anti-CD3 Fab Research Articles

Abstract LB128: A novel tri-specific T cell engager targeting BCMA and GPRC5D for treatment of multiple myeloma

Abstract Introduction Multiple myeloma (MM) accounts for 10% of all hematologic cancers. Recent advances in MM therapy have greatly increased the overall response and survival rate. However, almost all patients eventually relapse. The prognosis still remains poor. BCMA and GPRC5D are overexpressed in myeloma cells. Although CAR-T and T cell engager (TCE) targeting BCMA or GPRC5D have been efficacious in MM patients, resistance does occur. Since the expression of BCMA and GPRC5D in MM are heterogeneous, to further improve the overall response and survival, we have recently generated a novel tri-specific T-cell engager, GBD218, targeting both BCMA and GPRC5D. GBD218 has demonstrated potent in vitro and in vivo activity against myeloma cells. Methods Anti-BCMA and GPRC5D nanobodies were screened from alpaca immune libraries, and anti-CD3 antibody was engineered from mouse hybridoma clone. The tri-specific antibodies were constructed in a “1+1+1” format through “knob into hole” technology fused with silenced IgG1 Fc. The format of the tri-specific antibodies was optimized by multiple rounds of in vitro activity and druggability evaluation. The in vivo tumor growth inhibition effects were evaluated in PBMC-humanized xenograft mouse models. Results GBD218 has been designed to potently bind hBCMA (KD=0.4nM) and hGPRC5D (cell binding EC50 ~ 2nM). To reduce CRS and other potential AEs associated with TCEs, a low affinity of anti-CD3 Fab was used. In cell-based functional assays, GBD218 showed efficient cytotoxicity against single and double positive MM cell lines with various expression levels of BCMA and GPRC5D. T cell activation and cytokine release induced by GBD218, in the presence or absence of MM cancer cells, is nicely balanced for great killing efficacy and the low risk of CRS. Importantly, the results showed that GBD218 exhibited superior in vitro killing activity compared to benchmarks, including teclistamab, talquetamab, the combination of teclistamab and talquetamab, suggesting a synergistic effect of GBD218 by targeting BCMA and GPRC5D. In xenograft models, GBD218 showed excellent anti-tumor activity, indicating great potential for GBD218 as a promising therapeutics for MM. Conclusion GBD218 is a novel tri-specific antibody that showed potent in vitro and in vivo anti-tumor activity. GBD218 efficiently kills both BCMA and/or GPRC5D expressing MM cells, which may hold promise to increase response rate and improve survival in MM patients in clinic. Citation Format: Yongcong Tan, Xuezhen Li, Fan Yu, Juehua Xu, Zhen Qian, Yiqi Cao, Xueyan Yang, Qinglin Du, Fei Peng, Shuhua Han, Qian Ding. A novel tri-specific T cell engager targeting BCMA and GPRC5D for treatment of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB128.

Abstract LB442: Novel antibodies against a KK-LC-1-derived peptide presented on HLA-A*01 on tumor cells

