A New Platform For Trivalent Bispecific Antibodies Used For T-Cell Redirected Killing Of B-Cell Malignancies

Abstract Background Various formats of bispecific antibodies (bsAbs) to redirect effector T cells for the targeted killing of tumor cells have shown considerable promise both pre-clinically and clinically. The scFv-based constructs, including BiTE and DART, which bind monovalently to CD3 on T cells and to the target antigen on tumor cells, exhibit fast blood clearance and neurological toxicity due to their small size (∼55 kDa). Herein, we describe the generation of novel T-cell redirecting trivalent bsAbs comprising an anti-CD3 scFv covalently conjugated to a stabilized F(ab)2. The design was initially characterized with a prototype construct designated (19)-3s, which specifically targets CD19 on B cells. A panel of trivalent bsAbs was evaluated for their potential use in targeted T-cell immunotherapy of various B-cell malignancies. Potential advantages of this design include bivalent binding to tumor cells, a larger size (∼130 kDa) to preclude rapid renal clearance and penetration of the blood-brain barrier, and potent T-cell mediated cytotoxicity. Methods The DOCK-AND-LOCKTM (DNLTM) method was used to generate a panel of B-cell targeting bsAbs, (19)-3s, (20)-3s, (22)-3s, and (C2)-3s, which target CD19, CD20, CD22, and HLA-DR, respectively. This was achieved by combining a stabilized anti-X F(ab)2 with an anti-CD3-scFv, resulting in a homogeneous covalent structure of the designed composition, as shown by LC-MS, SE-HPLC, ELISA, SDS-PAGE, and immunoblot analyses. Each construct can mediate the formation of immunological synapses between T cells and malignant B cells, resulting in T-cell activation. At an E:T ratio of 10:1, using isolated T cells as effector cells, the bsAbs induced potent T-cell-mediated cytotoxicity in various B-cell malignancies, including Burkitt lymphomas (Daudi, Ramos, Namalwa), mantle cell lymphoma (Jeko-1), and acute lymphoblastic leukemia (Nalm-6). A non-tumor binding control, (14)-3s, induced only moderate T-cell killing at >10 nM. The nature of the antigen/epitope, particularly its size and proximity to the cell surface, appears to be more important than antigen density for T-cell retargeting potency (Table 1). It is likely that (20)-3s is consistently more potent than (19)-3s and (C2)-3s, even when the expression of CD19 or HLA-DR is considerably higher than CD20, as seen with Namalwa and Jeko-1, respectively. This is likely because the CD20 epitope comprises a small extracellular loop having close proximity to the cell surface. When compared directly using Daudi, (22)-3s was the least potent. Compared to CD19 and CD20, CD22 is expressed at the lowest density, is a rapidly internalizing antigen, and its epitope is further away from the cell surface; each of these factors may contribute to its reduced potency. Finally, sensitivity to T-cell retargeted killing is cell-line-dependent, as observed using (19)-3s, where Raji (IC50 >3 nM) is largely unresponsive yet Ramos (IC50 = 2 pM) is highly sensitive, even though the former expresses higher CD19 antigen density. Conclusions (19)-3s, (20)-3s, (22)-3s, and (C2)-3s can bind T cells and target B cells simultaneously and induce T-cell-mediated killing in vitro. The modular nature of the DNL method allowed the rapid production of several related conjugates for redirected T-cell killing of various B-cell malignancies, without the need for additional recombinant engineering and protein production. The close proximity of the CD20 extracellular epitope to the cell surface results in the highest potency for (20)-3s, which is an attractive candidate bsAb for use in this platform. We are currently evaluating the in vivo activity of these constructs to determine if this novel bsAb format offers additional advantages. Disclosures: Rossi: Immunomedics, Inc.: Employment. Rossi:Immunomedics, Inc.: Employment. Goldenberg:Immunomedics: Employment, stock options, stock options Patents & Royalties. Chang:Immunomedics, Inc: Employment, Stock option Other; IBC Pharmaceuticals, Inc.: Employment, Stock option, Stock option Other.

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T Cell Receptor Gene Therapy Targeting the Intracellular Transcription Factor Bob1 for the Treatment of Multiple Myeloma and Other B Cell Malignancies

Blood ◽

10.1182/blood.v126.23.3002.3002 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3002-3002 ◽

Cited By ~ 1

Author(s):

