scholarly journals P06.06 Enhancing trafficking and resistance to immunosuppression of synthetic agonistic receptor-transduced T cells in solid tumor models

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A21.2-A22
Author(s):  
M Schwerdtfeger ◽  
M Benmebarek ◽  
F Märkl ◽  
CH Karches ◽  
A Öner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Chemokine Receptors ◽  
Cell Activation ◽  
Target Cells ◽  
Tumor Associated Antigen ◽  
Overlap Extension ◽  
Part Time

BackgroundChimeric antigen receptor therapy – although very efficacious in B cell malignancies – is facing many challenges which limit its success in solid tumors, e.g. on-target off-tumor toxicities, antigen heterogeneity, lack of T cell migration into tumors and an immunosuppressive tumor microenvironment. To better control on-target off-tumor effects and address antigen heterogeneity we developed a modular approach where we equipped T cells with a synthetic agonistic receptor (SAR). The SAR is only activated in the presence of a bispecific antibody (BiAb) cross-linking the receptor with a tumor-associated antigen. While we could show efficacy of the SAR platform in different models, limited infiltration and immune suppression still hamper its function. We could previously demonstrate that T cell infiltration can be enhanced by transduction with carefully chosen chemokine receptors like CXCR6, CCR4 and CCR8. At the same time, gene silencing of checkpoint molecules like PD-1 can make T cells more resistant to immunosuppression, thus we assumed that combining these approaches might generate a desired T cell product.Materials and MethodsAll constructs had been generated previously by overlap-extension cloning. The EGFRvIII (E3) SAR consists of extracellular EGFRvIII, transmembrane CD28 and intracellular CD28 and CD3ζ. Human CXCR6-GFP, CCR4-GFP and CCR8-GFP are composed of the chemokine receptors fused to GFP via a 2A sequence. Primary human T cells were retrovirally transduced to stably express the SAR and chemokine receptors. We analyzed migration, cytotoxicity and activation of the single and double (E3 SAR and chemokine receptor) transduced T cells. In addition, PD-1 was knocked out using CRISPR-Cas9 and killing kinetics of target cells and T cell activation were assessed.ResultsCo-transduction with chemokine receptors significantly increased migration of E3 SAR T cells to their respective ligand while lysis of target-expressing tumor cell and T cell activation in the presence of BiAb were not affected in vitro. Additionally knocking out PD-1 enhanced killing kinetics and activation of E3 SAR and E3 SAR + CXCR6-GFP transduced T cells compared to corresponding mock electroporated T cells.ConclusionsUsing the controllable and modular SAR – BiAb platform SAR T cell activation can be limited by stopping BiAb dosing if adverse events occur. In addition, SAR T cells can be redirected to an alternative tumor-associated antigen by exchanging the BiAb in the case of antigen escape. Here we present add-ons to this approach for increased tumor infiltration and resistance to immunosuppression. Since migration is enhanced upon co-transduction with chemokine receptors and target cell lysis is accelerated upon PD-1 knockout in vitro these two additional modifications seem very promising options to further improve tumor control in vivo.Disclosure InformationM. Schwerdtfeger: None. M. Benmebarek: None. F. Märkl: None. C.H. Karches: A. Employment (full or part-time); Significant; Daiichi Sankyo Deutschland GmbH. A. Öner: None. M. Geiger: A. Employment (full or part-time); Significant; Roche. B. Cadilha: None. S. Endres: None. V. Desiderio: None. C. Klein: A. Employment (full or part-time); Significant; Roche. S. Kobold: None.