Abstract The prognostic benefit of immune checkpoint inhibitors (CPIs) is mainly restricted to specific cancers, such as melanoma and lung adenocarcinoma. For patients with cancers which are noninflamed (“cold”), or CPI-resistant, therapeutic options are currently limited. Recently, the US FDA approved the first bispecific molecule targeting a tumor peptide MHC. This highlights the potential of targeting MHC-presented tumor antigens to empower T cells to specifically recognize and eliminate solid tumors. While there is a growing number of agents targeting pMHCs, most of them are restricted to HLA-A*02. There is an urgent need to identify effective pMHC targeted therapies for solid tumors, especially on other alleles to increase the proportion of eligible patients. Kita-Kyushu lung cancer antigen-1 (KK-LC-1/CT83) peptide presented on HLA-A*01 is a promising tumor target as its expression is restricted to tumors and it is broadly expressed in many cancers including gastric, lung, breast, and cervical cancer. However, targeting the KK-LC-1/HLA-A*01 complex is challenging due to the high levels of similar off-target peptides presented in healthy tissues. Through in silico analysis we identified peptides within the human genome that share a high sequence identity to the target. These peptides were found in a database containing >1 million mass spectrometry (MS)-identified peptides presented by healthy tissues, cancer cell lines, and tumor tissue samples. A peptide derived from 6-pyruvoyltetrahydropterin synthase (PTS) was found to be abundantly presented in healthy tissues. MS confirmed that this peptide occurs at extremely high natural levels (620 - 3500 copies per cell) on HLA-A*01 cell lines and healthy tissue. Here, we describe a set of pMHC T-cell engager (TCE) antibodies with high specificity towards HLA-A*01 restricted KK-LC-1 epitope. ScFv phage display libraries were rigorously screened for binding to the pMHC target and counter-screened against predicted off-target peptides including PTS. Specific scFv hits were reformatted into TCEs using an anti-CD3 Fab effector arm. The TCEs showed efficient killing of KK-LC-1/HLA-A*01-positive human cell lines NCI-H1703 and EKVX in co-cultures with human PBMCs, while KK-LC-1-negative/HLA-A*01-positive cell lines SK-MEL-30 and PC-3 (known to present the risk peptides on the surface) were minimally affected. TCE-dependent T cell activation measured by IFNγ was significantly higher in presence of TAP-deficient, (empty) HLA-A*01-expressing T2 cells pulsed with the target peptide compared to the risk peptides. Alanine scans of isolated binders confirmed that at least 6 amino acids of the KK-LC-1 peptide are required for binding. Overall, the data show that these antibodies have promising anti-tumor activity and specificity for KK-LC-1/HLA-A*01 positive tumors. Future activities will focus on lead optimization for therapeutic development. Citation Format: Fabian Scheifele, Stephanie Jungmichel, Nagjie Alijaj, Athanasia Dasargyri, Romina Doerig, Philip Knobel, Hannes Merten, Philipp Richle, Thomas Schleier, Anna Sobieraj, Alessio Vantellini, Tim Fugmann, Rom Leidner, Swethajit Biswas, Leonardo Borras. Novel antibodies against a KK-LC-1-derived peptide presented on HLA-A*01 on tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB442.

CD38-Directed, Single-Chain T Cell-Engager Targets Leukemia Stem Cells through IFNγ-Induced CD38 Expression

Leukemia stem cells (LSCs), enriched in the CD34+CD38- AML population, are considered the main drivers of relapse in acute myeloid leukemia (AML). While more differentiated progenitors and bulk AML blasts reside in the CD34+CD38+ fraction, targeting CD38 in AML is less pursued, as LSCs are mainly CD38-. We show IFNγ (10 ng/ml) induced an increase in CD38 mRNA (mean fold change IFNγ over vehicle = 7.7, p=0.03, n=3) and surface CD38 (mean CD38 High% population IFNγ 56%±17 vs vehicle 16%±10, p<0.0001) in both CD34+ and CD34- AML primary blasts, and decreased the clonogenic activities of primary AML cells (colony forming unit IFNγ 50±36 vs vehicle 216±117, p=0.002, n=4) regardless of cytogenetics. scRNA and total RNA sequencing data in CD34+ AML primary blasts showed that upon IFNγ treatment, CD38 and Interferon Regulatory Factor 1 (IRF-1) were consistently the most upregulated genes compared to vehicle (>2.5 Log 2 Fold Change; p=0; FDR=0). Furthermore, gene set enrichment analysis (FDR=0) indicated IFNγ induced an upregulation of genes involved in apoptosis and DNA repair, and a suppression of genes in the pro-oncogenic Wnt beta catenin pathways. CD38 luciferase-based reporter assays, chromatin immunoprecipitation, and IRF-1 knockdown experiments in AML cells showed that, upon IFNγ treatment, IRF1 is pivotal in CD38 transcriptional regulation. Because CD4+T helper type 1 and CD8+ cytotoxic T cells release IFNγ once engaged on their target, we hypothesized a CD38-directed T cell engager will convert CD34+CD38- LSC into CD34+CD38+ blasts via the secreted IFNγ to kill LSCs and blasts. We created a single chain construct (BN-CD38), inserting a CD38 nanobody between the light and heavy chains of an anti-CD3 Fab using two short peptide linkers. Based on modeling, BN-CD38 reproduces the distance between T cell receptor and the major histocompatibility complex, mimicking the natural immunological synapse. Surface plasmon resonance and cytometry studies showed BN-CD38 had strong binding affinity to both CD38 (K D 4.77xE -10 M) and CD3 (K D 4.27xE -8 M). The IC 50 for BN-CD38 at 1:1 E:T at 24hrs in different CD38+ AML cell lines (n=6) was within the 0.01 to 0.1 ng/ml range, whereas the IC 50 of a non-binding CD38 construct (BN-CD38Mut) was 100-fold higher. By 24hrs of treatment, we noted >60% CD4+ and CD8+ T cell activation (CD69+, CD25+, IFNγ+) with BN-CD38 (e.g., CD69: 60%±27) in contrast to controls (e.g., CD69: IgG 7%±0.8 and BN-CD38Mut 11%±10, p<0.0001). In primary AML samples (n=10) BN-CD38 activated >70% T cells against autologous leukemia cells (CD69: BN-CD38 73%±16, BN-CD38Mut 10%±9, IgG 11%±10, p<0.0001). CyTOF analysis showed BN-CD38 but not controls significantly reduced CD34+CD38+ AML blasts (%change to IgG range: BN-CD38 -27 to -82% vs BN-CD38Mut 1% to 7%, p = 0.01) and CD34+CD38- LSCs (%change to IgG range: BN-CD38 -78 to -99% vs BN-CD38Mut -7 to 11%, p= 0.0003) and expanded CD8+ effector memory (p<0.01), CD8+ terminally differentiated effector memory CD45RA+ (p<0.05), NKT cells (p<0.01), and central memory Tregs (p<0.0001). Anti-human-IFNγ neutralizing antibody reverted BN-CD38 induced expression of CD38 and BN-CD38 killing activity on CD34+CD38- AML cells (p<0.05). BN-CD38 (2.5 mg/kg, i.v, n=6/group) co-injected weekly with healthy 3x10 6 T cells induced near-complete disease eradication in mice xenografted with luciferase-CD38+ THP1 (p=0.003) and U937 (p=0.004) cells. Long-term survival analysis showed 38% of BN-CD38-treated THP-1 engrafted mice achieved complete cure compared to IgG (OS median: BN-CD38 61.5d, control IgG 29d, p=0.0005). Consistently, BN-CD38 suppressed AML cell engraftment, splenomegaly, and prolonged survival in three patient-derived xenografts (PDX) with complex karyotypes including TP53 mutation (PDX1: OS median BN-CD38 undefined, BN-CD38Mut 28d (p=0.002), and IgG 27d (p=0.002); PDX2: OS median BN-CD38 39d, BN-CD38Mut and control IgG 32d (p<0.01); PDX3: OS median BN-CD38 undefined, BN-CD38Mut 92d (p=0.001), and IgG 81d (p=0.0007)). BN-CD38 did not affect normal hematopoietic stem cell clonogenicity and the development of multi-lineage human immune cells in CD34+ humanized mice. In sum, BN-CD38 induces an effective T cell synapse and IFNγ release, while concomitantly blocking the clonogenicity and inducing CD38 expression on LSCs. These studies suggest a new mechanism to unmask and target LSCs, providing the rationale for using BN-CD38 to effectively treat AML.