Lorenz Jahn ◽

Renate S. Hagedoorn ◽

Pleun Hombrink ◽

Michel G.D. Kester ◽

Dirk M. van der Steen ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

Transcription Factor ◽

T Cell ◽

Gene Transfer ◽

Cell Surface ◽

B Cell ◽

Cell Lines ◽

B Cell Malignancies ◽

T Cell Clone

Abstract Therapeutic reactivity of CD20-specific monoclonal antibodies (mAb) or CD19-specific chimeric antigen receptor (CAR)-transduced T cells is exerted by targeting extracellular antigens. In contrast to mAbs and CARs, T cell receptors (TCRs) recognize antigen-derived peptides that are bound to human leukocyte antigen (HLA) molecules on the cell surface. Since HLA molecules constantly sample the entire endogenous proteome of a cell, extracellular and intracellular antigens are presented and can thus be recognized by a TCR. Here, we identified the intracellular transcription factor Bob1 encoded by gene POU2AF1 as a suitable target for immunotherapy. Bob1 is highly expressed in CD19+ B cells, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and multiple myeloma (MM) and is absent in the non-B lineages including CD34+ hematopoietic progenitor cells (HPCs), T cells, fibroblasts, keratinocytes and gastrointestinal tract. Bob1 is localized intracellularly but HLA-presented Bob1-derived peptides are accessible on the cell surface to TCRs and can thus be recognized by T cells. From the HLA-presented ligandome (Mol Cell Proteomics, 2013;12:1829) we identified naturally processed Bob1-derived peptides displayed in HLA-A*0201 (HLA-A2) and in HLA-B*0702 (HLA-B7). Since auto-reactivity towards self-antigens such as Bob1 is prevented by depleting high-avidity T cells recognizing self-antigens in self-HLA, we exploited the immunogenicity of these peptides presented in allogeneic HLA. From a HLA-A2/B7-negative healthy individual we isolated T cell clone 4G11 demonstrating high sensitivity and specificity for Bob1-derived peptide Bob144 presented in HLA-B7. Bob1-dependent recognition was demonstrated by transduction of Bob1 into cell lines that otherwise lack Bob1 expression. No harmful toxicities of clone 4G11 were observed against a wide panel of Bob1-negative stimulator cells including HLA-B7-positive CD34+ HPCs, T cells, monocytes, immature and mature dendritic cells, and fibroblasts even under simulated inflamed conditions. Furthermore, stringent HLA-B7-restricted recognition was observed for clone 4G11 when tested against a stimulator panel expressing a wide range of common and rare HLA class I and II molecules. Clone 4G11 demonstrated clinical applicability by efficiently recognizing HLA-B7+ primary ALL, CLL and MCL. Furthermore, reproducible strong recognition of purified primary HLA-B7+ MM could be demonstrated. Therefore, the TCR of clone 4G11 may be used for immunotherapy by administering TCR-transduced T cells to patients suffering from B cell malignancies including multiple myeloma. Retroviral gene transfer of TCR 4G11 led to efficient cell surface expression demonstrated by binding of TCR-transduced CD8+ T cells to pMHC-tetramer composed of peptide Bob144 bound to HLA-B7. TCR-modified CD8+ T cells strongly recognized Bob1-expressing HLA-B7+ multiple myeloma cell lines U266 and UM9, and ALL cell lines. TCR-modified T cells efficiently lysed HLA-B7+ primary ALL, CLL and MCL at very low effector-to-target ratios. In addition, highly purified primary multiple myeloma samples were also readily lysed. Furthermore, TCR-transduced T cells strongly proliferated in an antigen-specific manner when stimulated with primary malignant cell samples including ALL, CLL, and MCL or MM cell lines. As expected, TCR-transduced T cells also lysed autologous primary and CD40L-stimulated B cells since these targets cells also express Bob1. In contrast, no lysis of Bob1-negative autologous primary and activated T cells, or monocytes was observed when co-cultured with TCR-transduced T cells. In summary, we identified the intracellular transcription factor Bob1 encoded by gene POU2AF1 as a suitable target for TCR-based immunotherapies of B cell malignancies. Bob1-specific T cell clone 4G11 efficiently recognized primary B cell leukemia and multiple myeloma. Gene transfer of TCR of clone 4G11 installed Bob1-reactivity and specificity onto recipient T cells shown here by cytolytic capacity and proliferation upon antigen encounter. TCR gene transfer approaches using this Bob1-specific TCR can bring novel treatment modalities and possibly curative therapy to patients with B cell malignancies including multiple myeloma. Disclosures No relevant conflicts of interest to declare.

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Enhanced T cell responses to antigenic peptides targeted to B cell surface Ig, Ia, or class I molecules.

Journal of Experimental Medicine ◽

10.1084/jem.168.1.171 ◽

1988 ◽

Vol 168 (1) ◽

pp. 171-180 ◽

Cited By ~ 40

Author(s):

L A Casten ◽

P Kaumaya ◽

S K Pierce

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Cytochrome C ◽

Cell Surface ◽

B Cell ◽

Peptide Fragment ◽

Peptide Antibody ◽

Antibody Conjugates ◽

Antibody Conjugate

The helper T cell recognition of soluble globular protein antigens requires that the proteins be processed by an APC, releasing a peptide that is transported to and held on the APC surface where it is recognized by the specific T cell in conjunction with Ia. When cellular processing functions are blocked, APC lose their ability to present native antigens while retaining the capacity to activate T cells when provided with a cognate peptide fragment that contains the T cell antigenic determinant. In this report, we show that a peptide fragment of the soluble globular protein antigen tobacco hornworm moth cytochrome c, residues 92-103 containing an additional NH2-terminal cysteine residue (THMcCys92-103), is effectively presented by B cells to an I-Ek-restricted, THMc-specific T cell hybrid when covalently coupled to antibodies specific for B cell surface Ig, Ia (Ak), or class I (Kk). Maximal activation of the T cells to the THMcCys92-103-antibody conjugates is achieved with 1/100-1/1,000th of the peptide required using unconjugated THMcCys92-103 or THMcCys92-103 coupled to nonspecific antibody. The T cell response to the peptide antibody conjugates is MHC restricted, but unlike native cytochrome c-antibody conjugates, THMcCys92-103-antibody conjugates do not require processing and can be presented by paraformaldehyde-fixed B cells. The THMcCys92-103-antibody conjugate are nearly as effective when incubated with B cells, and the unbound conjugates washed away before addition of T cells as when continuously present in culture with T cells and B cells, indicating that the active peptide antibody conjugate is associated at the B cell surface. The presentation of THMcCys92-103 coupled to monovalent Fab fragments of rabbit anti-Ig antibodies is less effective than that of the peptide coupled to bivalent antibody when either live or fixed B cells are APC, indicating that the avidity for the APC surface afforded by bivalent binding may be important in the conjugate's antigenicity. The results presented here indicate that a T cell-antigenic peptide, covalently coupled to a larger antibody molecule, can be readily recognized by an Ia-restricted helper T cell in the absence of processing. Moreover, the ability of the peptide to bind to B cell surfaces greatly augments the peptide's antigenicity, even when the binding is to structures distinct from the Ia molecule required for T cell activation.