A T Cell-Engaging CD3 Recruit-Tandab Potently Kills CD19+ Tumor B Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3721-3721
Author(s):  
Eugene Zhukovsky ◽  
Uwe Reusch ◽  
Carmen Burkhardt ◽  
Stefan Knackmuss ◽  
Ivica Fucek ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Target Cells ◽  
Cytotoxicity Assays

Abstract Abstract 3721 Background: 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. T cells are potent tumor-killing effector cells that cannot be recruited by native antibodies. The CD3 RECRUIT-TandAb AFM11, a humanized bispecific tetravalent antibody with two binding sites for both CD3 and CD19, is a novel therapeutic for the treatment of NHL that harnesses the cytotoxic nature of T cells. Methods: We engineered a bispecific anti-CD19/anti-CD3e tetravalent TandAb with humanized and affinity-matured variable domains. The TandAb's binding properties, T cell-mediated cytotoxic activity, and target-mediated T cell activation were characterized in a panel of in vitro assays. In vivo efficacy was evaluated in a murine NOD/scid xenograft model reconstituted with human PBMC. Results: AFM11 mediates highly potent CD19+ tumor cell lysis in cytotoxicity assays performed on a panel of cell lines (JOK-1, Raji, Nalm-6, MEC-1, VAL, Daudi) and primary B-CLL tumors: EC50 values are in the low- to sub-picomolar range and do not correlate with the expression density of CD19 on the target cell lines. The cytotoxic activity of tetravalent AFM11 is superior to that of alternative bivalent antibody formats possessing only a single binding site for both CD19 and CD3. High affinity binding of AFM11 to CD19 and to CD3 is essential for efficacious T cell recruitment. Both CD8+ and CD4+ T cells mediate cytotoxicity however the former exhibit much faster killing. We observe that AFM11 displays similar cytotoxic efficacy at different effector to target ratios (from 5:1 to 1:5) in cytotoxicity assays; this suggests that T cells are engaged in the serial killing of CD19+ target cells. In the absence of CD19+ target cells in vitro, AFM11 does not elicit T cell activation as manifested by cytokine release (from a panel of ten cytokines associated with T cell activation), their proliferation, or their expression of activation markers. AFM11 activates T cells exclusively in the presence of its targets and mediates lysis of CD19+ cells while sparing antigen-negative bystanders. In the absence of CD19+ target cells, AFM11 concentrations in excess of 500-fold over EC50 induce down-modulation of the CD3/TCR complex. Yet, AFM11-treated T cells can be re-engaged for target cell lysis. All of these features of AFM11-induced T cell activation may contribute additional safety without compromising its efficacy. In vivo AFM11 demonstrates a robust dose-dependent inhibition of subcutaneous Raji tumors in mice. At 5 mg/kg AFM11 demonstrates a complete suppression of tumor growth, and even at 5 ug/kg tumor growth is reduced by 60%. Moreover, we observe that a single administration of AFM11 produces inhibition of tumor growth similar to that of 5 consecutive administrations. Conclusions: In summary, our in vitro and in vivo experiments with AFM11 demonstrate the high potency and efficacy of its anti-tumor cytotoxicity. Thus, AFM11 is a novel highly efficacious drug candidate for the treatment of B cell malignancies with an advantageous safety profile. 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.

Enhancing The Efficacy and Specificity Of Antibody-Based T Cell Retargeting Strategies Against Hematological Malignancies

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 930-930 ◽  
Author(s):  
Claudia Arndt ◽  
Marc Cartellieri ◽  
Roberta Aliperta ◽  
Stefanie Koristka ◽  
Irene Michalk ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Side Effects ◽  
T Cell Activation ◽  
Cell Activation ◽  
Modular System ◽  
Target Cells ◽  
Cross Linkage ◽  
Target Module