Targeting NKG2DL with Bispecific NKG2D-CD16 and NKG2D-CD3 Fusion Proteins on Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a particularly aggressive subtype of breast cancer with a poor response rate to conventional systemic treatment and high relapse rates. Members of the natural killer group 2D ligand (NKG2DL) family are expressed on cancer cells but are typically absent from healthy tissues; thus, they are promising tumor antigens for novel immunotherapeutic approaches. We developed bispecific fusion proteins (BFPs) consisting of the NKG2D receptor domain targeting multiple NKG2DLs, fused to either anti-CD3 (NKG2D-CD3) or anti-CD16 (NKG2D-CD16) Fab fragments. First, we characterized the expression of the NKG2DLs (MICA, MICB, ULBP1-4) on TNBC cell lines and observed the highest surface expression for MICA and ULBP2. Targeting TNBC cells with NKG2D-CD3/CD16 efficiently activated both NK and T cells, leading to their degranulation and cytokine release and lysis of TNBC cells. Furthermore, PBMCs from TNBC patients currently undergoing chemotherapy showed significantly higher NK and T cell activation and tumor cell lysis when stimulated with NKG2D-CD3/CD16. In conclusions, BFPs activate and direct the NK and T cells of healthy and TNBC patients against TNBC cells, leading to efficient eradication of tumor cells. Therefore, NKG2D-based NK and T cell engagers could be a valuable addition to the treatment options for TNBC patients.