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Co-Expression of SYK and ZAP70 Subverts Negative B-Cell Selection and Enables Oncogenic Signaling in Multiple B-Cell Malignancies

Blood ◽

10.1182/blood-2019-128999 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 295-295

Author(s):

Teresa Sadras ◽

Mickaël Martin ◽

Lauren Kim-Sing ◽

Jevon Cutler ◽

Gal Lenz ◽

...

Keyword(s):

T Cells ◽

Cell Death ◽

B Cells ◽

B Cell ◽

Cell Lymphoma ◽

Cell Selection ◽

B Cell Malignancies ◽

Close Proximity ◽

Mantle Cell ◽

Bcr Signaling

B-cells are under intense selective pressure to eliminate autoreactive or premalignant clones. B-cell receptor (BCR) signals are required for survival, however, BCR-signaling exceeding maximum thresholds often reflects signaling from an autoreactive BCR or a transforming oncogene and triggers negative selection and cell death. The tyrosine kinase SYK initiates BCR-downstream signaling in B-cells while its close relative ZAP70 is almost exclusively expressed in T-cells. Interestingly, the segregation of SYK to B-cells and ZAP70 to T-cells is less confined in malignant lymphopoiesis suggesting that the balance of these related kinases may alter signaling output in disease and contribute to development of leukemia. As previously shown in B-cell chronic lymphocytic leukemia (B-CLL), we identified aberrant ZAP70 expression as a frequent feature in multiple other B-cell malignancies that depend on survival signals from a functional (pre-) BCR (E2A-PBX1+ pre-B ALL, and mantle cell lymphoma) or harbor oncogenic mimics of the BCR (BCR-ABL1+ B-ALL). Studying SYK and ZAP70 expression by single-cell Western blot, co-expression of the two tyrosine kinases was extremely rare in normal B- and T-cell populations. In contrast, >50% of tumor B-cells in mantle cell lymphoma, pre-B ALL and CLL co-expressed SYK and ZAP70. Despite their structural similarities, genetic deletion and engineered reconstitution of SYK and ZAP70 in human B-cell lymphoma cells revealed striking functional differences. Proximity-dependent biotin identification (BioID) analyses identified that SYK, but not ZAP70, engaged the PI3K pathway via interaction with CD19. Consistent with this, reconstitution with SYK and SYK-ZAP70 but not ZAP70 alone promoted survival and proliferation. Detailed analysis of BCR-mediated cascades in lymphoma cells expressing SYK, ZAP70 or SYK-ZAP70 established that ZAP70 is only weakly efficient at propagating BCR-mediated calcium and downstream pathway activation in B-cells. Strikingly, co-expression of ZAP70 with SYK resulted in re-wired BCR-signaling of intermediate strength: compared to cells expressing only SYK, SYK-ZAP70 co-expressing cells had markedly reduced activation of the BLNK-BTK-PLCγ pathway, further reflected in BCR-induced Ca2+ signaling with delayed onset, lower amplitude but longer duration. In this way, we speculated that SYK and ZAP70 may be present within close proximity at the apex of BCR-initiated interactions, and hence compete for downstream substrates resulting in a re-wiring of classic signaling programs propagated normally by SYK. To explore this, we utilized proximity ligation assays (PLA) to monitor the proximity of SYK and ZAP70 in resting or BCR-stimulated B-cells, and found that SYK and ZAP70 co-exist within close proximity consistent with the view that varying levels of these kinases may alter B-cell signaling output. Functional experiments further showed that phosphomimetic activation of SYK, but not ZAP70, induced hyperactivation of PI3K-signaling and acute BTK-mediated cell death in pre-B ALL cells. In line with altered BCR-signaling strength and quality in SYK and ZAP70 co-expressing cells, over-expression of Zap70 in pre-B ALL cells rescued auto-immune checkpoint activation induced by hyper-activation of BCR-associated signaling. To study functional consequences of SYK-ZAP70 co-expression during normal B-cell development, we generated a novel knock in Zap-70+/Mb1-Cre+mouse model, to induce conditional expression of Zap70 in the B cell compartment from the proB stage. Consistent with compromised central tolerance checkpoints, Syk-Zap70 co-expressing pro/pre-B and immature B-cells had reduced spontaneous apoptosis rates and gave rise to autoantibody production against multiple self-antigens. Importantly, our findings highlight a previously unrecognized role for ZAP70 in oncogenic BCR-signaling and we conclude that the co-expression of ZAP70 mitigates the ability of SYK, downstream of an autoreactive BCR or a transforming oncogene, to trigger negative B-cell selection and cell death (Figure 1). Disclosures Weinstock: Celgene: Research Funding. Meffre:AbbVie: Consultancy, Other: Grant.