Abstract New promising candidates for cancer immunotherapy are bispecific antibodies (bsAbs) which have already shown a convincing anti-tumor effect in first clinical trials. BsAbs are composed of two single-chain fragments variable (scFvs), derived from the variable heavy and light chain of a monoclonal Ab (mAb), which bind to the activating CD3-complex on T cells and a tumor-associated antigen (TAA) on cancer cells. Consequently, the cross-linkage of effector and target cells by bsAbs results in an efficient T cell-mediated cancer cell killing. However, several serious adverse effects were observed in patients after application of Ab constructs. Most critical is an overall activation of T cells which bears the risk of a systemic release of pro-inflammatory cytokines. With respect to the idea that the CD3 binding of an Ab construct is critical for T cell activation, the CD3 domain has to be optimized in order to prevent or even reduce unspecific T cell responses. For this reason, we cloned several anti-CD3 scFvs derived from different anti-CD3 mAb clones and compared their functionality and safety. The anti-CD3 scFv with lowest risk of side effects was introduced in different anti-TAA bsAbs. Functional and safety studies in vitro revealed that development of each bsAb requires extensive and time-consuming optimization steps to gain high efficacy but low risk of side effects. To improve the process of Ab engineering, we recently introduced a novel modular Ab platform that can be rapidly and cost-effectively adapted for redirection of T cells to any TAA (Arndt and Feldmann et al. accepted for publication in Leukemia 2013). In this novel modular system the dual specificity of a conventional bsAb is distributed to two separate Ab modules, (i) the effector module and (ii) the target module. Only the complex of both Ab modules mediates the cross-linkage of effector and target cells resulting in T cell activation and redirected cancer cell lysis similar to conventional T cell engaging bsAbs. The universal effector module is a well optimized bsAb balancing efficacy and safety. It binds CD3 on T cells and the E5B9 tag of the target module. The individual target module comprises an anti-TAA scFv and the peptide epitope E5B9. For treatment of lymphoid or myeloid malignancies a series of different target modules were designed. In vitro and in vivo data clearly underline that the combination of the established effector module with different target modules efficiently activated T cells against hematological malignancies. Both CD4+ and CD8+ T cells from healthy donors were efficiently activated to kill TAA-positive tumor cell lines at low effector to target cell ratios and Ab concentrations in a tumor-specific manner. Most importantly, we could demonstrate that patient-derived T cells were able to kill autologous malignant cells upon Ab-mediated cross-linkage. Another unique feature of the modular system is that multispecific or multifunctional target modules could be easily included to further improve the therapeutic effect. In order to increase anti-tumor specificity and reduce the risk of tumor escape variants, bispecific target modules that recognize different TAAs at the same time, were constructed. Cytotoxicity assays investigating dual targeting of double-positive tumor cells via the modular system demonstrated that target cell lysis can be considerably improved in comparison to single targeting modules. Moreover, proliferation and cytokine release of redirected T cells could be enhanced by supplying costimulatory immunoligands (e.g. 4-1BBL and Ox40L) via target modules. In summary, we developed a multifunctional and highly flexible modular system based on an anti-CD3 domain with lowest risk of side effects. In contrast to conventional anti-TAA-anti-CD3 bsAbs, the development of a novel modular system for different clinical indications is much easier and less time-consuming. Once the effector module containing the critical CD3 domain is optimized, the modular system can be flexibly applied to target any TAA simply by replacing the target module. Application of bispecific target modules or providing costimulatory signals via the modular system might prolong immune responses and further increase anti-tumor specificity and activity. Finally, the novel modular platform represents a valuable and promising tool for cancer immunotherapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Novel T Cell-Engaging Bispecific Antibody for Treating Mesothelin-Positive Solid Tumors

Biomolecules ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 399
Author(s):  
Aerin Yoon ◽  
Shinai Lee ◽  
Sua Lee ◽  
Sojung Lim ◽  
Yong-Yea Park ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
T Cell Activation ◽  
Target Cell ◽  
Cell Activation ◽  
Cell Killing ◽  
Bispecific Antibodies ◽  
Target Cells