Varied T cell repertoire bearing intact TCR/CD3 complexes required to observe maximal therapeutic anti-tumor effects of CD3 Fab fragments when binding to T cells

Abstract A cognate antigen engagement of TCR induces a conformation change in the CD3 complex (CD3Δc) that is required for productive T cell activation. CD3Δc fails when TCRs interact with poorly immunogenic antigens, like many tumor-associated-antigens (TAAs), which do not active T cells. Anti-CD3 monovalent Fab fragments (Mono-Fabs) bound to the CD3 complex mimic CD3Δc, producing T cell “co-potentiation” when associated with TCRs bound to weak antigens that enhances T cell activation, neither stimulating non-engaged T cells, nor interrupting T cell responses to strong antigens. Previously, we showed T cell co-potentiation can be exploited as a novel immunotherapeutic principle by reducing melanoma burden in B6 mice treated with anti-CD3 Mono-Fabs. TCR signaling initiation requires CD3Δc for the exposure of a conserved sequence in the cytosolic domain of CD3ɛ rich in prolines (PRS) that enables the recruitment of adaptor protein Nck. The mutation of cysteines in the CXXC motif of CD3ɛ extracellular domain inhibits the transmission of TCR signaling across transmembrane that defects CD3Δc and PRS accessibility. Here, using the B6 syngeneic melanoma cell lines Yumm 1.7 and Yummer1.7, which differ in the number of somatic mutations they carry, in B6 mouse strains either wild or mutated in either the CXXC or the PRS motifs in CD3ɛ, we have established the need of tumor cells to carry potential tumor neoantigens, and a more varied repertoire of T cells responding to the tumor with intact TCR/CD3 complexes to observe full anti-tumor effects by anti-CD3 Mono-Fab treatments. MU Start-up funds NIH, NCI, U01 CA244314 NIH, NIAID, R01 AI097187

Abstract 2891: Enhanced anti-tumor responses with a novel dual pMHC T-cell engager bispecific antibody

Abstract Intracellular tumor antigens presented as peptides on MHC (pMHC) class I molecules are attractive targets for more tumor-selective immunotherapeutic approaches with promising data already emerging from clinical trials. pMHCs have been targeted by TCR-engineered T cells or soluble recombinant T-cell receptors (TCRs) fused to an anti-CD3 fragment. Naturally occurring cancer-reactive TCRs have weak affinity and require substantial affinity enhancements for their cognate pMHC. However, the outcome of this process is difficult to predict and bears the risk for off-target cross reactivities in normal tissues, which may lead to severe adverse events in the clinic. Here, we describe a highly potent dual pMHC T-cell engager (TCE) antibody format with high specificity towards tumor-specific pMHCs utilizing an HLA-A*02:01 restricted MAGE-A4 epitope to validate the concept. A series of monovalent and bivalent antibody constructs composed of anti-MAGE-A4 binding arms, ranging in affinities from 30 nM to 100 pM, were fused to an anti-CD3 Fab fragment with lower affinity compared to that commonly used for TCR-fusions. The different antibody constructs were evaluated for selective killing of MAGE-A4/HLA-A*02 positive human U2OS osteosarcoma and A375 melanoma cancer cells versus a panel of different MAGE-A4-negative/HLA-A*02-positive human cell lines. Bivalent bispecific antibody variants mediated a 7-fold greater degree of cancer cell killing and similarly increased T cell activation compared to their monovalent bispecific counterparts. EC50 values ranged as low as single digit picomolar, while the overall cross reactivity against MAGE-A4-negative/HLA-A*02-positive cells was not substantially affected. These results prove that dual targeting of pMHCs on cancer cells provides selective and efficient T cell-mediated target cell killing and T cell activation, even at very low levels of pMHC on the cell surface, highlighting the pivotal roles played by the affinity of the individual arms, valency, and epitope densities. We further analyzed the dual pMHC TCE for potential off-target effects by recognition of similar and physiologically relevant non-MAGE-A4 peptides. T2 cells pulsed with similar peptides and co-cultured with PBMC effector cells showed no significant T cell activation or IFNg release in the presence of the dual pMHC TCE in comparison to MAGE-A4 peptide-pulsed T2 cells. Finally, we compared the dual pMHC TCE against a recombinant TCR fused to an anti-CD3 scFv, a construct that is currently in clinical development. Interestingly, the dual pMHC TCE resulted in a 3-fold more potent cancer cell killing while having significantly lower effect on cytokine production. In conclusion, dual pMHC targeting with TCE antibodies is an attractive alternative to soluble affinity-enhanced TCR-based cancer immunotherapies as they facilitate potent tumor targeting without the need for extensive affinity enhancements. Citation Format: Stephanie Jungmichel, Fabian Scheifele, Anna Sobieraj, Severin Wendelspiess, Thomas Schleier, Philip Knobel, Camilla Winnewisser, Melissa Vrohlings, Philipp Richle, Hannes Merten, Jacqueline Blunschi, Christian Leisner, Leonardo Borras. Enhanced anti-tumor responses with a novel dual pMHC T-cell engager bispecific antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2891.

