scholarly journals A scDb-based trivalent bispecific antibody for T-cell-mediated killing of HER3-expressing cancer cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nadine Aschmoneit ◽  
Sophia Steinlein ◽  
Lennart Kühl ◽  
Oliver Seifert ◽  
Roland E. Kontermann
Keyword(s):  
T Cell ◽  
Binding Site ◽  
Cancer Cell ◽  
Cancer Progression ◽  
T Cell Activation ◽  
Cell Activation ◽  
Egf Receptor ◽  
Bispecific Antibodies ◽  
Target Cells ◽  
Single Chain

AbstractHER3 is a member of the EGF receptor family and elevated expression is associated with cancer progression and therapy resistance. HER3-specific T-cell engagers might be a suitable treatment option to circumvent the limited efficacy observed for HER3-blocking antibodies in clinical trials. In this study, we developed bispecific antibodies for T-cell retargeting to HER3-expressing tumor cells, utilizing either a single-chain diabody format (scDb) with one binding site for HER3 and one for CD3 on T-cells or a trivalent bispecific scDb-scFv fusion protein exhibiting an additional binding site for HER3. The scDb-scFv showed increased binding to HER3-expressing cancer cell lines compared to the scDb and consequently more effective T-cell activation and T-cell proliferation. Furthermore, the bivalent binding mode of the scDb-scFv for HER3 translated into more potent T-cell mediated cancer cell killing, and allowed to discriminate between moderate and low HER3-expressing target cells. Thus, our study demonstrated the applicability of HER3 for T-cell retargeting with bispecific antibodies, even at moderate expression levels, and the increased potency of an avidity-mediated specificity gain, potentially resulting in a wider safety window of bispecific T-cell engaging antibodies targeting HER3.

scholarly journals CD18 Antibody Application Blocks Unwanted Off-Target T Cell Activation Caused by Bispecific Antibodies

Cancers ◽  
2021 ◽  
Vol 13 (18) ◽  
pp. 4596
Author(s):  
Joseph Kauer ◽  
Fabian Vogt ◽  
Ilona Hagelstein ◽  
Sebastian Hörner ◽  
Melanie Märklin ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Lymphoid Cells ◽  
Bispecific Antibodies ◽  
Target Cells ◽  
Cytokine Release ◽  
Life Threatening ◽  
Blocking Antibodies

T cell-recruiting bispecific antibodies (bsAbs) are successfully used for the treatment of cancer. However, effective treatment with bsAbs is so far hampered by severe side effects, i.e., potentially life-threatening cytokine release syndrome. Off-target T cell activation due to binding of bispecific CD3 antibodies to T cells in the absence of target cells may contribute to excessive cytokine release. We report here, in an in vitro setting, that off-target T cell activation is induced by bsAbs with high CD3 binding affinity and increased by endothelial- or lymphoid cells that act as stimulating bystander cells. Blocking antibodies directed against the adhesion molecules CD18/CD54 or CD2/CD58 markedly reduced this type of off-target T cell activation. CD18 blockade—in contrast to CD2—did not affect the therapeutic activity of various bsAbs. Since CD18 antibodies have been shown to be safely applicable in patients, blockade of this integrin holds promise as a potential target for the prevention of unwanted off-target T cell activation and allows the application of truly effective bsAb doses.

Activation of T Cells by Bispecific Single-Chain Construct MT103 (MEDI-538) Is Strictly Target Cell-Dependent.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3889-3889
Author(s):  
Klaus Brischwein ◽  
Scott A. Hammond ◽  
Larissa Parr ◽  
Schlereth Bernd ◽  
Mathias Locher ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Target Cell ◽  
Cell Activation ◽  
Cell Lysis ◽  
Target Cells ◽  
Single Chain ◽  
Activation Markers ◽  
Human T Cells