As mesothelin is overexpressed in various types of cancer, it is an attractive target for therapeutic antibodies. T-cell bispecific antibodies bind to target cells and engage T cells via binding to CD3, resulting in target cell killing by T-cell activation. However, the affinity of the CD3-binding arm may influence CD3-mediated plasma clearance or antibody trapping in T-cell-containing tissues. This may then affect the biodistribution of bispecific antibodies. In this study, we used scFab and knob-into-hole technologies to construct novel IgG-based 1 + 1 MG1122-A and 2 + 1 MG1122-B bispecific antibodies against mesothelin and CD3ε. MG1122-B was designed to be bivalent to mesothelin and monovalent to CD3ε, using a 2 + 1 head-to-tail format. Activities of the two antibodies were evaluated in mesothelin-positive tumor cells in vitro and xenograft models in vivo. Although both antibodies exhibited target cell killing efficacy and produced regression of xenograft tumors with CD8+ T-cell infiltration, the antitumor efficacy of MG1122-B was significantly higher. MG1122-B may improve tumor targeting because of its bivalency for tumor antigen. It may also reduce systemic toxicity by limiting the activation of circulating T cells. Thus, MG1122-B may be useful for treating mesothelin-positive solid tumors.

High affinity CD3 RECRUIT TandAb for T cell-mediated lysis of CD19+ tumor B cells.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 8059-8059 ◽  
Author(s):  
Eugene Zhukovsky ◽  
Uwe Reusch ◽  
Carmen Burkhardt ◽  
Stefan Knackmuss ◽  
Ivica Fucek ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
In Vitro Assays ◽  
Target Cells ◽  
B Cell Malignancies ◽  
High Affinity

8059 Background: CD19 is expressed from early B cell development to the differentiation into plasma cells and is an attractive target for B cell malignancies either lacking CD20 expression or refractory to anti-CD20 antibody therapies. T cells are potent tumor killing effector cells that are not recruited by native antibodies. The CD3 RECRUIT-TandAb AFM11, a human bispecific tetravalent antibody with two binding sites for both CD3 and CD19, is a novel therapeutic for the treatment of NHL that harnesses the cytotoxic nature of T cells. Methods: A bispecific anti-CD19/anti-CD3 tetravalent TandAb with humanized and affinity matured variable domains was constructed. The TandAb’s binding, T-cell mediated cytotoxic activity, and cytokine release were characterized in a panel of in vitro assays. In vivo efficacy was evaluated in a murine NOD/scid xenograft model reconstituted with human PBMC. Results: AFM11 mediates highly potent target tumor cell lysis in cytotoxicity assays: EC50 values are low to sub-picomolar range in a panel of CD19+ cell lines and primary B-CLL tumor cells. The cytotoxic activity of tetravalent AFM11 is superior to that of alternative bivalent antibody formats possessing only a single binding site for both CD19 and CD3. High affinity binding of AFM11 to CD19, and more so to CD3 (low to sub-nanomolar Kd), is essential for efficacious T cell recruitment. The high affinity bivalent binding of AFM11 to CD3 does not trigger T cell activation in the absence of CD19+ target cells in functional in vitro assays. AFM11 activates T cells only in the presence of its targets and mediates lysis while sparing antigen-negative bystanders. AFM11 induces down-modulation of the CD3/TCR complex in the absence of target cells and at high concentrations. Also, AFM11-treated T cells can be re-engaged for target cell lysis. These features of AFM11-induced T cell activation may contribute additional safety with no compromise of efficacy. Finally, AFM11 demonstrates a robust dose-dependent inhibition of subcutaneous Raji tumors in mice. Conclusions: AFM11 is a novel highly efficacious drug candidate for the treatment of B cell malignancies with an advantageous safety profile.

scholarly journals Immunomodulatory effects of different intravenous immunoglobulin preparations in chronic lymphocytic leukemia

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ana Colado ◽  
Esteban Enrique Elías ◽  
Valeria Judith Sarapura Martínez ◽  
Gregorio Cordini ◽  
Pablo Morande ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
T Cell Activation ◽  
Cell Activation ◽  
Lymphocytic Leukemia ◽  
Immunomodulatory Effects ◽  
Immunoglobulin Preparations