Dissecting molecular mechanisms underlying T cell co-potentiation when targeting the TCR/CD3 complex with anti-CD3 Fab fragments

Abstract A cognate antigen engagement of TCR induces a conformational change in the CD3 complex (CD3Δc) that is required for productive T cell activation. CD3Δc fails when TCRs interact with poorly immunogenic antigens that do not active T cells. Anti-CD3 monovalent Fab fragments bound to the CD3 complex mimic CD3Δc, producing T cell “co-potentiation” when associated with TCRs bound to weak antigens that enhances T cell activation, neither stimulating non-engaged T cells, nor interrupting T cell responses to strong antigens. Previously, we showed T cell co-potentiation can be exploited as a novel immunotherapeutic principle by reducing melanoma burden in B6 mice treated with anti-CD3 Fabs. TCR signaling initiation requires CD3Δc for the exposure of a conserved sequence in the cytosolic domain of CD3ɛ rich in prolines (PRS) that enables the recruitment of adaptor protein Nck. The mutation of cysteines in the CXXC motif at the extracellular domain of CD3ɛ inhibits the conformational change across the plasma membrane that results on PRS accessibility. Here, using two strains of B6 mice that express mutations in either the CXXC or the PRS motifs in CD3ɛ, we study the potential contribution of each motif in T cell co-potentiation. Our results from these studies favor the involvement of the CXXC and PRS motifs in the molecular mechanism supporting the co-potentiation of T cell activation by anti-CD3 Fab fragments. Supported by MU Start-up funds and NIH grants: NCI, U01 CA244314; NIAID, R01 AI097187

Surface Ligand Valency and Immunoliposome Binding: when More Is Not Always Better.

Nano-drug delivery systems are designed to contain surface ligands including antibodies for "active targeting". The number of ligands on each nanoparticle, known as the valency, is considered a critical determinant of the "targeting" property. We sought to understand the correlation between valency and binding properties using antibody conjugated liposomes, i.e. immunoliposomes (ILs), as the model. Anti-CD3 Fab containing a terminal cysteine residue were conjugated to DSPE-PEG-maleimide and incubated with preformed liposomes at 60°C. The un-incorporated antibodies were removed and the obtained ILs were characterized to contain in average 2-22 copies of anti-CD3 Fabs per liposome. The Biolayer Interferometry (BLI) probe surface was coated with various densities of CD3 epsilon&delta heterodimer (CD3D/E) to imitate different CD3 expression levels on target cells. The inference wavelength shifts upon anti-CD3 liposome binding were monitored and analyzed. The data indicated ILs may bind either monovalently or multivalently, determined mainly by the surface ligand density rather than the ILs antibody valency. The ILs valency indeed correlated with the dissociation rate constant (Koff), but not with the association rate constant (Kon). Their binding capabilities also did not necessarily increase with the surface anti-CD3 valency. We proposed a model for understanding the binding properties of ILs with different ligand valencies. The binding mode may change when the targeted surfaces had different antigen densities. The model should be important for the designing and optimization of active targeting drug delivery systems to fit different applications.

Development of an antibody-like T-cell engager based on VH-VL heterodimer formation and its application in cancer therapy

Following the clinical success of immunotherapeutic antibodies, bispecific antibodies for cytotoxic effector cell redirection, tumor-targeted immunomodulation and dual immunomodulation, have received particular attentions. Here, we developed a novel bispecific antibody platform, termed Antibody-Like Cell Engager (ALiCE), wherein the Fc domain of each heavy chain of immunoglobulin G (IgG) is replaced by the VH and VL domains of an IgG specific to a second antigen while retaining the N-terminal Fab of the parent antibody. Because of specific interactions between the substituted VH and VL domains, the C-terminal stem Fv enables ALiCE to assemble autonomously into hetero-tetramers, thus simultaneously binding to two distinct antigens but with different avidities. This design strategy was used to generate ACE-05 (two anti-PD-L1 Fab × anti-CD3 Fv) and ACE-31 (two anti-CD3 Fab × anti-PD-L1 Fv), both of which bound PD-L1 and CD3. However, ACE-05 was more effective than ACE-31 in reducing off-target toxicity caused by the indiscriminate activation of T cells. Moreover, in cell-based assays and PBMC-reconstituted humanized mice harboring human non-small-cell lung cancer tumors, ACE-05 showed marked antitumor efficacy, causing complete tumor regression at a dose of 0.05 mg/kg body weight. The dual roles of ACE-05 in immune checkpoint inhibition and T-cell redirection, coupled with reduced off-target toxicity, suggest that ACE-05 may be a promising anti-cancer therapeutic agent. Moreover, the bispecific ALiCE platform can be further used for tumor-targeted or multiple immunomodulation applications.