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Donor-Derived Anti-CD19 Chimeric-Antigen-Receptor-Expressing T Cells Cause Regression Of Malignancy Persisting After Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood.v122.21.151.151 ◽

2013 ◽

Vol 122 (21) ◽

pp. 151-151 ◽

Cited By ~ 4

Author(s):

James N Kochenderfer ◽

Mark E. Dudley ◽

Robert O. Carpenter ◽

Sadik H Kassim ◽

Jeremy J. Rose ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

B Cell Malignancies ◽

Hematopoietic Stem ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Grade 3

Abstract Progressive malignancy is a leading cause of death in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT). To improve treatment of B-cell malignancies that persist despite alloHSCT, we conducted a clinical trial of allogeneic T cells genetically modified to express a chimeric antigen receptor (CAR) targeting the B-cell antigen CD19. Ten patients were treated on this trial. Four patients were recipients of human-leukocyte-antigen (HLA)-matched unrelated donor (URD) transplants and 6 patients were recipients of HLA-matched sibling transplants. T cells for genetic modification were obtained from each patient’s healthy alloHSCT donor. Patients received a single infusion of anti-CD19-CAR T cells. Cell doses ranged from 1x106 to 10x106 T cells/kg. A mean of 58% of the infused cells expressed the CAR. Patients did not receive chemotherapy or other anti-malignancy therapy with the CAR-T-cell infusions, so the responses observed in these patients are not confounded by the effects of chemotherapy. In contrast to other reports of successful treatment of B-cell malignancies with anti-CD19-CAR T cells, the patients on this study were not lymphocyte-depleted at the time of the CAR-T-cell infusions. Two patients with chronic lymphocytic leukemia (CLL) refractory to standard unmanipulated allogeneic donor lymphocyte infusions (DLIs) had regressions of large malignant lymph node masses after infusion of allogeneic anti-CD19-CAR T cells. One of these CLL patients obtained a complete remission that is ongoing 9 months after treatment with allogeneic anti-CD19-CAR T cells. This patient also had complete eradication of blood B cells within 9 days after her CAR-T-cell infusion. Another patient had tumor lysis syndrome requiring rasburicase treatment as his CLL dramatically regressed in lymph nodes, bone marrow, and blood within 2 weeks of his anti-CD19-CAR-T-cell infusion. A patient with mantle cell lymphoma obtained a partial remission that is ongoing 3 months after infusion of anti-CD19-CAR T cells. A fourth patient with diffuse large B-cell lymphoma has ongoing stable disease 11 months after infusion of anti-CD19-CAR T cells. The other 6 treated patients all had short periods of stable malignancy or progressive disease after their CAR-T-cell infusions. Specific eradication of blood B cells occurred after infusion of CAR T cells in 3 of 4 patients with measurable blood B cells pretreatment. None of the patients treated on this study developed GVHD after their anti-CD19-CAR-T-cell infusions, despite the fact that 6 of 10 treated patients had experienced GVHD at earlier time-points after their most recent alloHSCT. One patient, who had a history of cardiac dysfunction with prior acute illnesses, had temporary cardiac dysfunction after infusion of anti-CD19-CAR T cells. The most prominent toxicities experienced by patients were fever and hypotension; these peaked 5 to 12 days after CAR-T-cell infusions and resolved within 14 days after the T-cell infusions. Two patients had Grade 3 fever, and 2 patients had Grade 3 hypotension. No patients experienced Grade 4 toxicities that were attributable to the CAR-T-cell infusions. Elevated levels of serum interferon gamma were detected in 3 patients at the time that they were experiencing toxicities. We detected cells containing the anti-CD19-CAR gene in the blood of 8 of 10 patients. The peak blood levels of CAR T cells varied from undetec to 2.8% of peripheral blood mononuclear cells. The persistence of the CAR T cells in the blood of patients was limited to one month or less. When we assessed T cells from the blood of patients ex vivo, we found elevated levels of the T-cell inhibitory molecule programmed cell death protein-1 (PD-1) on CAR+ T cells compared to CAR-negative T cells. These results show for the first time that small numbers of donor-derived allogeneic anti-CD19-CAR T cells can cause regression of highly treatment-resistant B-cell malignancies after alloHSCT without causing GVHD. Malignancies that were resistant to standard DLIs regressed after anti-CD19-CAR-T-cell infusions. Future goals for improving this approach include enhancing the persistence of anti-CD19-CAR T cells and reducing toxicities. Infusion of allogeneic T cells genetically modified to recognize malignancy-associated antigens is a promising approach for treating residual malignancy after alloHSCT. Disclosures: No relevant conflicts of interest to declare.

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Pre-Clinical Characterization of T Cell-Dependent Bispecific Antibody Anti-CD79b/CD3 As a Potential Therapy for B Cell Malignancies

Blood ◽

10.1182/blood.v124.21.4507.4507 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4507-4507 ◽

Cited By ~ 2

Author(s):

L. Laura Sun ◽

Xiaocheng Chen ◽

Yvonne Chen ◽

Mark S. Dennis ◽

Diego Ellerman ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Cell Receptor ◽