Abstract Background: Bispecific antibodies have been extensively studied in vitro and in vivo for their use in redirected tumor cell lysis. A particular challenge of bispecific antibody constructs recognizing the CD3 signaling complex is to achieve a controlled polyclonal activation of T-cells that, ideally, is entirely dependent on the presence of target cells. If this is not the case, systemic production of inflammatory cytokines and secondary endothelial reactions may occur as side effects, as are observed with the murine anti-human CD3e antibody OKT-3 (muromab, Orthoclone®). Here we present evidence that MT103 (or MEDI-538), a bispecific single chain antibody of the BiTE class that targets CD19 and CD3, induces T-cell activation exclusively in the presence of target cells. Material and methods: Peripheral blood mononuclear cells from healthy donors were prepared by Ficoll density centrifugation. PBMC were incubated for 24 hours with MT103 in presence or absence of specific target cells. Target cell lysis was determined by measurement of adenylate kinase activity released from lysed cells. De novo expression of activation markers CD69 and CD25 on T-cells was assessed by flow cytometry using directly conjugated monoclonal antibodies, and the concentration of cytokines in the supernatant was determined by a commercial FACS-based bead array. Results: MT103 was analyzed for conditional T-cell activation. In the presence of target-expressing cell lines, low picomolar concentrations of MT103 were sufficient to stimulate a high percentage of peripheral human T-cells to express cytokines and surface activation markers, to enter into the cell cycle and to induce redirected lysis of target cells. However, in the absence of target cells, the BiTE molecules no longer detectably activated human T-cells even at concentrations exceeding the ED50 for redirected lysis and conditional T-cell activation by more than five orders of magnitude. Conclusion: Our data show that T-cell activation by MT103 is highly conditional in that it is strictly dependent on the presence.

scholarly journals P07.01 A modular and controllable T cell therapy platform for AML

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A22.2-A23
Author(s):  
M Benmebarek ◽  
B Loureiro Cadilha ◽  
M Herrmann ◽  
S Schmitt ◽  
S Lesch ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
T Cell Activation ◽  
Tumour Cell ◽  
Cell Activation ◽  
Cell Lysis ◽  
Target Cells ◽  
Single Chain

BackgroundTargeted immunotherapies have shown limited success in the context of acute myeloid leukemia (AML). The mutational landscape, heterogeneity attributed to this malignancy and toxicities associated with the targeting of myeloid lineage antigens, it has become apparent that a modular and controllable cell therapy approach with the potential to target multiple antigens is required. We propose a controlled ACT approach, where T cells are equipped with synthetic agonistic receptors (SARs) that are selectively activated only in the presence of a target AML-associated antigen, and a cross-linking tandem single chain variable fragment (taFv) specific for both (SAR) T cell and tumour cell.Materials and MethodsA SAR composed of an extracellular EGFRvIII, trans- membrane CD28, and intracellular CD28 and CD3z domains was fused via overlap- extension PCR cloning. T cells were retrovirally transduced to stably express our SAR construct. SAR-specific taFvs that target AML-associated antigens were designed and expressed in Expi293FTM cells and purified by nickel affinity and size exclusion chromatography (SEC). We validated our approach in three human cancer models and patient-derived AML blasts expressing our AML-associated target antigens CD33 and CD123.ResultsAnti-CD33-EGFRvIII and anti-CD123 EGFRvIII taFv, monovalently selective for our SAR, induced conditional antigen-dependent activation, proliferation and differentiation of SAR-T cells. Further, SAR T cells bridged to their target cells by taFv could form functional immunological synapses, resulting in efficient tumor cell lysis with specificity towards CD33-expressing AML cells. SAR-taFv combination could also mediate specific cytotoxicity against patient-derived AML blasts and leukemic stem cells whilst driving SAR T cell activation. In vivo, treatment with SAR-taFv combination could efficiently eradicate leukemia and enhance survival in an AML xenograft models. Furthermore, we could show selective activation of SAR T cells, as well as a controllable reversibility and modularity of said activation upon depletion of the T cell engaging molecule, both in vitro and in vivo.ConclusionsHere we apply the SAR-taFv platform in efforts to deliver specific and conditional activation of SAR-transduced T cells, and targeted tumour cell lysis. The modularity of our platform will allow for a multi-targeting ACT approach with the potential to translate the ACT successes of B cell malignancies to AML. With a lack of truly specific AML antigens, it is invaluable that this approach possesses an intrinsic safety switch via its taFv facet. Moreover, we are able to circumvent pan-T cell activation due to the specific targeting and activation of SAR T cells.Disclosure InformationM. Benmebarek: None. B. Loureiro Cadilha: None. M. Herrmann: None. S. Schmitt: None. S. Lesch: None. S. Stoiber: None. A. Darwich: None. C. Augsberger: None. B. Brauchle: None. M. Schwerdtfeger: None. A. Gottschlich: None. A. Gottschlich Rataj: None. N.C. Fenn: None. C. Klein: None. M. Subklewe: None. S. Endres: None. K. Hopfner: None. S. Kobold: None.

