scholarly journals Engineered FVIII-expressing cytotoxic T cells target and kill FVIII-specific B cells in vitro and in vivo

2018 ◽  
Vol 2 (18) ◽  
pp. 2332-2340 ◽  
Author(s):  
Kalpana Parvathaneni ◽  
David W. Scott
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cd8 T Cells ◽  
Hemophilia A ◽  
Cytotoxic T Cells ◽  
Cell Antibody ◽  
Engineered T Cells

Abstract Hemophilia A is an X-linked bleeding disorder caused by mutations in the factor VIII (FVIII) gene (F8). Treatment with recombinant or plasma-derived FVIII replacement therapy is standard therapy. A major problem in treating hemophilia A patients with therapeutic FVIII is that 20% to 30% of these patients produce neutralizing anti-FVIII antibodies (inhibitors) because they are not immunologically tolerant to this human protein. Hence, there is a need to establish tolerogenic protocols to FVIII epitopes. To specifically target FVIII-specific B cells, we engineered immunodominant FVIII domains (A2 and C2) as a chimeric antigen receptor expressed by both human and murine cytotoxic T cells. This FVIII domain engineered B-cell antibody receptor (BAR) that expresses T cells was capable of killing FVIII-reactive B-cell hybridomas in vitro and in vivo. Moreover, FVIII BAR CD8 T cells blocked the development of specific antibody from unimmunized spleen cells stimulated polyclonally with lipopolysaccharide in vitro. In addition, adoptive transfer of FVIII A2- and C2-BAR CD8 T cells significantly reduced the anti-FVIII antibody formation in hemophilic mice. These data suggest that BAR-engineered T cells are a promising approach for future prophylactic treatment for patients with severe hemophilia A who are at high risk of developing inhibitors.

scholarly journals Factor VIII BAR T-Cell Recognition By Antibodies to Different Domains

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1171-1171
Author(s):  
Kalpana Parvathaneni ◽  
Katherine Pohida ◽  
David W. Scott
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Chimeric Receptors ◽  
Flow Cytometry Data ◽  
Cell Antibody ◽  
Engineered T Cells

Abstract Up to 30% of patients with hemophilia A develop inhibitory antibodies to therapeutic FVIII and these antibodies block the efficacy of this critical protein. To target the B cells specific for FVIII, we previously generated cytotoxic cells expressing the FVIII C2 or A2 immunodominant domains as chimeric receptors. We termed these antigen-expressing engineered T cells, "BARs", for B-cell Antibody Receptor. These could directly interact and kill with A2 or C2 FVIII-specific B cells as well as FVIII-specific hybridomas in vitro and in vivo. To increase the number of B-cell targets that could be killed, we recently engineered human and murine BAR CD8 T cells to express the FVIII light chain (LC), which includes A3 and C1, as well as C2 domains. In addition, since C2-expressing BAR T cells showed tonic signaling, we hypothesized that LC expressing BARs might not. Our data suggest that LC-expressing BAR T cells do not display tonic signaling but they can be stimulated to proliferate by anti-LC monoclonal antibody (mAb 5G12), for example. Flow cytometry data showed that LC-BAR T cells are stained by anti-C2 mAb (3G6) but this mAb did not stimulate them to proliferate. These data reflect the accessibility of different FVIII domains in BAR T cells, and are important in terms of the utility of FVIII domain expressing BARs to target specific B cells in vivo in the presence of inhibitors. (Supported by NIH grant R01 HL126727) Disclosures No relevant conflicts of interest to declare.

Low Incidence of Post-Transplant EBV-Related Disease After Alemtuzumab-Mediated T Cell Depletion Is Explained by the Differential Susceptibility to Alemtuzumab of B Cells and Protective CD8 and CD4 T Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2330-2330
Author(s):  
Constantijn J.M. Halkes ◽  
Inge Jedema ◽  
Judith Olde Wolbers ◽  
Esther M van Egmond ◽  
Peter A. Von Dem Borne ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Cell Depletion ◽  
T Cell Depletion