Antibody Fragments ◽

Bispecific Antibodies ◽

Cell Targeting ◽

B Cell Malignancies

Abstract T-cell recruiting bispecific antibodies and antibody fragments have been used to harness the cytotoxic potential of T cells for cancer treatment. As an example, encouraging clinical responses have been reported with the B cell targeting Blinatumomab, a 55-kDa fusion protein composed of two single-chain antibody fragments (scFvs). However, the therapeutic promise of many reported bispecific antibodies and fragments is often limited by unfavorable pharmacokinetics and administration schedule, immunogenicity, and a propensity towards aggregation. We have adopted a knobs-into-holes (KIH) antibody format and produced T-cell dependent bispecific antibodies (TDB), which allow one arm to target various B cell antigens while the other arm recruits T cells by binding to the CD3e subunit of the T-cell receptor. These B cell targeting TDBs are full length, humanized IgG1 antibodies with natural antibody architecture. Single dose pharmacokinetic/pharmacodynamic studies in cynomolgus monkeys show the KIH format TDBs are well tolerated in life, result in potent B cell depletion in peripheral and lymphoid tissue, and demonstrate pharmacokinetic properties resembling conventional antibody therapy. One B cell antigen targeted is CD79b, a component of the B cell receptor complex. CD79b is restricted to B cells, is highly prevalent on B cell leukemia and lymphomas, and has been clinically validated by an anti-CD79b antibody-drug conjugate as a safe and effective therapeutic target for B cell malignancies (ASCO 2014 abstract#8519). In our present work, we show that anti-CD79b/CD3 TDB can be produced and purified from E.coli, free of homodimer and aggregates. Anti-CD79b/CD3 TDB is a conditional agonist, activating CD3+T cells only in the presence of CD79b expressing B cells. In vitro, it induces potent B cell killing in a T-cell dependent manner, and is broadly active against lymphoma cell lines with a wide range of CD79b antigen levels. Compared to bispecific antibodies targeting some other B cell antigens, anti-CD79b/CD3 TDB appears to be more potent in autologous B cell killing assays with human PBMCs isolated from healthy donors. Taking advantage of antibodies with a range of binding affinities, we show that the B cell cytotoxic potency of anti-CD79b/CD3 TDB can be enhanced with increased binding affinity of either the anti-CD79b arm or the anti-CD3 arm in vitro. To assess the therapeutic potential of anti-CD79b/CD3 TDB, we further demonstrate that it is active in killing B lymphoma cells isolated from leukemia and lymphoma patients. Collectively, these preclinical data suggest anti-CD79b/CD3 TDB may be a promising agent for clinical development in B cell malignancies. Disclosures Sun: Genentech: Employment. Chen:Genentech: Employment. Chen:Genentech: Employment. Dennis:Genentech: Employment. Ellerman:Genentech: Employment. Johnson:Genentech: Employment. Mathieu:Genentech: Employment. Oldendorp:Genentech: Employment. Polson:Genentech: Employment. Reyes:Genentech: Employment. Stefanich:Genentech: Employment. Wang:Genentech: Employment. Wang:Genentech: Employment. Zheng:Genentech: Employment. Ebens:Genentech: Employment.

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Development of Novel Non-Immunoglobulin Centyrin-Based Cars (CARTyrins) Targeting Human Bcma

Blood ◽

10.1182/blood.v128.22.4557.4557 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4557-4557 ◽

Cited By ~ 3

Author(s):

Burton Earle Barnett ◽

Xinxin Wang ◽

David L. Hermanson ◽

Yening Tan ◽

Eric M. Osertag ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Surface ◽

B Cell ◽

Tumor Cells ◽

Surface Expression ◽

Domain Swapping ◽

Target Cells ◽

High Quality ◽

Car T

Abstract Chimeric-antigen receptor (CAR)-T cell immunotherapy is a promising type of cancer therapy and substantial progress has been made in developing adoptive T cell approaches for B cell malignancies. B cell maturation antigen (BCMA) is an attractive target for patients with multiple myeloma (MM) due to its high level of expression on tumor cells and restricted expression on normal tissues. Traditionally, the antigen-binding domain of a CAR is a single chain variable fragment (scFv) comprised of heavy chain (HC) and light chain (LC) variable fragments joined by a flexible linker that has been derived from a non-human monoclonal Ab (mAb). However, there are a number of disadvantages to scFv-based CARs including the limited availability of scFv, their potential to elicit antibody responses, and their association with tonic signaling due, in part, to inherent instability and flexibility of the structure and the potential for both HC/LC domain swapping and multimer formation through framework region interactions. Thus, replacement with alternative binding technologies may improve CAR-T efficacy in the clinic. Centyrins are alternative scaffold molecules that bind protein targets with high affinity and specificity, similar to scFv molecules. However, unlike scFv, Centyrins are smaller, derived from human consensus tenascin FN3 domains and are predicted to have decreased immunogenicity. Additionally, a monomeric Centryin in CAR format (i.e. CARTyrin molecule) is less likely to engage in domain swapping or interact with other Centyrins at the cell surface, thereby limiting the potential for the tonic signaling that drives the functional exhaustion of CAR T cells. Centyrins can be isolated against virtually any antigen through ex vivo panning of an extensive Centyrin library, yielding many distinct binders with a range of affinities and target epitopes. Panning with soluble BCMA protein yielded a large pool of BCMA-specific Centyrins, from which 11 distinct monomeric binders and 1 non-monomeric binder were selected for further study in CAR format. In addition, we tested numerous signal peptides, linkers, transmembrane domains and signaling domains to determine optimal configuration. We then created all CARTyrins by fusing each Centyrin with a CD8a leader peptide, spacer and transmembrane domain, as well as an intracellular signaling domain derived from both 4-1BB and CD3ζ. High quality mRNA of each CARTyrin construct was produced in order to rapidly screen CARTyrin cell surface expression and functionality in human pan T cells against BCMA+ targets. We also constructed scFv-based CARs against CD19 and BCMA for comparison. Previously CD3/CD28-stimulated T cells were electroporated (EP) with mRNA encoding each of the 12 anti-BCMA CARTyrins and, the following day, analyzed for surface expression of CARTyrin and their ability to degranulate against BCMA+ tumor cells. All 12 CARTyrins were detected on the cell surface and the 11 monomeric CARTyrins imparted BCMA-specific killing capacity to T cells. Notably, in these assays, CARTyrins were functionally comparable to scFv-based CARs against BCMA or to CD19-specific scFv-based CARs in a parallel assay with CD19+ tumor cells. The 11 functional anti-BCMA CARTyrins were further characterized for functional avidity by determining their activity against a panel of target cells with titrated levels of surface BCMA expression. To create this panel, various amounts of high quality BCMA mRNA were electroporated into BCMA- K562 tumor cells. After 4 hours of co-culture with the panel of BCMA expressing cells, CARTyrin+ T cell activity was measured as a function of CD107a expression. We observed a range of activities by each CARTyrin and show that this assay can be utilized to determine the minimal effective dose of BCMA needed to induce killing by CARTyrin+ cells. Furthermore, we establish that certain BCMA-specific CARTyrins are responsive to target cells with extremely low levels of surface BCMA expression. These results confirm that Centyrins are viable replacements for scFv in the construction of functional CARs and establish their potential utility in generating novel BCMA-specific CAR molecules, as well as other novel targetable tumor antigens. Disclosures Barnett: Poseida Therapeutics: Employment. Wang:Poseida Therapeutics: Employment. Hermanson:Poseida Therapeutics: Employment. Tan:Poseida Therapeutics: Employment. Osertag:Poseida Therapeutics: Employment, Equity Ownership. Shedlock:Poseida Therapeutics: Employment.