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.

scholarly journals 697 Tumor-targeted CD28 costimulatory bispecific antibodies enhance T cell activation in solid tumors

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A739-A739
Author(s):  
Michael Hedvat ◽  
Veronica Zeng ◽  
Juan Diaz ◽  
Christine Bonzon ◽  
Kendra Avery ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Cancer Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Bispecific Antibodies ◽  
Anti Tumor Activity

BackgroundT cells in the tumor micro-environment require TCR/MHC engagement and co-stimulatory receptor engagement to achieve complete activation. Solid tumors often lack expression of CD28 ligands, so we hypothesized that activation of CD28 signaling could be beneficial in solid tumors. We designed tumor-associated-antigen (TAA) x CD28 bispecific antibodies that conditionally costimulate CD28 only in the presence of TAA and TCR engagement. Clinical application of this class of antibodies has potential to enhance activity of either anti-PD(L)1 antibodies or TAA x CD3 T cell engagers.MethodsWe designed a stability and affinity optimized anti-CD28 antibody that can be paired with TAA of choice to engage CD28 monovalently using Xencor’s XmAb 2+1 and 1+1 platforms. In vitro T cell activation with these bispecifics was measured by T cell proliferation, cytokine production, and cytotoxicity, in co-cultures of human cancer cell lines mixed with primary human CD3-stimulated T cells. In vitro activity was validated in a CMV recall assay measuring CMV+ T cell proliferation of CMV+ PBMC co-cultured with cancer cell lines ectopically treated with pp65-derived NLV-peptide. In vivo anti-tumor and T cell proliferative activity of B7H3 x CD28 bispecific antibodies were determined in tumor-bearing huPBMC-NSG mice treated simultaneously with TAA x CD3 bispecific antibody. In vivo activity of PDL1 x CD28 antibodies was determined with hCD28 KI mice inoculated with MC38 tumors expressing hPDL1-antigen. Finally, safety and tolerability of B7H3 x CD28 and PDL1 x CD28 was determined in cynomolgus monkeys.ResultsB7H3 x CD28 and PDL1 x CD28 antibodies enhanced T cell degranulation, cytokine secretion, and cancer cell cytotoxicity in concert with CD3 stimulation only in the presence of target antigen. B7H3 x CD28, alone or in combination with anti-PD1 antibody, enhanced proliferation of CMV+ T cells recognizing cancer cells loaded with pp65-derived NLV peptide. PDL1 x CD28 also enhanced CMV+ cell expansion but did not synergize with anti-PD1 antibody treatment. B7H3 x CD28 significantly enhanced in vivo anti-tumor activity of TAA x CD3 antibodies while also promoting greater T cell expansion. In hCD28 mice inoculated with MC38 tumors expressing hPDL1, PDL1 x CD28 antibody inhibited tumor growth greater than an anti-PDL1 antibody alone. B7H3 x CD28 and PDL1 x CD28 were well tolerated in cynomolgus monkeys.ConclusionsB7H3 x CD28 and PDL1 x CD28 bispecific antibodies show promising anti-tumor activity and warrant further development.

scholarly journals Fc-comprising scDb-based trivalent, bispecific T-cell engagers for selective killing of HER3-expressing cancer cells independent of cytokine release

2021 ◽  
Vol 9 (11) ◽  
pp. e003616
Author(s):  
Nadine Aschmoneit ◽  
Lennart Kühl ◽  
Oliver Seifert ◽  
Roland E Kontermann
Keyword(s):  
T Cell ◽  
Cancer Cells ◽  
T Cell Activation ◽  
Target Cell ◽  
Cell Activation ◽  
Cell Killing ◽  
Bispecific Antibodies ◽  
Target Cells ◽  
Cytokine Release ◽  
Her3 Expression