Abstract Abstract 2330 In vivo T cell depletion with anti-thymocyte globulin (ATG) or alemtuzumab (anti-CD52) before reduced intensity allogeneic stem cell transplantation (alloSCT) in combination with in vitro T cell depletion with alemtuzumab reduces the risk of GVHD. Detectable levels of circulating antibodies are present up to several months after the alloSCT, leading to a delayed immune reconstitution which is associated with an increased incidence of opportunistic infections and early relapses. Prior to 2007, combined in vitro (Alemtuzumab 20 mg added “to the bag”) and in vivo T cell depletion with horse-derived ATG (h-ATG) resulted in good engraftment without GVHD in the absence of GVHD prophylaxis after reduced intensity alloSCT using conditioning with fludarabine and busulphan. Due to the unavailability of h-ATG, rabbit-derived ATG (r-ATG) 10–14 mg/kg was introduced in the conditioning regimen in 2007. Strikingly, in this cohort of patients, early EBV reactivation and EBV-associated post-transplantation lymphoproliferative disease (PTLD) was observed in 10 out of 18 patients at a median time of 6 weeks after alloSCT (range 5 to 11 weeks) in the absence of GVHD or immunosuppressive treatment. Analysis of T and B cell recovery early after transplantation revealed preferential depletion of T cells as compared to B cells, thereby allowing unrestricted proliferation of EBV infected B cells. Due to this unacceptable high incidence of EBV-related complications, in the conditioning regimen r-ATG was replaced by low dose alemtuzumab (15 mg i.v. day -4 and -3) in 2008. In this cohort of 60 patients, only 2 patients experienced transient EBV reactivation during the first 3 months after alloSCT and one patient developed an EBV-associated lymphoma 4 weeks after alloSCT. To investigate the mechanisms underlying the low incidence of EBV reactivation using alemtuzumab for T cell depletion, we studied the in vivo and in vitro effects of alemtuzumab on different lymphocyte subsets. First, lineage-specific reconstitution was studied in 20 patients from the alemtuzumab cohort with known CD52 negative diseases (11 AML and 9 multiple myeloma) to exclude the confounding effect of antibody absorption by malignant cells. Whereas at 3 weeks after alloSCT detectable numbers of circulating NK cells and T cells were observed (medians 71 (range 6–378), and 12 (range 1–1164)E6/L, respectively), no circulating B cells could be detected (median 0, range 0–1 E6/L). At 6 weeks after alloSCT, NK and T cell numbers further increased (medians 212 (52-813), and 130 (range 25–1509)E6/L, respectively), whereas B cell numbers still remained low in the majority of patients (median 15, range 0–813E6/L). In all patients, T cells were detectable before the appearance of circulating B cells. Furthermore, the expression of CD52 and the sensitivity to alemtuzumab-mediated complement-dependent cell lysis (CDC) of B cells, T cells and NK cells was measured in vitro. The highest CD52 expression was observed on B cells (mean fluorescence intensity (MFI) 120), resulting in 95% lysis after incubation with 10ug/mL alemtuzumab and rabbit complement. NK cells showed a significantly lower CD52 expression (MFI 41), which was also reflected by a lower susceptibility to alemtuzumab-mediated CDC (62% lysis). Interestingly, differential expression of CD52 was observed on CD4 and CD8 T cells (MFI 120 and 101, respectively). Cytotoxicity analysis revealed relative protection of CD8 compared to CD4 T cells against alemtuzumab-mediated CDC, resulting in 52% and 90% lysis, respectively. Based on these results, we investigated in detail the presence and phenotype of the CD4 and CD8 subsets and EBV-specific CD8 T cells using tetramer staining at 6 weeks after alloSCT. In accordance with the in-vitro expression and susceptibility data, circulating CD52+ CD8 T cells including EBV-specific T cells were detectable. Interestingly, the majority of circulating CD4 T cells (64-93%, n=4) lacked CD52 expression, explaining their capacity to persist in the presence of alemtuzumab. We conclude that in vivo and in vitro T cell depletion with alemtuzumab is associated with a relatively low risk of EBV-associated PTLD because of efficient B cell depletion and persistent EBV immunity allowed by the relative insusceptibility for alemtuzumab of CD8 T cells and the development of CD52 negative escape variants of CD4 T cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Suppression of FVIII Antibody Production By FVIII BAR T-Cell in Vitro in the Presence of Inhibitors

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3789-3789
Author(s):  
Kalpana Parvathaneni ◽  
Ai-Hong Zhang ◽  
David W. Scott
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antibody Response ◽  
B Cell ◽  
Cd8 T Cells ◽  
Conflicts Of Interest ◽  
Spleen Cells ◽  
Engineered T Cells