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CpG-Containing Oligonucleotides Are Efficient Adjuvants for Induction of Protective Antiviral Immune Responses with T-Cell Peptide Vaccines

Journal of Virology ◽

10.1128/jvi.73.5.4120-4126.1999 ◽

1999 ◽

Vol 73 (5) ◽

pp. 4120-4126 ◽

Cited By ~ 91

Author(s):

Annette Oxenius ◽

Marianne M. A. Martinic ◽

Hans Hengartner ◽

Paul Klenerman

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

B Cells ◽

Cell Surface ◽

B Cell ◽

Challenge Infection ◽

Cpg Odn ◽

Surface Binding ◽

Cpg Odns

ABSTRACT Synthetic nonmethylated oligonucleotides containing CpG dinucleotides (CpG-ODNs) have been shown to exhibit immunostimulatory activity. CpG-ODNs have the capacity to directly activate B cells, macrophages, and dendritic cells, and we show here that this is reflected by cell surface binding of oligonucleotides to these cell subsets. However, T cells are not directly activated by CpG-ODNs, which correlates with the failure to bind to the T-cell surface. Efficient competition for CpG-induced B-cell activation by non-CpG-containing oligonucleotides suggests that oligonucleotides might bind to an as yet undefined sequence-nonspecific receptor prior to cellular activation. Induction of protective T-cell responses against challenge infection with lymphocytic choriomeningitis virus (LCMV) or with recombinant vaccinia virus expressing the LCMV glycoprotein was achieved by immunizing mice with the immunodominant major histocompatibility complex class I-binding LCMV glycoprotein-derived peptide gp33 together with CpG-ODNs. In these experiments, B cells, potentially serving as CpG-ODN-activated antigen-presenting cells (APCs), were not required for induction of protective immunity since CpG-ODN–gp33-immunized B-cell-deficient mice were equally protected against challenge infection with both viruses. This finding suggested that macrophages and/or dendritic cells were sufficiently activated in vivo by CpG-ODNs to serve as potent APCs for the induction of naive T cells. Furthermore, treatment with CpG-ODN alone induced protection against infection with Listeria monocytogenes via antigen-independent activation of macrophages. These data suggest that CpG activation of macrophages and dendritic cells may provide a critical step in CpG-ODN adjuvant activity.

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Angioimmunoblastic T-Cell Lymphoma: Molecular Characterization of Clonal T and B-Cells and a Patient Derived Xenograft Model of Coexisting T and B-Cell Proliferations

Blood ◽

10.1182/blood-2019-129131 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1572-1572

Author(s):

Xiaoxian Zhao ◽

Bartlomiej Przychodzen ◽

Juraj Bodo ◽

Lisa Durkin ◽

Daniel Lindner ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Tumor Cells ◽

