterns of APM component expression in plasma cells from the 3 groups. Compared with control patients, MM samples had lower expression of proteasome subunits and peptide transporters and greater expression of chaperones, considering both percentages of stained cells and molecular equivalents of soluble fluorochrome. MGUS samples had intermediate percentages of stained cells but molecular equivalents of soluble fluorochrome similar to control patients. Real-time polymerase chain reaction documented that APM changes occurred at the transcriptional level. Cytotoxicity assays demonstrated that MGUS CD8+ T cells lysed autologous transformed plasma cells more than MM CD8+ T cells did. MGUS progression correlated directly with calnexin, calreticulin, and tapasin and indirectly with δ, LMP2, and LMP10 expression levels; MM disease status did not correlate with APM levels. APM changes may allow transformed plasma cells to elude immunesurveillance in the MGUS-MM pathogenetic sequence.

Download Full-text

PD-1 Blockade Reinvigorates Bone Marrow CD8+ T Cells from Patients with Multiple Myeloma in the Presence of TGF-β Inhibitors

Blood ◽

10.1182/blood-2019-128182 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3241-3241

Author(s):

Minsuk Kwon ◽

Eui-Cheol Shin ◽

Yoon Seok Choi

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Immune Checkpoint ◽

Cytokine Production ◽

Plasma Cells ◽

Myeloma Cells ◽

Tgf Β1

Programmed cell death (PD)-1/PD-Ligand 1(PD-L1) blockade that reinvigorates exhausted T cells has been approved for the treatment of various solid tumors and hematological malignancies. However, in a clinical trial of multiple myeloma (MM) patients, anti-PD-1 monotherapy did not result in a clinical response. Furthermore, clinical trials of combining PD-1 blockade with immunomodulatory drugs or anti-CD38 monoclonal antibody failed to demonstrate clinical benefits in MM patients. To overcome the limitation of anti-PD-1 therapy in MM, the phenotype and differentiation of CD8+ T cells need to be characterized in the bone marrow (BM) of MM patients, particularly by analyzing myeloma antigen-specific CD8+ T cells. In addition, the role of immunosuppressive factors abundant in the MM microenvironment should be considered, including TGF-β. First, we confirmed the upregulation of PD-1 and PD-L1 expression in CD8+ T cells and myeloma cells, respectively, from the BM of MM patients. PD-1-expressing CD8+ T cells from the BM of MM patients co-expressed other checkpoint inhibitory receptors including Tim-3, LAG-3, and TIGIT. We also investigated the expression of T-cell transcription factors, such as T-bet, and EOMES, which are related to T-cell differentiation. In BM from MM patients, PD-1+CD8+ T cells had a higher percentage of EomeshiT-betlo cells than PD-1-CD8+ T cells. These data demonstrate that PD-1-expressing CD8+ T cells from the BM of MM patients exhibit a terminally differentiated phenotype with co-expression of multiple immune checkpoint inhibitory receptors. These results were also observed in BM CD8+ T cells specific to myeloma antigens NY-ESO-1 and HM1.24. Next, we investigated proliferation and cytokine production of BM CD8+ T cells from MM patients. BM CD8+ T cells from MM patients exhibited reduced proliferation and cytokine production upon T cell receptor (TCR) stimulation, compared to BM CD8+ T cells from other control group such as of undetermined significance. However, both anti-PD-1 alone and combined blockade of PD-1 with other immune checkpoint receptors, such as Tim-3, Lag-3, or TIGIT, did not increase the proliferation of BM CD8+ T cells from MM patients. Likewise, anti-PD-1 treatment failed to induce reinvigoration of BM CD8+ T cells stimulated with HLA-A*0201-restricted myeloma antigen peptides, including NY-ESO-1157-165 and HM1.2422-30 peptides. These data demonstrate that blocking PD-1 is not sufficient to restore the function of BM CD8+ T cells from MM patients. It has been known that TGF-β, which is actively secreted by malignant plasma cells and BM stromal cells, can inhibit T-cell responses. We confirmed that the major source of TGF- β1 is plasma cells including myeloma cells among BMMCs from MM patients, and the number of TGF- β1-producing plasma cells, including myeloma cells, is increased in the BM of MM patients. We investigated whether blocking TGF-β signaling enhances reinvigoration of BM CD8+ T cells from MM patients. The combined blockade of PD-1 and TGF- β significantly increased the proliferation of BM CD8+ T cells from MM patients in the presence of TCR stimulation. The production of IFN-γ and TNF by BM CD8+ T cells was also rescued by combined blockade of PD-1 and TGF-β. Moreover, combination of anti-PD-1 antibody and TGF-β inhibitors increased proliferative responses of BM CD8+ T cells from HLA-A2+ MM patients stimulated with a mixture of HLA-A*0201-restricted myeloma antigen peptides (NY-ESO-1157-165 and HM1.2422-30 peptides). Thus, PD-1 blockade reinvigorates BM CD8+ T cells from MM patients in the presence of TGF-β inhibitors. Taken together, BM CD8+ T cells and myeloma antigen-specific CD8+ T cells express increased levels of PD-1 and have a terminally exhausted phenotype in MM patients. Under TGF-β inhibition, anti-PD-1 reinvigorates BM CD8+ T cells from MM patients, but PD-1 blockade alone does not restore the function of BM CD8+ T cells. Blocking both TGF-β and PD-1 can be a promising therapeutic strategy for the treatment of MM. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Superior Efficacy of CAR-T Cells Using Marrow-Infiltrating Lymphocytes (MILsTM) As Compared to Peripheral Blood Lymphocytes (PBLs)