Abstract To modulate B-cell responsiveness to FVIII, we previously generated cytotoxic cells expressing FVIII C2 or A2 immunodominant domains as chimeric receptors. We termed these antigen-expressing engineered T cells, "BARs", for B-cell Antibody Receptor. These CD8 T cells directly interact and kill FVIII-specific B cells and anti-FVIII hybridomas in prophylactic experiments in vitro and in vivo. It was not known whether these BAR CD8s could function or would be blocked in the presence of circulating antibodies to the expressed BAR domains. To test this, we cultured FVIII C2 or A2 BAR CD8 T cell with a mixture of monoclonal antibodies specific for these domains (up to 10 BU), and then added them to spleen cells from FVIII-immunized mice. These spleen cells were then re-stimulated with FVIII and the antibody response was determined after 5 days. Our results showed that these BAR CD8 T cells were not blocked in their ability to suppress the antibody response to FVIII under these conditions. Coupled with the observation that BAR-T cells can be stimulated to proliferate by anti-FVIII monoclonals, these results suggest that BAR cytotoxic activity may still be effective in the presences of inhibitors. (Supported by NIH grant R01 HL126727) Disclosures No relevant conflicts of interest to declare.

scholarly journals Hematopoietic lineage-converted T cells carrying tumor-associated antigen-recognizing TCRs effectively kill tumor cells

2020 ◽  
Vol 8 (2) ◽  
pp. e000498
Author(s):  
Fangxiao Hu ◽  
Dehao Huang ◽  
Yuxuan Luo ◽  
Peiqing Zhou ◽  
Cui Lv ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Tumor Cells ◽  
Cell Receptor ◽  
Histocompatibility Complex ◽  
Tumor Associated Antigen ◽  
Engineered T Cells ◽  
Antitumor Immunotherapy

Tumor-associated antigen (TAA) T-cell receptor (TCR) gene-engineered T cells exhibit great potential in antitumor immunotherapy. Considering the high costs and low availability of patient-derived peripheral blood T cells, substantial efforts have been made to explore alternatives to natural T cells. We previously reported that enforced expression of Hoxb5 converted B cells into induced T (iT) cells in vivo. Here, we successfully regenerated naive OT1 (major histocompatibility complex I restricted ovalbumin antigen) iT cells (OT1-iT) in vivo by expressing Hoxb5 in pro-pre-B cells in the OT1 transgenic mouse. The OT1-iT cells can be activated and expanded in vitro in the presence of tumor cells. Particularly, these regenerated OT1-iT cells effectively eradicated tumor cells expressing the TAA (ovalbumin) both in vitro and in vivo. This study provides insights into the translational applications of blood lineage-transdifferentiated T cells in immunotherapy.

Hepatic Stellate Cell Conditioned Myeloid Cells Provide a Novel Therapy for Prevention of Factor VIII Antibody Formation in the Hemophilia A Mouse Model

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4117-4117
Author(s):  
Sumantha Bhatt ◽  
Kathleen Brown ◽  
Feng Lin ◽  
Michael P Meyer ◽  
Margaret V. Ragni ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Hemophilia A ◽  
Myeloid Cells ◽  
Gm Csf ◽  
Cox 2

