Ex vivo modelling of PD-1/PD-L1 immune checkpoint blockade under acute, chronic, and exhaustion-like conditions of T-cell stimulation

AbstractBlockade of PD-1/PD-L1 interactions is proving an exciting, durable therapeutic modality in a range of cancers whereby T cells are released from checkpoint inhibition to revive their inherent anti-tumour activity. Here we have studied various ways to model ex vivo T cell function in order to compare the impact of the clinically utilised anti-PD-1 antibody, pembrolizumab (Keytruda) on the activation of human T cells: focussing on the release of pro-inflammatory IFNγ and anti-inflammatory IL-10 to assess functionality. Firstly, we investigated the actions of pembrolizumab in an acute model of T-cell activation with either immature or mature allogeneic dendritic cells (DCs); pembrolizumab enhanced IFNγ and IL-10 release from purified CD4+ T-cells in the majority of donors with a bias towards pro-inflammatory cytokine release. Next, we modelled the impact of pembrolizumab in settings of more chronic T-cell activation. In a 7-day antigen-specific response to EBV peptides, the presence of pembrolizumab resulted in a relatively modest increase in both IFNγ and IL-10 release. Where pembrolizumab was assessed against long-term stimulated CD4+ cells that had up-regulated the exhaustion markers TIM-3 and PD-1, there was a highly effective enhancement of the otherwise exhausted response to allogeneic DCs with respect to IFNγ production. By contrast, the restoration of IL-10 production was considerably more limited. Finally, to assess a direct clinical relevance we investigated the consequence of PD-1/PD-L1 blockade in the disease setting of dissociated cells from lung and colon carcinomas responding to allogeneic DCs: here, pembrolizumab once more enhanced IFNγ production from the majority of tumour preparations whereas, again, the increase in IL-10 release was modest at best. In conclusion, we have shown that the contribution of PD-1—revealed by using a canonical blocking antibody to interrupt its interaction with PD-L1—to the production of an exemplar pro- and anti-inflammatory cytokine, respectively, depends in magnitude and ratio on the particular stimulation setting and activation status of the target T cell. We have identified a number of in vitro assays with response profiles that mimic features of dissociated cell populations from primary tumours thereby indicating these represent disease-relevant functional assays for the screening of immune checkpoint inhibitors in current and future development. Such in vitro assays may also support patient stratification of those likely to respond to immuno-oncology therapies in the wider population.

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Allergen-Induced C5a/C5aR1 Axis Activation in Pulmonary CD11b+ cDCs Promotes Pulmonary Tolerance through Downregulation of CD40

Cells ◽

10.3390/cells9020300 ◽

2020 ◽

Vol 9 (2) ◽

pp. 300 ◽

Cited By ~ 4

Author(s):

Konstantina Antoniou ◽

Fanny Ender ◽

Tillman Vollbrandt ◽

Yves Laumonnier ◽

Franziska Rathmann ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cd4 T Cells ◽

Cell Activation ◽

T Cell Proliferation ◽

C5a Receptor ◽

The Impact

Activation of the C5/C5a/C5a receptor 1 (C5aR1) axis during allergen sensitization protects from maladaptive T cell activation. To explore the underlying regulatory mechanisms, we analyzed the impact of C5aR1 activation on pulmonary CD11b+ conventional dendritic cells (cDCs) in the context of house-dust-mite (HDM) exposure. BALB/c mice were intratracheally immunized with an HDM/ovalbumin (OVA) mixture. After 24 h, we detected two CD11b+ cDC populations that could be distinguished on the basis of C5aR1 expression. C5aR1− but not C5aR1+ cDCs strongly induced T cell proliferation of OVA-reactive transgenic CD4+ T cells after re-exposure to antigen in vitro. C5aR1− cDCs expressed higher levels of MHC-II and CD40 than their C5aR1+ counterparts, which correlated directly with a higher frequency of interactions with cognate CD4+ T cells. Priming of OVA-specific T cells by C5aR1+ cDCs could be markedly increased by in vitro blockade of C5aR1 and this was associated with increased CD40 expression. Simultaneous blockade of C5aR1 and CD40L on C5aR1+ cDCs decreased T cell proliferation. Finally, pulsing with OVA-induced C5 production and its cleavage into C5a by both populations of CD11b+ cDCs. Thus, we propose a model in which allergen-induced autocrine C5a generation and subsequent C5aR1 activation in pulmonary CD11b+ cDCs promotes tolerance towards aeroallergens through downregulation of CD40.