Public and private human T-cell clones respond differentially to HCMV antigen when boosted by CD3 copotentiation

AbstractHuman cytomegalovirus (HCMV) induces long-lasting T-cell immune responses that control but do not clear infection. Typical responses involve private T-cell clones, expressing T-cell antigen receptors (TCRs) unique to a person, and public T-cell clones with identical TCRs active in different people. Here, we report the development of a pretherapeutic immunostimulation modality against HCMV for human T cells, CD3 copotentiation, and the clonal analysis of its effects in recall assays at single-cell resolution. CD3 copotentiation of human T cells required identification of an intrinsically inert anti-CD3 Fab fragment that conditionally augmented signaling only when TCR was coengaged with antigen. When applied in recall assays, CD3 copotentiation enhanced the expansion of both public and private T-cell clones responding to autologous HLA-A2(+) antigen-presenting cells and immunodominant NLVPMVATV (NLV) peptide from HCMV pp65 protein. Interestingly, public vs private TCR expression was associated with distinct clonal expansion signatures in response to recall stimulus. This implied that besides possible differences in their generation and selection in an immune response, public and private T cells may respond differently to pharmacoimmunomodulation. Furthermore, a third clonal expansion profile was observed upon CD3 copotentiation of T-cell clones from HLA-A2(−) donors and 1 HLA-A2(+) presumed-uninfected donor, where NLV was of low intrinsic potency. We conclude that human T-cell copotentiation can increase the expansion of different classes of T-cell clones responding to recall antigens of different strengths, and this may be exploitable for therapeutic development against chronic, persistent infections such as HCMV.

Alternative Approaches to Oral Tolerance Induction to Factor FVIII

A serious complication in the treatment X-linked bleeding disorder hemophilia A is the formation of inhibitory antibodies against factor VIII (FVIII), which compromise traditional replacement therapy. We previously developed an Oral immunotherapy (OTI) based on repeated uptake of a mixture of lettuce plant cells transgenic for heavy chain (HC) or C2 domain of human FVIII fused to cholera toxin B (CTB) subunit [Blood 124:1659; Plant Biotechnol J. 16:1148]. Fusion proteins were transgenically expressed in the chloroplasts. Repeated oral uptake of a mixture of freeze-dried powder of lettuce cells accomplished antigen delivery to the immune system of the small intestine by targeting of the GM1 receptor that is highly expressed on the surface of the gut epithelium, resulting in induction of regulatory T cells (Treg) that suppress inhibitor formation upon subsequent intravenous (iv) FVIII replacement therapy. An alternative to oral antigen delivery is the oral delivery of immune modulatory antibodies. Here, we compared the plant cell-based method with oral delivery of anti-CD3, which has been successful in murine models of autoimmune disease and is currently evaluated in clinical trials. Unlike in iv administration, oral anti-CD3 does not systemically deplete T cells. Hemophilia A BALB/c mice (F8 e16 gene deletion) received oral gavage of a mixture of CTB-FVIII-HC/-C2 (1.5 µg/antigen) expressing lettuce leaf cells 2x/week for 9 weeks. Starting at 4 weeks into the experiment, 1 IU/mouse of BDD-FVIII (Xyntha) was given iv, once per week for 5 weeks. Alternatively, following a published protocol that was successful in other models, 5 µg of hamster anti-murine CD3 was given by oral gavage daily for 5 straight days, followed by 5 weekly iv injections of BDD-FVIII. Control animals (no OTI) developed inhibitors with an average titer of 18 ± 3 BU/ml (n=16). Of these, 88% were high-titer (i.e >5 BU/ml, up to 43 BU/ml). Inhibitor formation was significantly reduced in plant cell-treated mice (10 ± 2.5 BU/ml, n=17), with 47% showing no or low-titer inhibitors (<5 BU/ml). Interestingly, inhibitor formation in anti-CD3 treated mice showed a unique pattern, with two distinct outcomes. One subset of animals developed inhibitors of 0-15 BU/ml (on average 2.5-fold lower than control group), while the second subset showed increased titers of 41-89 BU/ml. There were no animals with intermediate titers. In general, FVIII-specific IgG titers followed the same patterns as inhibitor responses. When the 2 approaches (plant cells and anti-CD3) were combined, 50% of mice developed <5 BU/ml (as compared to 36% for anti-CD3 only). However, this did not represent an improvement over giving lettuce cells only. Finally, we tested anti-CD3 Fab fragment in place of FL anti-CD3. While a daily dose of 5 µg anti-CD3 Fab/mouse was unsuccessful in suppressing inhibitor formation, a dose of 0.5 µg marginally decrease titers. In summary, oral anti-CD3 delivery was not as effective in reducing inhibitor formation as plant cells expressing CTB-FVIII antigen fusions. While FL anti-CD3 showed promise in a subset of animals, it also posed a risk of an increased response in others, which complicates development of this approach. Distinct from all other treatment groups, lettuce-fed mice showed elevated frequencies in Foxp3+ Treg, a reduction in Teff cells, and the highest increase in LAP+ Treg. Further studies on induced CD4+LAP+FoxP3- Treg cells from lettuce-fed mice showed high expression of ICOS, CD69 and Ki-67, but not CTLA-4. Among the different subsets of Treg, LAP+ T cell contained the highest proportion of IL-10 expressing cells, and oral antigen delivery induced IL-10+LAP+ Treg, which are likely key to the suppression of the immune response against FVIII. Disclosures Daniell: Takeda Pharmaceuticals: Patents & Royalties. Herzog:Takeda Pharmaceuticals: Patents & Royalties.