Abstract Abstract 4117 Background: Hemophilia is an X-linked bleeding disorder resulting from a mutation in coagulation factor VIII (F.VIII). A major drawback of current plasma-derived or recombinant F.VIII therapy is the formation of F.VIII antibodies (inhibitors). Inhibitor formation is a T cell-dependent, B cell-mediated immune response to foreign infused F.VIII. Myeloid derived suppressor cells (MDSCs) are potent suppressors of T cell and B cell responses and are currently under study for therapeutic applications in transplantation and autoimmune diseases. However, the mechanisms of MDSC development and function remain unknown, and in vitro propagation of MDSCs has been a challenge. We hypothesized that MDSCs might be effective in inhibiting F.VIII inhibitor formation in the hemophilia A model. Methods: We developed a novel method for generating MDSCs in vitro by culturing bone marrow cells from hemophilia A mice with hepatic stellate cells (HSCs), hereafter referred to as HSC-conditioned myeloid cells (H-MCs). DCs were propagated from the bone marrow with GM-CSF and IL-4, whereas H-MCs were propagated from the bone marrow with GM-CSF and HSCs. Granulocyte contaminants were removed on day 2 and the remaining monocytic populations were harvested on day 5. Expression of cell surface antigens was analyzed by flow cytometry. Arginase1 and iNOS levels were compared by qPCR, with or without LPS stimulation. The in vitro suppressive capacity of the H-MCs was determined by a mixed leukocyte reaction culture. Splenic T cells from hemophilia A mice were stimulated by irradiated DCs (at a 1–20 ratio, APC to T cell) and recombinant F.VIII. Additional irradiated DCs or H-MCs were added in graded numbers as regulators. The proliferative response was determined by 3H-thymidine incorporation. The phenotype of cultured CD4+ T cells was characterized by intracellular staining for Foxp3 and IFN-gamma and analyzed by flow cytometry. Inhibition of B cells by H-MCs was determined by a CFSE dilution assay. Purified splenic B cells were labeled with CFSE and stimulated by Ig-M and IL-4. APCs (spleen cells) or H-MCs were added at a ratio of 1:10 (APC to B cell). The proportion of proliferating B cells was determined by CFSE dilution of B220 stained cells. In the COX-2 suppression assay, CFSE labeled B cells were treated with varying concentrations of the selective inhibitor of COX-2, NS398. The suppressive effect of H-MCs on B cells in vivo was determined by simultaneously administering H-MCs (I.V) and F.VIII (I.V.) to hemophila A mice on day 0 and rechallenging with recombinant F.VIII on days 2 and 4. WT B6 mice and hemophilia A mice without H-MC transfer served as controls. Plasma anti-F.VIII antibody titers were measured on day 12 by a modified ELISA assay. Results: H-MCs expressed low levels of costimulatory molecules but high levels of the inhibitory molecule B7-H1 and immunoregulatory enzyme arginase-1. In contrast, DCs expressed high levels of costimulatory molecules and MHC class II. In vitro studies demonstrated that the H-MCs markedly inhibited antigen specific T cell proliferation induced by dendritic cells in response to recombinant F.VIII (Fig. 1). H-MCs altered the T cell response in hemophilia A mice by promoting the expansion of regulatory T cells and inhibiting IFN-γ producing CD4+ T cells. When the H-MCs were cocultured with B cells isolated from hemophilia A mice, in the presence of Ig-M and IL-4, the H-MCs abrogated B cell activation and proliferation directly (Fig. 2). H-MCs may be modulating the B cell response through the Cox-2 pathway, as inhibition of Cox-2 through NS398 led to the restoration of B cell proliferation. More importantly, adoptive transfer of H-MCs into hemophilia Amice, at the time of F.VIII infusion, markedly suppressed anti-F.VIII antibody formation (Fig. 3). Conclusion: These results suggest that HSC conditioned myeloid cells may represent a potential therapeutic approach to induction of immune tolerance in patients with hemophilia A andother immune disorders. Disclosures: No relevant conflicts of interest to declare.

Preventing restimulation of memory B cells in hemophilia A: a potential new strategy for the treatment of antibody-dependent immune disorders

Blood ◽  
2004 ◽  
Vol 104 (1) ◽  
pp. 115-122 ◽  
Author(s):  
Christina Hausl ◽  
Rafi U. Ahmad ◽  
Hans Peter Schwarz ◽  
Eva M. Muchitsch ◽  
Peter L. Turecek ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Hemophilia A ◽  
Memory B Cells ◽  
Third Party ◽  
Activated T Cells ◽  
Specific Memory ◽  
Inducible Costimulator

Abstract Memory B cells are responsible for the rapidly emerging antibody response after antigen reexposure. The signals required for the restimulation of memory B cells have not been fully explained. We used a murine model of anti–factor VIII (FVIII) antibody responses in hemophilia A to study the requirements for the restimulation of FVIII-specific memory B cells and their differentiation into anti-FVIII antibody-producing cells. We were particularly interested in the significance of activated T cells and costimulatory interactions. Our results indicate that the restimulation of FVIII-specific memory B cells is strictly dependent on interactions with activated T cells. These activated T cells can be specific for either FVIII or third-party antigens. Restimulation by T cells specific for third-party antigens requires the presence of FVIII, indicating that signals induced by B-cell receptor (BCR) triggering and by interactions with activated T cells are important. The blockade of B7-1 or B7-2 as well as the blockade of CD40L inhibits the restimulation and differentiation of FVIII-specific memory B cells in vitro and in vivo. The interference with inducible costimulator–inducible costimulator ligand (ICOS-ICOSL) interactions, however, does not cause any modulation. As expected, the production of anti-FVIII antibodies by plasma cells is not dependent on any of the costimulatory interactions tested.