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Melanoma reactive TCR-modified T cells generated without activation retain a less differentiated phenotype and mediate a superior in vivo response

Scientific Reports ◽

10.1038/s41598-021-92808-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Siao-Yi Wang ◽

Tamson V. Moore ◽

Annika V. Dalheim ◽

Gina M. Scurti ◽

Michael I. Nishimura

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Viral Vectors ◽

Cell Activation ◽

Adoptive T Cell Therapy ◽

In Vitro Assays ◽

T Cell Therapy

AbstractAdoptive T cell therapy with T cell receptor (TCR)-modified T cells has shown promise in treating metastatic melanoma and other malignancies. However, studies are needed to improve the efficacy and durability of responses of TCR-modified T cells. Standard protocols for generating TCR-modified T cells involve activating T cells through CD3 stimulation to allow for the efficient transfer of tumor-reactive receptors with viral vectors. T cell activation results in terminal differentiation and shortening of telomeres, which are likely suboptimal for therapy. In these studies, we demonstrate efficient T cell transduction with the melanoma-reactive TIL1383I TCR through culturing with interleukin 7 (IL-7) in the absence of CD3 activation. The TIL1383I TCR-modified T cells generated following IL-7 culture were enriched with naïve (TN) and memory stem cell populations (TSCM) while maintaining longer telomere lengths. Furthermore, we demonstrated melanoma-reactivity of TIL1383I TCR-modified cells generated following IL-7 culture using in vitro assays and a superior response in an in vivo melanoma model. These results suggest that utilizing IL-7 to generate TCR-modified T cells in the absence of activation is a feasible strategy to improve adoptive T cell therapies for melanoma and other malignancies.

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Antigen Presenting Cells from Tumor and Colon of Colorectal Cancer Patients Are Distinct in Activation and Functional Status, but Comparably Responsive to Activated T Cells

Cancers ◽

10.3390/cancers13205247 ◽

2021 ◽

Vol 13 (20) ◽

pp. 5247

Author(s):

Frank Liang ◽

Azar Rezapour ◽

Louis Szeponik ◽

Samuel Alsén ◽

Yvonne Wettergren ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Functional Status ◽

T Cell Activation ◽

Cell Activation ◽

Ex Vivo ◽

Activated T Cells ◽

Ifn Γ ◽

Colorectal Cancer Patients

Although mouse models of CRC treatments have demonstrated robust immune activation, it remains unclear to what extent CRC patients’ APCs and TILs interact to fuel or quench treatment-induced immune responses. Our ex vivo characterization of tumor and adjacent colon cell suspensions suggest that contrasting environments in these tissues promoted inversed expression of T cell co-stimulatory CD80, and co-inhibitory programmed death (PD)-ligand1 (PD-L1) on intratumoral vs. colonic APCs. While putative tumor-specific CD103+CD39+CD8+ TILs expressed lower CD69 (early activation marker) and higher PD-1 (extended activation/exhaustion marker) than colonic counterparts, the latter had instead higher CD69 and lower PD-1 levels. Functional comparisons showed that intratumoral APCs were inferior to colonic APCs regarding protein uptake and upregulation of CD80 and PD-L1 after protein degradation. Our attempt to model CRC treatment-induced T cell activation in vitro showed less interferon (IFN)-γ production by TILs than colonic T cells. In this model, we also measured APCs’ CD80 and PD-L1 expression in response to activated co-residing T cells. These markers were comparable in the two tissues, despite higher IFN- γ exposure for colonic APCs. Thus, APCs within distinct intratumoral and colonic milieus showed different activation and functional status, but were similarly responsive to signals from induced T cell activation.