Protease-activation using anti-idiotypic masks enables tumor specificity of a folate receptor 1-T cell bispecific antibody

T-cell bispecific antibodies (TCBs) crosslink tumor and T-cells to induce tumor cell killing. While TCBs are very potent, on-target off-tumor toxicity remains a challenge when selecting targets. Here, we describe a protease-activated anti-folate receptor 1 TCB (Prot-FOLR1-TCB) equipped with an anti-idiotypic anti-CD3 mask connected to the anti-CD3 Fab through a tumor protease-cleavable linker. The potency of this Prot- FOLR1-TCB is recovered following protease-cleavage of the linker releasing the anti-idiotypic anti-CD3 scFv. In vivo, the Prot-FOLR1-TCB mediates antitumor efficacy comparable to the parental FOLR1-TCB whereas a noncleavable control Prot-FOLR1-TCB is inactive. In contrast, killing of bronchial epithelial and renal cortical cells with low FOLR1 expression is prevented compared to the parental FOLR1-TCB. The findings are confirmed for mesothelin as alternative tumor antigen. Thus, masking the anti-CD3 Fab fragment with an anti-idiotypic mask and cleavage of the mask by tumor-specific proteases can be applied to enhance specificity and safety of TCBs.

Molecular mechanisms underlying T cell co-potentiation by anti-CD3 Fab fragments

Abstract Cognate antigen binding to the T cell receptor (TCR) induces a conformational change of the CD3 complex (CD3Δc), which is required but not sufficient to trigger productive signaling from the TCR/CD3. CD3Δc fails to occur when the TCR ligates poorly immunogenic antigens. In the absence of CD3Δc, T cells do not display productive immune responses. CD3Δc involves the exposure of a conserved proline-rich-sequence (PRS) in CD3ɛ cytoplasmic tail that functions as a docking site for cytoplasmic adaptor proteins like Nck, which participate in the CD3 signaling cascade. The opening of the PRS is controlled by a CXXC motif in CD3ɛ located adjacent to the plasma membrane. We previously described it is possible to mimic CD3Δc on T cells when adding Fab fragments capable to bind to the CD3 complex. When combining TCR engagement to weak antigens with binding of Fabs to the CD3 complex that not interfere with antigen binding, T cell co-potentiation occurs. In this instance, T cells become capable to display responses to poorly immunogenic stimulation. As an example of the consequences of T cell co-potentiation in vivo, we studied anti-mouse CD3 Fabs as a novel immunotherapeutic against poorly immunogenic types of cancer. Anti-CD3 Fabs reduced melanoma burden in B6 mice bearing B16-F10 tumor in lungs. Now, we have explored the molecular mechanisms supporting T cell co-potentiation in monoclonal and polyclonal mouse models. In B6 mice expressing null mutations for either the CXXC or the PRS motifs, anti-CD3 Fabs fail to induce CD3Δc, as well as to co-potentiate murine T cells in vitro and in vivo. These data suggest PRS opening by anti-CD3 Fabs may be linked to their capacity to co-potentiate T cells responses to poorly immunogenic antigens.