Leukemia-associated monoclonal and oligoclonal TCR-BV use in patients with B-cell chronic lymphocytic leukemia

Blood ◽  
2003 ◽  
Vol 101 (3) ◽  
pp. 1063-1070 ◽  
Author(s):  
Mohammad-Reza Rezvany ◽  
Mahmood Jeddi-Tehrani ◽  
Hans Wigzell ◽  
Anders Österborg ◽  
Håkan Mellstedt
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cd8 T Cells ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Cell Chronic Lymphocytic Leukemia

Abstract T-cell receptor–B-variable (TCR-BV) gene usage and the CDR3 size distribution pattern were analyzed by reverse transcription–polymerase chain reaction (RT-PCR) in patients with B-cell chronic lymphocytic leukemia (B-CLL) to assess the T-cell repertoire. The use of TCR-BV families in CD4 and CD8 T cells stimulated with autologous activated leukemic cells was compared with that of freshly obtained blood T cells. Overexpression of individual TCR-BV families was found in freshly isolated CD4 and CD8 T cells. Polyclonal, oligoclonal, and monoclonal TCR-CDR3 patterns were seen within such overexpressed native CD4 and CD8 TCR-BV families. In nonoverexpressed TCR-BV families, monoclonal and oligoclonal populations were noted only within the CD8 subset. After in vitro stimulation of T cells with autologous leukemic B cells, analyses of the CDR3 length patterns showed that in expanded TCR-BV populations, polyclonal patterns frequently shifted toward a monoclonal/oligoclonal profile, whereas largely monoclonal patterns in native overexpressed TCR-BV subsets remained monoclonal. Seventy-five percent of CD8 expansions found in freshly obtained CD8 T cells further expanded on in vitro stimulation with autologous leukemic B cells. This suggests a memory status of such cells. In contrast, the unusually high frequency of CD4 T-cell expansions found in freshly isolated peripheral blood cells did not correlate positively to in vitro stimulation as only 1 of 9 expansions continued to expand. Our data suggest that leukemia cell–specific memory CD4 and CD8 T cells are present in vivo of patients with CLL and that several leukemia cell–associated antigens/epitopes are recognized by the patients' immune system, indicating that whole leukemia cells might be of preference for vaccine development.

scholarly journals Automated Generation and Phenotypic Characterization of Clinical Grade CD19 CAR-T Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1675-1675
Author(s):  
Ashish Sharma ◽  
Anne Roe ◽  
Filipa Blasco Lopes ◽  
Ruifu Liu ◽  
Jane Reese ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Cd8 T Cells ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Central Memory ◽  
Car T Cells ◽  
Car T