AbstractHypogammaglobulinemia is the most frequently observed immune defect in chronic lymphocytic leukemia (CLL). Although CLL patients usually have low serum levels of all isotypes (IgG, IgM and IgA), standard immunoglobulin (Ig) preparations for replacement therapy administrated to these patients contain more than 95% of IgG. Pentaglobin is an Ig preparation of intravenous application (IVIg) enriched with IgM and IgA (IVIgGMA), with the potential benefit to restore the Ig levels of all isotypes. Because IVIg preparations at high doses have well-documented anti-inflammatory and immunomodulatory effects, we aimed to evaluate the capacity of Pentaglobin and a standard IVIg preparation to affect leukemic and T cells from CLL patients. In contrast to standard IVIg, we found that IVIgGMA did not modify T cell activation and had a lower inhibitory effect on T cell proliferation. Regarding the activation of leukemic B cells through BCR, it was similarly reduced by both IVIgGMA and IVIgG. None of these IVIg preparations modified spontaneous apoptosis of T or leukemic B cells. However, the addition of IVIgGMA on in vitro cultures decreased the apoptosis of T cells induced by the BCL-2 inhibitor, venetoclax. Importantly, IVIgGMA did not impair venetoclax-induced apoptosis of leukemic B cells. Overall, our results add new data on the effects of different preparations of IVIg in CLL, and show that the IgM/IgA enriched preparation not only affects relevant mechanisms involved in CLL pathogenesis but also has a particular profile of immunomodulatory effects on T cells that deserves further investigation.

Modulation of T-Cell Functions in KIR2DL3 (CD158b) Transgenic Mice

Blood ◽  
1999 ◽  
Vol 94 (7) ◽  
pp. 2396-2402 ◽  
Author(s):  
Anna Cambiaggi ◽  
Sylvie Darche ◽  
Sophie Guia ◽  
Philippe Kourilsky ◽  
Jean-Pierre Abastado ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Transgenic Mice ◽  
T Cell Activation ◽  
Cell Activation ◽  
Killer Cell ◽  
In Vitro Proliferation ◽  
Cell Functions ◽  
Double Transgenic Mice

In humans, a minor subset of T cells express killer cell Ig-like receptors (KIRs) at their surface. In vitro data obtained with KIR+ β and γδ T-cell clones showed that engagement of KIR molecules can extinguish T-cell activation signals induced via the CD3/T-cell receptor (TCR) complex. We analyzed the T-cell compartment in mice transgenic for KIR2DL3 (Tg-KIR2DL3), an inhibitory receptor for HLA-Cw3. As expected, mixed lymphocyte reaction and anti-CD3 monoclonal antibody (MoAb)-redirected cytotoxicity exerted by freshly isolated splenocytes can be inhibited by engagement of transgenic KIR2DL3 molecules. In contrast, antigen and anti-CD3 MoAb-induced cytotoxicity exerted by alloreactive cytotoxic T lymphocytes cannot be inhibited by KIR2DL3 engagement. In double transgenic mice, Tg-KIR2DL3 × Tg-HLA-Cw3, no alteration of thymic differentiation could be documented. Immunization of double transgenic mice with Hen egg white lysozime (HEL) or Pigeon Cytochrome-C (PCC) was indistinguishable from immunization of control mice, as judged by recall antigen-induced in vitro proliferation and TCR repertoire analysis. These results indicate that KIR effect on T cells varies upon cell activation stage and show unexpected complexity in the biological function of KIRs in vivo.

scholarly journals Costimulation by B7 Modulates Specificity of Cytotoxic T Lymphocytes: A Missing Link That Explains Some Bystander T Cell Activation

1997 ◽  
Vol 186 (10) ◽  
pp. 1787-1791 ◽  
Author(s):  
Pan Zheng ◽  
Yang Liu
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
Influenza Virus ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
T Cell Proliferation ◽  
Target Cells ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation

It has been proposed that some bystander T cell activation may in fact be due to T cell antigen receptor (TCR) cross-reactivity that is too low to be detected by the effector cytotoxic T lymphocyte (CTL). However, this hypothesis is not supported by direct evidence since no TCR ligand is known to induce T cell proliferation and differentiation without being recognized by the effector CTL. Here we report that transgenic T cells expressing a T cell receptor to influenza virus A/NT/68 nucleoprotein (NP) 366-374:Db complexes clonally expand and become effector CTLs in response to homologous peptides from either A/PR8/34 (H1N1), A/AA/60 (H2N2), or A/NT/68 (H3N2). However, the effector T cells induced by each of the three peptides kill target cells pulsed with NP peptides from the H3N2 and H2N2 viruses, but not from the H1N1 virus. Thus, NP366–374 from influenza virus H1N1 is the first TCR ligand that can induce T cell proliferation and differentiation without being recognized by CTLs. Since induction of T cell proliferation was mediated by antigen-presenting cells that express costimulatory molecules such as B7, we investigated if cytolysis of H1N1 NP peptide–pulsed targets can be restored by expressing B7-1 on the target cells. Our results revealed that this is the case. These data demonstrated that costimulatory molecule B7 modulates antigen specificity of CTLs, and provides a missing link that explains some of the bystander T cell activation.

scholarly journals T cell receptor zeta/CD3-p59fyn(T)-associated p120/130 binds to the SH2 domain of p59fyn(T).

1993 ◽  
Vol 178 (6) ◽  
pp. 2107-2113 ◽  
Author(s):  
A J da Silva ◽  
O Janssen ◽  
C E Rudd
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Intracellular Signaling ◽  
Cell Activation ◽  
Cell Receptor ◽  
Sh2 Domain ◽  
T Complex

Intracellular signaling from the T cell receptor (TCR)zeta/CD3 complex is likely to be mediated by associated protein tyrosine kinases such as p59fyn(T), ZAP-70, and the CD4:p56lck and CD8:p56lck coreceptors. The nature of the signaling cascade initiated by these kinases, their specificities, and downstream targets remain to be elucidated. The TCR-zeta/CD3:p59fyn(T) complex has previously been noted to coprecipitate a 120/130-kD doublet (p120/130). This intracellular protein of unknown identity associates directly with p59fyn(T) within the receptor complex. In this study, we have shown that this interaction with p120/130 is specifically mediated by the SH2 domain (not the fyn-SH3 domain) of p59fyn(T). Further, based on the results of in vitro kinase assays, p120/130 appears to be preferentially associated with p59fyn(T) in T cells, and not with p56lck. Antibody reprecipitation studies identified p120/130 as a previously described 130-kD substrate of pp60v-src whose function and structure is unknown. TCR-zeta/CD3 induced activation of T cells augmented the tyrosine phosphorylation of p120/130 in vivo as detected by antibody and GST:fyn-SH2 fusion proteins. p120/130 represents the first identified p59fyn(T):SH2 binding substrate in T cells, and as such is likely to play a key role in the early events of T cell activation.

scholarly journals CCL21 mediates CD4+ T-cell costimulation via a DOCK2/Rac-dependent pathway

Blood ◽  
2009 ◽  
Vol 114 (3) ◽  
pp. 580-588 ◽  
Author(s):  
Kathrin Gollmer ◽  
François Asperti-Boursin ◽  
Yoshihiko Tanaka ◽  
Klaus Okkenhaug ◽  
Bart Vanhaesebroeck ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Early Time ◽  
Cell Receptor ◽  
Tcr Signaling ◽  
Activation Markers