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Augmentation of T-cell activation by oscillatory forces and engineered antigen-presenting cells

10.1101/580704 ◽

2019 ◽

Author(s):

Fatemeh S. Majedi ◽

Mohammad Mahdi Hasani-Sadrabadi ◽

Timothy J. Thauland ◽

Song Li ◽

Louis-S. Bouchard ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Signal Strength ◽

Antigen Presenting Cells ◽

Oscillatory Movement ◽

Antigen Presenting ◽

The Impact

AbstractActivation of T cells by antigen presenting cells allows them to proliferate, produce cytokines, and kill infected or cancerous cells. We and others have shown that T cell receptors receive and in fact require mechanical forces from their own movements and the movements of antigen presenting cells. Emulation of T cell activation in vitro allows for the massive expansion of T cells necessary for clinical applications. In this paper, we studied the impact of augmenting novel artificial antigen presenting cells of various sizes and antigenic signal strength with mechanical, oscillatory movement. We showed that dynamic culture roughly doubles signal strength as compared to conventional, static culture. We demonstrated that tuning the strength of signal to a “sweet spot” allows for robust expansion of induced regulatory T cells, which is impeded by approaches that simply maximize activation.

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T Cell Pathway Deficiencies in Chronic Lymphocityc Leukemia: Partial Restoration with OKT3/IL-2 Activation.

Blood ◽

10.1182/blood.v110.11.4692.4692 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4692-4692

Author(s):

Mauro Di Ianni ◽

Lorenzo Moretti ◽

Beatrice Del Papa ◽

Maria De Ioanni ◽

Adelmo Terenzi ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

T Cell Activation ◽

Cell Activation ◽

Activated T Cells ◽

Mutational Status ◽

The Impact

Abstract As Chronic Lymphocytic Leukemia (CLL) is associated with several defects in the T cell compartment, the impact of tumour burden on the autologous immune system was studied. Gene expression profiles (using Applied Biosystem Human Genome Microarray) identified 237 genes with significantly increased expression and 221 genes with significantly decreased expression (p<0.05) in CD3+ cells from CLL patients compared with healthy donors. Panther software analysis identified 34/237 upregulated genes and 26/221 downregulated genes that were involved in specific pathways, mainly cell differentiation and proliferation, survival, apoptosis, cytoskeleton formation, vesicle trafficking and T cell activation. The 26 dowregulated genes included Zap70, a member of the syk family protein tyrosine kinase, which is involved in T-cell activation. Zap-70 results were validated by mRNA quantification by RT-PCR (−1.77 fold in comparison with healthy controls) and by flow-cytometric analysis (Mean Intensity Fluorescence=33±12 vs 80±23.62 in controls, p<0.05). To test the hypothesis that activation with OKT3 /IL-2 could bypass these T cell deficiencies, activated T cells from 20 patients with CLL were tested in vitro for cytotoxicity (using the 51chromium release assay) against mutated and unmutated (according to IgVH mutational status) autologous B cells, DAUDI, K562 and P815 cell lines. After 10 days’ culture, the T cell count remained unchanged; CD8 cells expanded more than CD4; TCR spectratyping analysis indicated no differences in TCR repertoires. Activation restored the ZAP-70 mRNA (+1.67 fold). The 51chromium release cytotoxicity assay showed an index > 30% in 5/20 patients. The other 15 were partially cytotoxic against P815, K562 and Daudi. Cell line analysis in all 20 confirmed prevalently T cell-mediated cytotoxicity and poor NK/LAK activity. Cytotoxicity did not correlate with B cell mutational status. We tested the cytotoxic activity of autologous activated T cells in NOD/SCID mice co-transplanted with leukaemic B cells. Only activated T cells exerting cytotoxicity vs autologous B-cell CLL prevent CLL in human-mouse chimera, as confirmed by PCR and FACS analysis which visualised only CD3+ cells. In conclusion, in patients with CLL, activating autologous T cells with OKT3 /IL-2 bypasses, at least in part, the T cell immunological deficiencies. These in vitro and in vivo findings might serve to throw light on new mechanisms that could be exploited in immunotherapy designed to exert disease control.