Blood ◽

10.1182/blood-2019-130629 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4437-4437 ◽

Cited By ~ 2

Author(s):

Eric R. Lutz ◽

Srikanta Jana ◽

Lakshmi Rudraraju ◽

Elizabeth DeOliveira ◽

Jing Zhou ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Employment Equity ◽

Car T Cells ◽

Car T ◽

Equity Ownership

Background The type of T cell used in generating chimeric antigen receptor (CAR) T cells is an important choice. Evidence suggests that T cells that are early in the effector/memory differentiation pathway with more stemness and greater potential to persist are better than more differentiated T cells with less stemness that are more readily exhausted and have less potential to persist. Marrow-infiltrating Lymphocytes (MILsTM) is a novel form of adoptive T cell therapy composed of patient-autologous, polyclonal CD4 and CD8 T cells that are activated and expanded from the bone marrow. Genetically unmodified MILsTM have demonstrated antitumor activity in patients with multiple myeloma and are being developed for several other tumor types, including non-small cell lung cancer and other solid tumors. Distinguishing features of bone marrow T cells used to produce MILsTM include their memory phenotype, inherent tumor antigen-specificity, higher CD8:CD4 ratio and ability to persist long-term when compared to peripheral blood lymphocytes (PBLs) which is the T cell source used to produce currently approved CAR-T therapies. Based on these differences, we hypothesize that MILsTM provide a more robust and better fit platform for CAR-T therapy compared to PBLs. Using a CD38-specific, 4-1BB/CD3z-signaling CAR as an initial model, we have demonstrated the feasibility of producing CAR-modified MILsTM (CAR-MILsTM) and showed that CAR-MILsTM demonstrate superior killing in vitro compared to CAR-T cells generated from patient-matched PBLs (CAR-PBLs). Herein, we build on our previous data and add a second BCMA-specific CAR model. We use the two multiple myeloma model systems to compare cytolytic potential, functionality, and expression of phenotypic markers of memory, stemness and exhaustion between patient-matched CAR-MILsTM and CAR-PBLs. Methods Matched pairs of CAR-MILsTM and CAR-PBLs were produced from the bone marrow and blood of multiple myeloma patients. Two different in vitro cytotoxicity assays, the RTCA xCelligence real-time impedance and FACS assays, were used to evaluate antigen-specific killing of target tumor cells. Functionality of CD4 and CD8 CAR-T cells, at the single-cell level, was evaluated by measuring the secretion of 32 cytokines and chemokines following in vitro antigen-specific stimulation using IsoPlexis IsoCode chips and analyzed using IsoPeak. Expression of markers of T cell memory (CD45RO & CCR7/CD62L), stemness (CD27) and exhaustion (PD1 & TIM3) on CAR-MILsTM and CAR-PBLs prior to and following antigen-specific stimulation was evaluated by flow-cytometry (FACS). Results CAR-MILsTM demonstrated superior killing of tumor target cells in vitro, regardless of the antigen specificity of the CAR, when compared to matched CAR-PBLs and this superiority persisted even upon repeated antigen encounter - a factor that may be critical in guaranteeing better anti-tumor efficacy and persistence. CAR-MILsTM demonstrated increased polyfunctionality (secretion of 2+ cytokines per cell) and an increased polyfunctional strength index (PSI) following antigen-stimulation compared to CAR-PBL in both CD4 and CD8 T cells. The enhanced PSI in CAR-MILsTM was predominately mediated by effector, stimulatory and chemoattractive proteins associated with antitumor activity including Granzyme B, IFNg, IL-8, MIP1a and MIP1b. Coincidentally, increased PSI and enhanced secretion of these same proteins was reported to be associated with improved clinical responses in patients with Non-Hodgkin lymphoma treated with CD19-specific CAR-T therapy. Expression of memory markers on CD4 and CD8 T cells were similar in CAR-MILsTM and CAR-PBLs both prior to and following antigen-stimulation. Although expression of CD27, PD1 and TIM3 were similar at baseline, CAR-MILs maintained higher levels of CD27 and lower levels of PD1 and TIM3 compared to CAR-PBLs following antigen-stimulation in both CD4 and CD8 T cells. Conclusions Collectively, our data suggest that CAR-MILsTM have several advantages over CAR-PBLs, including increased cytolytic potential, enhanced polyfunctionality, increased stemness and less exhaustion. Based on these differences and the inherent antitumor properties of MILsTM, we speculate that CAR-MILsTM would be more potent and effective than currently approved CAR-T products derived from PBLs. Disclosures Lutz: WindMIL Therapeutics: Employment, Equity Ownership. Jana:WindMIL Therapeutics: Employment, Equity Ownership. Rudraraju:WindMIL Therapeutics: Employment, Equity Ownership. DeOliveira:WindMIL Therapeutics: Employment, Equity Ownership. Zhou:Isoplexis: Employment, Equity Ownership. Mackay:Isoplexis: Employment, Equity Ownership. Borrello:Aduro: Patents & Royalties: intellectual property on allogeneic MM GVAX; BMS: Consultancy; WindMIL Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Celgene: Honoraria, Research Funding, Speakers Bureau. Noonan:WindMIL Therapeutics: Employment, Equity Ownership, Patents & Royalties; Aduro: Patents & Royalties: intellectual property on allogeneic MM GVAX.