Abstract CD4+ T cells use the chemokine receptor CCR7 to home to and migrate within lymphoid tissue, where T-cell activation takes place. Using primary T-cell receptor (TCR)–transgenic (tg) CD4+ T cells, we explored the effect of CCR7 ligands, in particular CCL21, on T-cell activation. We found that the presence of CCL21 during early time points strongly increased in vitro T-cell proliferation after TCR stimulation, correlating with increased expression of early activation markers. CCL21 costimulation resulted in increased Ras- and Rac-GTP formation and enhanced phosphorylation of Akt, MEK, and ERK but not p38 or JNK. Kinase-dead PI3KδD910A/D910A or PI3Kγ-deficient TCR-tg CD4+ T cells showed similar responsiveness to CCL21 costimulation as control CD4+ T cells. Conversely, deficiency in the Rac guanine exchange factor DOCK2 significantly impaired CCL21-mediated costimulation in TCR-tg CD4+ T cells, concomitant with impaired Rac- but not Ras-GTP formation. Using lymph node slices for live monitoring of T-cell behavior and activation, we found that G protein-coupled receptor signaling was required for early CD69 expression but not for Ca2+ signaling. Our data suggest that the presence of CCL21 during early TCR signaling lowers the activation threshold through Ras- and Rac-dependent pathways leading to increased ERK phosphorylation.

107 Effects of IL-2 and IL-15 on the proliferative and antitumor capacities of allogeneic CD20 CAR-engineered γδ T cells in a 3D B cell lymphoma spheroid assay

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A119-A119
Author(s):  
Lu Bai ◽  
Kevin Nishimoto ◽  
Mustafa Turkoz ◽  
Marissa Herrman ◽  
Jason Romero ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cytokine Production ◽  
Γδ T Cells ◽  
Γδ T Cell ◽  
Car T

BackgroundAutologous chimeric antigen receptor (CAR) T cells have been shown to be efficacious for the treatment of B cell malignancies; however, widespread adoption and application of CAR T cell products still face a number of challenges. To overcome these challenges, Adicet Bio is developing an allogeneic γδ T cell-based CAR T cell platform, which capitalizes on the intrinsic abilities of Vδ1 γδ T cells to recognize and kill transformed cells in an MHC-unrestricted manner, to migrate to epithelial tissues, and to function in hypoxic conditions. To gain a better understanding of the requirements for optimal intratumoral CAR Vδ1 γδ T cell activation, proliferation, and differentiation, we developed a three-dimensional (3D) tumor spheroid assay, in which tumor cells acquire the structural organization of a solid tumor and establish a microenvironment that has oxygen and nutrient gradients. Moreover, through the addition of cytokines and/or tumor stromal cell types, the spheroid microenvironment can be modified to reflect hot or cold tumors. Here, we report on the use of a 3D CD20+ Raji lymphoma spheroid assay to evaluate the effects of IL-2 and IL-15, positive regulators of T cell homeostasis and differentiation, on the proliferative and antitumor capacities of CD20 CAR Vδ1 γδ T cells.MethodsMolecular, phenotypic, and functional profiling were performed to characterize the in vitro dynamics of the intraspheroid CD20 CAR Vδ1 γδ T cell response to target antigen in the presence of IL-2, IL-15, or no added cytokine.ResultsWhen compared to no added cytokine, the addition of IL-2 or IL-15 enhanced CD20 CAR Vδ1 γδ T cell activation, proliferation, survival, and cytokine production in a dose-dependent manner but were only able to alter the kinetics of Raji cell killing at low effector to target ratios. Notably, differential gene expression analysis using NanoString nCounter® Technology confirmed the positive effects of IL-2 or IL-15 on CAR-activated Vδ1 γδ T cells as evidenced by the upregulation of genes involved in activation, cell cycle, mitochondrial biogenesis, cytotoxicity, and cytokine production.ConclusionsTogether, these results not only show that the addition of IL-2 or IL-15 can potentiate CD20 CAR Vδ1 γδ T cell activation, proliferation, survival, and differentiation into antitumor effectors but also highlight the utility of the 3D spheroid assay as a high throughput in vitro method for assessing and predicting CAR Vδ1 γδ T cell activation, proliferation, survival, and differentiation in hot and cold tumors.

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