Cell Lymphoma ◽

T And B Cells ◽

Pdx Model ◽

Cell Proliferations

Introduction: Angioimmunoblastic T-cell lymphoma (AITL) is a rare and aggressive type of lymphoma that accounts for about 20% of peripheral T-cell lymphomas with a 5 year overall survival rate of 30%. As most patients relapse after anthracycline-containing regimens and newer agents such as histone deacetylase inhibitors, other novel therapeutic approaches are needed. Signaling lymphocytic activation molecule F7 (SLAMF7), a molecule expressed on a subset of T-cells, activated B cells and myeloma cells, is an attractive target to explore based on our previous studies showing SLAMF7 expression in a subset of AITL cases. The association of AITL with Epstein Barr virus (EBV) positive B-cells is nearly always present and the efficacy of treatment in such patients with significant EBV viral load is not well-understood. In this study, we performed the molecular characterization of an aggressive EBV+ AITL case, established a patient-derived xenograft (PDX) AITL model of coexisting T and B-cell proliferations and evaluated novel therapeutic strategies. Methods: Primary tumor cells were injected into a NSG mouse. Flow cytometry, immunohistochemistry (IHC), CISH-EBER and BIOMED 2 PCR based clonality studies were used to confirm the engraftment and compared the consistency of engrafted tumor cells with the primary sample. Genomic DNA extracted from sorted T and B cells and from paired normal neutrophils of the original patient were subjected to Whole Exome sequencing (WES). In vivo AITL PDX model trials were tested for the efficacy of romidepsin (Rom), elotuzumab (Elo), rituximab (Rit) and in combinations of these drugs. Results: A 53 year old woman with AITL was treated with 6 cycles of CHOEP followed by autologous stem cell transplantation. 3 months after transplantation (9 months after diagnosis) she developed progressive fatigue and arthralgias. PET-CT scan showed new cervical, thoracic, abdominal and pelvic lymphadenopathy. A cervical lymph node biopsy was performed to confirm relapse. IHC staining showed atypical T cells expressing CD2, CD3, CD4, CD5, CD7, CD10, BCL6, PD1, SLAMF7 and CXCL13. Scattered CD20+/EBER+ B-immunoblasts were present with focal large clusters/small sheets. Primary tumor cells engrafted in NSG mouse via tail vein injection caused splenomegaly. Flow cytometry assay demonstrated the engraftment of tumor cells in peripheral blood, bone marrow and spleen tissue. CD3+CD19- cells dominated the engrafted cells in all three tissues. Histologic examination and immunophenotyping (IHC and EBER staining) of spleen were consistent with primary tumor tissue. Engrafted tumor cells were capable of serial passage in NSG mice with an increasing malignant B cell percentage that mimics the situation in which the B-cell component masks an underlying T-cell lymphoma in humans. T-cell receptor gene rearrangement assay confirmed the clonal identity of engrafted T-cells matched the primary relapsed tumor. A clonal IGH rearrangement of engrafted B-cells was also detected, while no monoclonal B-cell population was detected in the relapsed AITL sample, possibly due to the low number of EBV+ B-cells present in that biopsy. WES of sorted malignant T-cells showed 33 mutants in 31genes, including RhoA G17V, TET2,STAT3 and VAV1, previously described in AITL or other T-cell lymphomas. In parallel WES assay, 9 mutations were found in 9 genes from sorted EBV+ B immunoblasts. A PDX model using cells harvested from the second passage showed single agent, Elo or Rit, extended the survival of mice compared to the control group (p < 0.05). Rom alone had no such effect (p = 0.27). Combination of Rit with either Elo or Rom further improved survival compared to each single agent exposed cohort (p < 0.05). There was no significant difference between Rit/Elo and Rit/Rom (p = 0.067). PARP cleavage by IHC was higher in the Rit/Rom and Rit/Elo groups compared to other cohorts. Expression of SLAMF7 in a subset of engrafted T and B cells of the control mouse were confirmed via flow cytometry assay. Conclusions: To date, this is the first molecular characterization of AITL tumor cells in comparison with associated EBV+ B cells and use of such a PDX model for therapeutic evaluation of agents targeting both malignant T and B cells simultaneously. The in vivo data support further clinical investigation of applying elotuzumab or romidepsin in combination with rituximab in AITL containing EBV-positive B-cell proliferations. Disclosures Maciejewski: Novartis: Consultancy; Alexion: Consultancy. Hsi:Abbvie: Research Funding; Eli Lilly: Research Funding; Jazz: Consultancy; Cleveland Clinic&Abbvie Biotherapeutics Inc: Patents & Royalties: US8,603,477 B2.

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A Phase I Clinical Trial of Treatment of B-Cell Malignancies with Autologous Anti-CD19-CAR-Transduced T Cells

Blood ◽

10.1182/blood.v116.21.2865.2865 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2865-2865 ◽

Cited By ~ 1

Author(s):

James N. Kochenderfer ◽

Mark E. Dudley ◽

Maryalice Stetler-Stevenson ◽

Wyndham H. Wilson ◽

John E. Janik ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Adoptive Transfer ◽

B Cell Malignancies ◽

B Lineage

Abstract Abstract 2865 T cells can be genetically modified to express chimeric antigen receptors (CARs) that specifically recognize the B-cell antigen CD19. Adoptive transfer of autologous T cells expressing anti-CD19 CARs is an attractive new approach for treating B-cell malignancies. We have constructed a CAR that consists of the variable regions of a mouse-anti-human-CD19 antibody coupled to the signaling domains of CD28 and CD3-zeta. We have treated 5 patients with 2 doses of 60 mg/kg of cyclophosphamide and 5 doses of 25 mg/m2 of fludarabine followed by infusions of anti-CD19-CAR-transduced T cells and administration of high-dose IL-2. All of the patients received infusions of cells that produced cytokines in a CD19-specific manner. The percentage of the infused cells that expressed the anti-CD19 CAR as measured by flow cytometry ranged from 45% to 65%. The first patient enrolled on our trial has follicular lymphoma. He was treated twice. The patient obtained a partial remission (PR) from his first course of chemotherapy, 0.4×109 anti-CD19-CAR-transduced T cells, and IL-2 (reported in Kochenderfer et al. Blood First Edition); however, he subsequently developed progressive disease, and 40 weeks after his first CAR-transduced T cell infusion he received a second course of chemotherapy followed by 2×109 CAR-transduced T cells and IL-2. The second course of treatment resulted in an additional PR and was not associated with any toxicity that could be attributed to the CAR-transduced T cells. At last follow-up, a small amount residual disease detected only by positron emission tomography remained. In this first patient, the initial treatment course resulted in eradication of blood and bone marrow B-lineage cells for 39 weeks. In contrast to the prolonged eradication of B-lineage cells after the initial treatment course, the number of polyclonal blood B cells normalized 9 weeks after the second CAR-transduced T cell infusion. CAR-transduced T cells were present at a level of 0.1% of total peripheral blood mononuclear cells (PBMCs) one month after the first CAR-transduced T cell infusion. Despite the five-fold higher dose of CAR-transduced T cells administered with the second treatment, CAR-transduced T cells were not detected in the blood one month after the second CAR-transduced T cell infusion. The second patient treated on our protocol had follicular lymphoma and had received extensive prior therapy including autologous stem cell transplantation. After an initially uncomplicated course, this patient developed pneumonia caused by culture-proven influenza A virus and died 18 days after CAR-transduced T cell infusion. Quantitative PCR was used to measure the level of CAR-transduced cells in multiple tissues obtained from this patient at autopsy. CAR-transduced cells were widely distributed with the highest levels in the spleen and bone marrow. The third patient treated on our trial obtained a complete remission of advanced chronic lymphocytic leukemia (CLL) after treatment with chemotherapy, infusion of 2×109 anti-CD19-CAR-transduced T cells, and IL-2. At the time of last follow-up, three months after treatment, adenopathy had resolved, CLL cells were not detected by flow cytometry analysis of the blood and bone marrow, and the number of normal polyclonal B cells in the blood was below normal levels. This patient had a period of fever and hypotension 7 days after cell infusion that was associated with an elevated serum interferon-gamma level of 1532 pg/mL. At the time of the hypotensive episode 7 days after cell infusion, anti-CD19-CAR-transduced cells made up 2.1% of PBMCs. The fourth patient treated on our study obtained a PR of splenic marginal zone lymphoma that continues 2 months after treatment with chemotherapy, 2×109 CAR-transduced T cells, and IL-2. This patient did not have prolonged depletion of normal B cells after treatment, and he did not have any toxicity that could be attributed to the anti-CD19 CAR-transduced T cells. We recently treated a fifth patient who has CLL. Follow-up on this patient is too short to evaluate toxicity or response. In conclusion, we have shown that adoptive transfer of anti-CD19-CAR-transduced T cells with in vivo activity is feasible. The promising results obtained on this trial raise important questions for future research aimed at optimizing therapy with anti-CD19-CAR-transduced T cells. Disclosures: No relevant conflicts of interest to declare.