Download Full-text

Chemically Controlled, Immunosuppression-Resistant, Anti-Bcma CAR-T Cells for Treatment of Antibody-Mediated Autoimmunity

Blood ◽

10.1182/blood-2018-99-117630 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2203-2203 ◽

Cited By ~ 1

Author(s):

Sowndharya Rajavel ◽

Cade E. Ito ◽

Keith Abe ◽

Valerie Guerrero ◽

Gene I. Uenishi ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

Side Effect ◽

Plasma Cells ◽

Car T Cells ◽

Car T Cell ◽

Nsg Mice ◽

Car T ◽

Equity Ownership

Abstract Auto-reactive antibody production by plasma cells is the direct cause of many auto-immune diseases. In such cases elimination of plasma cells would ameliorate the disease. Chimeric antigen receptor T (CAR-T) cells with cytotoxicity toward cells expressing B-cell maturation antigen (BCMA) have shown remarkable promise for the treatment of multiple myeloma, a plasma cell neoplasm. Elimination of non-malignant plasma cells is a side-effect of anti-BCMA CAR-T treatment of multiple myeloma, suggesting the use of these anti-BCMA CAR T cells for auto-immune indications. Unfortunately, CAR-T administration requires use of lymphodepletion to achieve efficient cell engraftment, and is often accompanied by cytokine release syndrome (CRS), a potentially life-threatening side-effect. As lymphodepletion and CRS pose morbidity/mortality risks that are unacceptable for therapy of many auto-immune diseases, we have utilized CRISPR-Cas9 gene editing to develop a controllable CAR-T cell platform that provides for (1) engraftment with non-cytotoxic transient immunosuppression; and (2) small-molecule dependent CAR T-cell expansion. We have implemented this platform using a unique dual targeting approach in which a BCMA CAR transgene is integrated into the TRAC locus, and additional payloads are integrated into a second locus, thus also enabling an allogeneic manufacturing process. Transgene integration occurred in >50% of cells individually with several percent of cells targeted at two loci. TRAC-targeted, anti-BCMA CAR T cells demonstrated CAR-dependent, target-cell-BCMA-dependent cytotoxicity towards both high-BCMA- and low-BCMA-expressing cell lines and in multiple myeloma cells xenografted into NSG mice. Drug-regulation properties and immunosuppression resistance are the subject of ongoing experiments. Anti-BCMA CAR T cells that are chemically controlled, incapable of graft-versus-host disease, and insensitive to immunosuppression may be an attractive treatment option a variety of antibody-mediated auto-immune conditions. Disclosures Rajavel: Casebia Therapeutics: Employment. Ito:Casebia Therapeutics: Employment. Abe:Casebia Therapeutics: Employment. Guerrero:Casebia Therapeutics: Employment. Uenishi:Casebia Therapeutics: Employment. Scharenberg:Casebia Therapeutics: Employment; Generation Bio: Equity Ownership; Alpine Immune Sciences: Equity Ownership. Cost:Casebia Therapeutics: Employment.

Download Full-text