Abstract BACKGROUND: Chimeric antigen receptor (CAR) T cells have shown enormous promise in the treatment of certain B cell malignancies. Access to treatment is still limited due to a variety of issues, including pricing and centralized manufacturing models. Generation of CAR-T cells using an automated platform, followed by rigorous functional phenotyping, may contribute to the development of a robust long-lasting therapy. METHODS: Here, we used the Miltenyi Prodigy (Miltenyi Biotech, Bergisch Gladbach, Germany) to automate the process of manufacturing genetically manipulated T cells in a closed system. The system obviates the need for clean room infrastructure. We tested the feasibility of utilizing the Miltenyi Prodigy to manufacture CAR-T cells using a CD19 scFV vector with the 4-1BB co-stimulatory domain. (Lentigen Technology, Inc, Gaithersburg, MD). The purity, differentiation capacity and effector function of the enriched CAR-T cells was studied using high-dimensional flow cytometry. Finally, the functional potential of these cells was tested in vitro and by treating NOD-SCID-gamma (NSG) mice infused with B cell lymphoma cells (Raji B cell), with the CAR-T cells. RESULTS: Starting with 1 x 108 CD4 and CD8 cells from donor apheresis products, CAR-T cells were expanded for 12 days in culture media containing with TransAct (Miltenyi Biotech), IL7 and IL15. The mean fold-expansion at day 12 was 44 ± 5.6, range 39-50 (n=3). The mean transduction efficiency of CAR-T vector was 20%, range 10-25% (n=3), which is similar to other reported methods. The CD19 CAR-T product was enriched in both the CD4 and CD8 T cells subsets, and showed high-level of cytotoxicity against CD19+ cell lines in vitro and in vivo (Figure 1: Mice treated with the CD19-CAR T demonstrated a marked reduction in disease burden as compared to T cell control as measured by bioluminescence imaging and flow cytometric analysis). The CAR-T product was enriched in cell subsets with both effector (CD27-CCR7-; ~20% of total cells) and central memory phenotypes (CD27+CCR7+; ~30% of total T cells). The effector CD4 and CD8 T cells showed increased expression of major functional T cell differentiation transcription factors (i.e. T-bet and GATA3) critical for the development of anti-tumor responses. Whereas, the central CD4 and CD8 T cells were enriched for the expression of TCF7 (a stemness related member of the WNT signaling known to increase longevity of these cells). The frequencies and phenotypes of these cells were maintained in peripheral blood of NSG mice infused with B cell lymphoma cells (Raji B cells), 1 week after treatment. A significant expansion of CD8+ effector T cells and a dramatic reduction in tumor burden was observed over the next 4 weeks in all major organs. Interestingly, we observed that smaller proportion of central-memory like cells (with higher TCF7 levels) continued to persist 6 weeks post-treatment, potentially contributing to a long-lived recallable response. Based on these data we have initiated a phase 1 clinical trial to test the therapeutic potential of the CAR-T product in patients with advanced/refractory B cell lymphoma. The first clinical grade manufacturing run resulted in a CD19 + cell yield of 1.4 x109. CONCLUSION: Our data highlight that the automated CAR-T generation platform (i.e. Miltenyi Prodigy) is effective at the generating purified functionally competent CAR-T cells. Further, findings from our phenotyping analyses show that the CAR-T product is enriched in both effector and central memory subsets and is effective at tumor clearance in vivo. Thus far, we have treated one patient with CD19 CAR-T manufactured on this platform and 2 more have been enrolled. Though this initial study is based on CD19 CAR-T cells, the approach described here could easily be utilized to genetically engineer T cells with gene constructs that are more relevant for specific cancers and infectious diseases. Disclosures No relevant conflicts of interest to declare.

A CD19/CD3 Bispecific Tandab, AFM11, Recruits T Cells To Potently and Safely Kill CD19+ Tumor Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4405-4405
Author(s):  
Eugene Zhukovsky ◽  
Uwe Reusch ◽  
Carmen Burkhardt ◽  
Stefan Knackmuss ◽  
Ivica Fucek ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cancer Cells ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
Cross Reactivity ◽  
Equity Ownership