BackgroundBispecific T-cell engagers are an established therapeutic strategy for the treatment of hematologic malignancies but face several challenges when it comes to their application for the treatment of solid tumors, including on-target off-tumor adverse events. Employing an avidity-mediated specificity gain by introducing an additional binding moiety for the tumor-associated antigen can be achieved using formats with a 2+1 stoichiometry.MethodsBesides biochemical characterization and validation of target cell binding to cancer cells with different HER3 expression, we used in vitro co-culture assays with human peripheral blood mononuclear cells (PBMCs) and HER3-expressing target cells to determine T-cell activation, T-cell proliferation and PBMC-mediated cancer cell lysis of HER3-positive cell lines by the trivalent, bispecific antibodies.ResultsIn this study, we developed trivalent, bispecific antibodies comprising a silenced Fc region for T-cell retargeting to HER3-expressing tumor cells, combining a bivalent single-chain diabody (scDb) fused to a first heterodimerizing Fc chain with either an Fab or scFv fused to a second heterodimerizing Fc chain. All these HER3-targeting T-cell engagers comprising two binding sites for HER3 and one binding site for CD3 mediated target cell killing. However, format and orientation of binding sites influenced efficacy of target cell binding, target cell-dependent T-cell activation and T-cell-mediated target cell killing. Beneficial effects were seen when the CD3 binding site was located in the scDb moiety. These molecules showed efficient killing of medium HER3-expressing cancer cells with very low induction of cytokine release, while sparing target cells with low or undetectable HER3 expression.ConclusionOur study demonstrates that these trivalent, bispecific antibodies represent formats with superior interdomain spacing resulting in efficient target cell killing and a potential advantageous safety profile due to very low cytokine release.

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.

T-cell activation and B-cell depletion in chimpanzees treated with a bispecific anti-CD19/anti-CD3 single-chain antibody construct

2005 ◽  
Vol 55 (5) ◽  
pp. 503-514 ◽  
Author(s):  
Bernd Schlereth ◽  
Cornelia Quadt ◽  
Torsten Dreier ◽  
Peter Kufer ◽  
Grit Lorenczewski ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Depletion ◽  
B Cell Depletion ◽  
Single Chain ◽  
Single Chain Antibody

scholarly journals Brucella Peptide Cross-Reactive MHC I Presentation Activates SIINFEKL-Specific TCR Expressing T Cells

2018 ◽  
Author(s):  
Jerome S. Harms ◽  
Mike Khan ◽  
Cherisse Hall ◽  
Gary A. Splitter ◽  
E. Jane Homan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Pathogenic Bacteria ◽  
Cell Activation ◽  
Immune Surveillance ◽  
Cross Reactivity ◽  
Near Neighbor ◽  
Target Cells ◽  
Mhc I

ABSTRACTBrucella spp are intracellular pathogenic bacteria remarkable in their ability to escape immune surveillance and therefore inflict a state of chronic disease within the host. To enable further immune response studies, Brucella were engineered to express the well characterized chicken ovalbumin (OVA). Surprisingly, we found that CD8 T cells bearing T cell receptors (TCR) nominally specific for the OVA peptide SIINFEKL (OT-1) reacted to parental Brucella-infected targets as well as OVA-expressing Brucella variants in cytotoxicity assays. Furthermore, splenocytes from Brucella immunized mice produced IFN-γ and exhibited cytotoxicity in response to SIINFEKL-pulsed target cells. To determine if the SIINFEKL-reactive OT-1 TCR could be cross-reacting to Brucella peptides, we searched the Brucella proteome using an algorithm to generate a list of near-neighbor nonamer peptides that would bind to H2Kb. Selecting five Brucella peptide candidates, along with controls, we verified that several of these peptides mimicked SIINFEKL resulting in T cell activation through the “SIINFEKL-specific” TCR. Activation was dependent on peptide concentration as well as sequence. Our results underscore the complexity and ubiquity of cross-reactivity in T cell recognition. This cross-reactivity may enable microbes such as Brucella to escape immune surveillance by presenting peptides similar to the host, and may also lead to the activation of autoreactive T cells.

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