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Rituximab Directly Decreases T Cell Activation, Both In Vivo and In Vitro

Blood ◽

10.1182/blood.v116.21.3935.3935 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3935-3935 ◽

Cited By ~ 1

Author(s):

Tamar Katz ◽

Dina Stroopinsky ◽

Jacob M. Rowe ◽

Irit Avivi

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

T Cell Activation ◽

Inflammatory Cytokine ◽

Cell Activation ◽

Cell Function ◽

Activation Profile

Abstract Abstract 3935 Rituximab, a chimeric anti-C20 monoclonal antibody, has been extensively used over the last decade for the therapy of B cell malignancies. Recent clinical data suggest that rituximab may affect T cell function, increasing the risk of T cell dependent infections in heavily-treated patients. The current study was designed to investigate the effect of rituximab on T cell activation and assess T cell function following the addition of rituximab to purified T cells. The T cell activation profile, dependent on rituximab administration, was evaluated in vivo and in vitro. Peripheral blood mononuclear cells (PBMCs) generated from B-cell non-Hodgkin lymphoma (NHL) patients prior and immediately after the administration of 375 mg/m2 rituximab, were examined for the expression of inflammatory cytokines. The in vitro studies were performed by using CD25 depleted PBMCs or B cell depleted T cells (CD3+CD25-CD19-). The obtained cells were stimulated with allogeneic dendritic cells (DCs), in the absence or presence or 2 mg/ml rituximab. T cell activation was evaluated using immunophenotypic markers, cytokine profile and T cell proliferation assay. Eight NHL patients participated in the study. The level of T cells expressing inflammatory cytokines was significantly decreased following the administration of a single dose of rituximab. T cells expressing IL-2 declined from a mean level of 26.5% to 11.5% and the level of IFN- γ decreased from 22% to 4.2%. Further administration of rituximab, up to 4 weekly doses, resulted in an additional decline in the amount of inflammatory cytokine producing T cells to a level of 1.4% for IL-2 and 3.5% for IFN-g. However, repeated evaluation, performed at 4 months after completing rituximab, showed restoration of the inflammatory population. In accord with this inhibitory effect, in vitro stimulation of T cells with allogeneic DCs, in the presence of rituximab, resulted in a significant decrease in activation markers (CD25, GITR and CTLA-4) (Table 1). These changes were accompanied by a marked reduction in inflammatory cytokine production and proliferative capacity. Of interest, these inhibitory effects were also obtained whilst using B cell depleted T cells (CD3+CD25-CD19-). In conclusion, rituximab administration results in a transient T cell inactivation, demonstrated through the reduction in inflammatory cytokine production and T cell proliferation capacity. This effect appears to be non-B cell dependent, being obtained in the absence of B cell in the culture, and may account for clinical observations in ameliorating T-cell dependent disorders, such as graft-versus-host disease. Table 1. Activation profile depending on rituximab (in vitro) Without rituximab With rituximab *Activation marker (%) CD25 27 9 GITR 15.6 4.7 CTLA4 17.7 7 *Cytokines expression (%) IL-2 22 2 IL12 16 4 IFN-gamma 21 1.8 T cells proliferation (O.D.) DC stimulation 1.528 0.580 CMV stimulation 1.563 0.570 anti CD3/CD28 stimulation 0.705 0.407 * Gated out of lymphocytes Disclosures: No relevant conflicts of interest to declare.