Abstract To harness the potent tumor-killing capacity of T cells for the treatment of CD19+ malignancies, we developed a humanized bispecific tetravalent antibody, with two binding sites for CD3 and CD19, the CD19/CD3 RECRUIT-TandAb AFM11. CD19 is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for the development of therapeutic antibodies to treat B cell malignancies such as Non Hodgkin Lymphoma. Since native antibodies cannot recruit T cells, we engineered a bispecific anti-CD19/anti-CD3 TandAb. The tumor-specific CD19 antigen module targets the TandAb to cancer cells, while simultaneously, the CD3 effector module recruits and activates T cells, leading to cancer cell lysis. The advantages of the TandAb technology, relative to other bi-functional fragment antibody scaffolds, include: improved pharmacokinetics (PK) enabling intravenous dosing, more drug-like properties, and avidity-enhanced efficacy for the targeting and killing of tumor cells. We evaluated in vitro efficacy and safety using CD19+ cell lines, and in vivo efficacy in a murine NOD/scid xenograft model reconstituted with human PBMC. Further, we used standard preclinical IND enabling assays to evaluate tissue cross reactivity, PK, and toxicological profile (local tolerance, hematocompatibility, effects on hematopoesis, etc). In vitro assays demonstrated the higher potency and efficacy of target cell lysis by AFM11 relative to a bispecific tandem scFv (that is currently in clinical evaluation). CD8+ T cells dominate early AFM11-mediated cytotoxicity (4 hrs) while after 24 hrs both CD4+ and CD8+ T cells equally contribute to tumor lysis with EC50 between 0.5 – 5 pM; cytotoxicity was independent of CD19 cell-surface density. AFM11 exhibited similar cytotoxicity over effector:target ratios ranging from 5:1 to 1:5, and facilitated serial T cell-killing of its targets. The advantage of AFM11 over the bispecific tandem scFv was most pronounced at lower effector:target ratios. AFM11 activated T cells only in the presence of CD19+ cells. In PBMC cultures, AFM11 induced CD69 and CD25 expression, T cell proliferation, and production of IFN-γ, TNF-α, IL-2, IL-6, and IL-10. Depletion of CD19+ cells from PBMC abrogated these effects, demonstrating that the T cell activation is strictly CD19+ target-dependent. Thus, AFM11 should not elicit the devastating cytokine release observed when full-length antibodies bind CD3. Up to one week co-incubation with AFM11 did not inhibit T cell cytotoxicity, suggesting that the TandAb does not induce anergy. In vivo, AFM11 induced dose-dependent growth inhibition of Raji tumors; a single 0.5 mg/kg dose exhibited efficacy similar to 5 daily injections. In the tissue cross reactivity study, only tissues containing CD19+ and CD3+ cells were stained by AFM11; all other tissues, including vital organs, displayed no cross reactivity. Similarly, no local intolerance was observed in rabbits, and no effect on myeloid and erythroid progenitors was observed in a colony-forming assay. Strong accumulation of 125I-labeled AFM11 was observed in the tumors of mice engrafted with CD19+ cancer cells, and no unspecific organ accumulation was observed. Finally, evaluated on the basis of Cmax and the area under the curve (AUC), AFM11 exhibited dose linearity (20 – 500 mg AFM11 dose range) upon single i.v. bolus administration in mice; half-life (T1/2) ranged from 18.4 to 22.9 hr. In summary, AFM11 is a highly efficacious novel drug candidate for the treatment of CD19+ malignancies with an advantageous safety profile and anticipated dosing regimen. Disclosures: Zhukovsky: Affimed Therapeutics AG: Employment, Equity Ownership. Reusch:Affimed Therapeutics AG: Employment. Burkhardt:Affimed Therapeutics AG: Employment. Knackmuss:Affimed Therapeutics AG: Employment. Fucek:Affimed Therapeutics AG: Employment. Eser:Affimed Therapeutics AG: Employment. McAleese:Affimed Therapeutics AG: Employment. Ellwanger:Affimed Therapeutics AG: Employment. Little:Affimed Therapeutics AG: Consultancy, Equity Ownership.

In Vitro and in Vivo Imaging of Initial T-B Cell Interactions in the Setting of B Cell-Based Cancer Immunotherapy

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2740-2740
Author(s):  
Kerstin Wennhold ◽  
Nela Klein-Gonzalez ◽  
Michael von Bergwelt-Baildon ◽  
Alexander Shimabukuro-Vornhagen
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cancer Immunotherapy ◽  
Cell Interactions ◽  
Migration Behavior ◽  
Cell Zone

Abstract In recent years, there has been a growing interest in the use of B cells for cellular immunotherapy, since B cell-based cancer vaccines have yielded promising results in preclinical animal models. Contrary to dendritic cells (DCs), we know little about the migration behavior of B cells in vivo. Therefore, we investigated the interactions between CD40-activated (CD40) B cells and cytotoxic T cells in vitro and the migration behavior of CD40B cells in vivo. The dynamic interactions of human antigen-presenting cells and antigen-specific T cells were observed by time-lapse videomicroscopy. The migratory and chemoattractant potential of CD40B cells was analyzed by flow cytometry and standard transwell migration assays. GFP+ CD40B cells or CD40B cells isolated from Luciferase+mice were used for subsequent in vivo studies. Murine CD40B cells show similar migratory and chemotactic characteristics compared to human CD40B cells. Upon CD40-activation, B cells upregulate the important molecules involved in lymh node homing (CD62L, CCR7/CDCR4), which are functional and induce chemotaxis of T cells in vitro. Striking differences were observed for interactions of human CD40B cells or DCs with T cells. Antigen-loaded CD40B cells differ from immature and mature DCs by displaying a rapid migratory pattern undergoing highly dynamic, short-lived (7.5 min) and sequential interactions with cognate T cells. In vivo, CD40B cells migrate to the spleen and the lymph nodes, where they enrich in the B cell zone before traveling to B cell/ T cell boundary close to the T cell zone. CD40B cell interactions with T cells are dynamic and short-lived and thereby differ from DCs. Taken together, the migration behavior of CD40B cells and their interaction with T cells underline their potential as cellular adjuvant for cancer immunotherapy. Disclosures No relevant conflicts of interest to declare.

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