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Examining the Heterogeneity of Follicular Lymphoma By Multi-Parameter Flow Cyotmetry in Previously Untreated Patients

Blood ◽

10.1182/blood.v128.22.2947.2947 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2947-2947

Author(s):

Debra K Czerwinski ◽

Steven R Long ◽

Michael Khodadoust ◽

Matthew J. Frank ◽

Adel Kardosh ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Follicular Lymphoma ◽

B Cell ◽

Tumor Cells ◽

Immune Cells ◽

Research Funding ◽

T Cell Exhaustion ◽

Cell Exhaustion

Abstract BACKGROUND: Follicular lymphoma (FL) is an indolent form of Non-Hodgkin B cell lymphoma that remains incurable with present therapies. Derived from germinal center B cells, FL B cells experience ongoing hypermutation of the immunoglobulin variable region gene. In addition, Michael Green, et al (PNAS; 2015), reported the presence of numerous somatic mutations to include those of the chromatin-modifying genes. These mutations accumulate over the course of the disease and play an important role in regulating gene transcription, B cell development and immune interactions. Furthermore, FL tumors maintain a resemblance to primary lymphoid follicles, and as such, present with a number of infiltrating immune cells, especially T cells, the numbers of which vary from patient to patient. The close association and interaction of these immune cells with the tumor B cells play an important part in determining the disease biology (Dave SS, et al. N Engl J Med; 2004). For instance, tumor B cells, through cell-cell contact with these immune cells and/or through secretion of inhibitory cytokines such as TGF-b and IL-10, induce T cell exhaustion and apoptosis as well as suppressive T cell phenotypes (FoxP3+ T Regulatory cells) thus evading immune eradication (Yang Z-Z, et al. Blood 2007 and Ai WZ, et al. IntJ Cancer; 2009). They also promote their own survival and proliferation through their interaction with resident T follicular helper cells via CD40L/CD40 interactions (Ame'-Thomas P, et al. Blood; 2005). As a corollary to an ongoing clinical trial, we received fine needle aspirates (FNAs) of easily accessible tumors from 14 patients with FL prior to any treatment. 6 of these patients had samples taken from a second site simultaneously. All samples were processed within 24 hours into a single-cell suspension; red blood cells were lysed. Cells were then stained with antibodies to delineate T, B, NK, dendritic, and myeloid cells, as well as their subsets. Antibodies against activation antigens, T cell exhaustion, inhibition and function were also used to characterize these cells. Finally, the cells were run on a 17-parameter LSRII (Becton Dickinson) and data analyzed via Cytobank, a web-based data storage and analysis tool. PURPOSE: To better understand the biology of FL as represented by protein expression by the tumor cells and the immune cells that make up the microenvironment. We will especially look to evaluate the heterogeneity inherent in FL by flow cytometry across patients as well as within any one individual. RESULTS: Each sample is stained with 4 panels of antibodies, 13 antibodies each, allowing us to measure over 100 cell subsets. A quick preview of all data shows that there is a high variability between patients in the percentage of T cells within the microenvironment (37.7% + 16.6% of all cells collected from all samples). This variability is represented by the differences in the CD4 T cell compartment (27.6 + 12.9%) and to a lesser degree in the CD8 compartment (7.7 + 3.7%). To note, this variability in T cells does not correlate with time from diagnosis to sample collection which ranged from 3.4 years to approximately 5 months. Also, this is in contrast to the similar percentage of CD4 and CD8 T cells expressing PD-1 (55.5 + 8.8% and 46.0 + 8.9%, respectively) across patients. Notably, there is much less variability from site to site within each patient then between patients as demonstrated by Figure 1 where Site A and Site B are 2 separate lesions within each patient listed, sampled at the same time. Since FL presumably begins in a single site in the body and then becomes disseminated, the fact that a characteristic relationship exists between tumor cells and immune cells wherever the disease is found implies a mutual interdependence of the tumor cells in each case and their immune host component. CONCLUSION: Follicular lymphoma is a very heterogeneous disease as would be expected by the diversity of mutations seen at the genomic level. This heterogeneity is also apparent in the microenvironment from one patient to another. Conversely, different tumor sites within each patient have a characteristic and fixed relationship to their immune microenvironment. The emergence of novel therapies for FL, including checkpoint antibodies such as anti-PD-1 and anti-PD-L1 and small molecules such as Ibrutinib, will be informed by understanding the differences as well as the similarities in each case of FL. Disclosures Levy: Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding.

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