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Group BStreptococcusInduces a Robust IFN-γResponse by CD4+T Cells in anIn VitroandIn VivoModel

Journal of Immunology Research ◽

10.1155/2016/5290604 ◽

2016 ◽

Vol 2016 ◽

pp. 1-12 ◽

Cited By ~ 9

Author(s):

Damian Clarke ◽

Corinne Letendre ◽

Marie-Pier Lecours ◽

Paul Lemire ◽

Tristan Galbas ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Capsular Polysaccharide ◽

Ex Vivo ◽

Specific Cell ◽

Proinflammatory Response

Group BStreptococcus(GBS) serotype III causes life-threatening infections. Cytokines have emerged as important players for the control of disease, particularly IFN-γ. Although potential sources of this cytokine have been proposed, no specific cell line has ever been described as a leading contributor. In this study, CD4+T cell activation profiles in response to GBS were evaluated throughin vivo,ex vivo,andin vitroapproaches. Total splenocytes readily produce a type 1 proinflammatory response by releasing IFN-γ, TNF-α, and IL-6 and actively recruit T cells via chemokines like CXCL9, CXCL10, and CCL3. Responding CD4+T cells differentiate into Th1 cells producing large amounts of IFN-γ, TNF-α, and IL-2.In vitrostudies using dendritic cell and CD4+T cell cocultures infected with wild-type GBS or a nonencapsulated mutant suggested that GBS capsular polysaccharide, one of the major bacterial virulence factors, differentially modulates surface expression of CD69 and IFN-γproduction. Overall, CD4+T cells are important producers of IFN-γand might thus influence the course of GBS infection through the expression balance of this cytokine.

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Rapid and efficient generation of antigen-specific isogenic T cells from cryopreserved blood samples

10.1101/2021.11.12.468355 ◽

2021 ◽

Author(s):

Anneke Eerkens ◽

Annege Vledder ◽

Nienke van Rooij ◽

Floris Foijer ◽

Hans W Nijman ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Gene Editing ◽

Ex Vivo ◽

Immune Monitoring ◽

Functional Evaluation ◽

Starting Point

Objectives: CRISPR/Cas9-mediated gene editing has been leveraged for the modification of human and mouse T cells. However, limited experience is available on the application of CRISPR/Cas9 electroporation in cryopreserved T cells collected during e.g. clinical trials. Methods: PBMCs from healthy donors were used to generate knockout T cell models for interferon-gamma (IFNg), Cbl Proto-Oncogene B (CBLB), Fas cell surface death receptor (Fas) and T cell receptor (TCR alpha and beta) genes. The effect of CRISPR-cas9-mediated gene editing on T cells was evaluated using apoptosis assays, cytokine bead arrays and ex vivo and in vitro stimulation assays. Results: Our results demonstrate that CRISPR/Cas9-mediated gene editing of ex vivo T cells is efficient and does not overtly affect T cell viability. Cytokine release and T cell proliferation were not affected in gene edited T cells. Interestingly, memory T cells were more susceptible to CRISPR/Cas9 gene editing than naive T cells. Ex vivo and in vitro stimulation with antigens resulted in equivalent antigen-specific T cell responses in gene-edited and untouched control cells; making CRISPR/Cas9-mediated gene editing compatible with clinical antigen-specific T cell activation and expansion assays. Conclusion: Here, we report an optimized protocol for rapid, viable and highly efficient genetic modification in ex vivo human antigen specific T cells, for subsequent functional evaluation and/or expansion. Our platform extends CRISPR/Cas9-mediated gene editing for use in gold-standard clinically-used immune-monitoring pipelines and serves as a starting point for development of analogous approaches such as those including transcriptional activators and or epigenetic modifiers.

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The Impact of the Bone Marrow Microenvironment on T Cell Redirection Therapeutics

Blood ◽

10.1182/blood.v128.22.2752.2752 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2752-2752

Author(s):

Priyanka Nair-Gupta ◽

Stephen Rudnick ◽

Leopoldo Luistro ◽

Diana Chin ◽

Melissa Smith ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Stromal Cells ◽

Cell Activation ◽

Leukemic Cells ◽

Anti Cancer ◽

The Impact

Abstract Redirecting T cells to specifically target and kill malignant cells has been validated as an effective anti-cancer strategy in the clinic with the approval of CD19xCD3 BiTE (Blincyto) for acute lymphoblastic leukemia. However, the immunosuppressive nature of the tumor microenvironment potentially poses a significant hurdle to T cell therapies. For instance, the bone marrow (BM) niche is appreciated to be a site of immune privilege at steady state to allow for normal hematopoiesis and immune cell generation. Additionally, in hematological malignancies, the BM niche is protective to leukemic stem cells, a phenomenon that has minimized the efficacy of several anti-cancer drugs including chemotherapy, targeted small molecule inhibitors and antibody based therapies. In this study, we investigated the impact of the BM microenvironment on T cell redirection. Using antibodies made with the Genmab DuoBody® technology targeting specific tumor antigens (CD123 and BCMA) and CD3, we observed that co-culture of acute myeloid leukemia (AML; KG-1, MOLM-13 and OCI-AML5) or multiple myeloma (MM; H929 and RPMI8226) cell lines with bone marrow stromal cell lines (HS-5 and HS-27a) significantly protected leukemic cells from DuoBody®-T cell mediated lysis in vitro. Specifically, co-culture of bone marrow stromal cells in killing assays led to a 20-50% decrease in the maximum observed cytotoxicity and a 3-10 fold weaker EC50, reflecting an impact on both the efficacy and potency of bispecific DuoBody® antibodies. Similar results were also observed with primary stromal cells obtained from healthy donors. Furthermore, presence of stromal cells in a humanized xenograft AML model attenuated tumor growth inhibition (TGI) observed with DuoBody® treatment (78% TGI with MOLM-13 vs 15% TGI with MOLM-13+HS-5). Impaired TGI correlated with reduced T cell activation (7 fold decrease in CD25 upregulation) and production of granzyme B (8 fold reduction), providing one potential mechanism to explain loss of activity of the DuoBody® antibody. In vitro trans-well redirection assays revealed that cell-cell contact with stromal cells was crucial for reduced T cell activation and target cell survival relative to controls. Additionally, leukemic cells not killed by T cells were observed to preferentially cluster around stromal cells. We propose that target cells can evade T cell death by a stromal cell dependent mechanism involving activation of multiple pro-survival and anti-apoptotic pathways in leukemic cells in addition to suppressed activation of T cells. We are currently studying pathways mediating the cross-talk between cancer, immune and stromal cells. A better understanding of the mechanisms underlying the protective and immunosuppressive nature of the BM microenvironment will be instrumental to the design of more effective CD3 redirected therapeutics or novel combinatorial regimens for robust anti-cancer responses. Disclosures Nair-Gupta: Janssen: Employment. Rudnick:Janssen Pharmaceuticals R&D: Employment. Luistro:Janssen: Employment. Chin:Janssen: Employment. Smith:Janssen Research & Development, LLC: Employment, Equity Ownership. McDaid:Janssen Pharmaceuticals Research and Development: Employment. Li:Janssen: Employment. Pillarisetti:Janssen Research and Development, LLC: Employment. Baldwin:Janssen: Employment. Packman:Janssen: Employment. Elsayed:Janssen: Employment. Attar:Janssen: Employment. Gaudet:Janssen Pharmaceuticals R&D: Employment, Other: Stock options, Patents & Royalties: pending, not yet issued.

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

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.8059 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 8059-8059 ◽

Cited By ~ 1

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.

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