Bivalent anti-CD3 Fab dimers drive T cells into a highly efficient fratricide with potential clinical application

Abstract Non-covalent anti-CD3 Fab dimers (Bi-Fabs) bind to the TCR/CD3 complex and activate T cells. In the presence of proper co-stimulation, anti-CD3 Bi-Fabs promote T cell cycling and acquisition of effector functions on CD4 and CD8 T cells more robustly than other bivalent antibody species like F(ab′)2 and mAb with the same specificity. Surprisingly, the net effect of anti-CD3 Bi-Fab treatment on T cells in vitro is contraction of CD4 and CD8 T cells, via AICD. This is in contrast with the net T cell proliferation observed with soluble anti-CD3 mAbs that are stimulatory in the presence of proper co-stimulation. Bi-Fab binding to the TCR/CD3 complex, and direct cellular contact with Bi-Fab engaged T cells, are required to observe the net contraction of treated T cells. Using perforin-deficient T cells, and Fas-FasL blockade, we show this net contraction results from T cell apoptosis via Fas-FasL and CTL degranulation. Structural studies on Bi-Fabs suggest a rigid rod like structure. We hypothesize that the rigid rod allows for a potent crosslinking structure with both paratopes from the anti-CD3 Fabs capable to engage effector T cells on fratricide. In vivo, mice injected with anti-CD3 Bi-Fabs present depletion of T cells from blood, and absence of any adverse effects derived from T cell activation like cytokine storm. We have begun exploring the therapeutic potential of T cell depletion in mouse models of autoimmune disease with promising results.

Bp-Bs, a Novel T-cell Engaging Bispecific Antibody with Biparatopic Her2 Binding, Has Potent Anti-tumor Activities

Patients with Human epidermal growth factor receptor type 2 (Her2) overexpression are associated with aggressive tumor growth and poor clinical outcomes. Bispecific antibodies targeting Her2 have recently exhibited potent effects on Her2 signal inhibition. In this study, a novel biparatopic anti-Her2 bispecific antibody (Bp-Bs) was constructed by linking a single anti-CD3 Fab with two different anti-Her2 single-domain antibodies targeting non-overlapping epitopes of Her2. The Bp-Bs demonstrated strong binding on Her2-positive cells and potent cytotoxicity on Her2-positive tumor cells, even Her2-low expression cells, suggesting that biparatopic bispecific antibodies may have improved therapeutic benefits on broad Her2 patient populations.

Anti-CD3 Fab Fragments Enhance Tumor Killing by Human γδ T Cells Independent of Nck Recruitment to the γδ T Cell Antigen Receptor.

T lymphocytes expressing the γδ T cell receptor (γδ TCR) can recognize antigens expressed by tumor cells and subsequently kill these cells. γδ T cells are indeed used in cancer immunotherapy clinical trials. The anti-CD3ε antibody UCHT1 enhanced the in vitro tumor killing activity of human γδ T cells by an unknown molecular mechanism. Here, we demonstrate that Fab fragments of UCHT1, which only bind monovalently to the γδ TCR, also enhanced tumor killing by expanded human Vγ9Vδ2 γδ T cells or pan-γδ T cells of the peripheral blood. The Fab fragments induced Nck recruitment to the γδ TCR, suggesting that they stabilized the γδ TCR in an active CD3ε conformation. However, blocking the Nck-CD3ε interaction in γδ T cells using the small molecule inhibitor AX-024 neither reduced the γδ T cells’ natural nor the Fab-enhanced tumor killing activity. Likewise, Nck recruitment to CD3ε was not required for intracellular signaling, CD69 and CD25 up-regulation, or cytokine secretion by γδ T cells. Thus, the Nck-CD3ε interaction seems to be dispensable in γδ T cells.

Engineering Bifunctional Antibodies with Constant Region Fusion Architectures.

We report a method to generate bifunctional antibodies by grafting full-length proteins into constant region loops of a full-length antibody or an antigen-binding fragment (Fab). The fusion proteins retain the antigen binding activity of the parent antibody but have an additional activity associated with the protein insert. The engineered antibodies have excellent in vitro activity, physiochemical properties, and stability. Among these, a Her2 × CD3 bispecific antibody (BsAb) was constructed by inserting an anti-Her2 single-chain variable fragment (ScFv) into an anti-CD3 Fab. This bispecific antibody efficiently induces targeted cell lysis in the presence of effector cells at as low as sub-picomolar concentrations in vitro. Moreover, the Her2 × CD3 BsAb shows potent in vivo antitumor activity in mouse Her22+ and Her21+ xenograft models. These results demonstrate that insertion of a full-length protein into non-CDR loops of antibodies provides a feasible approach to generate multifunctional antibodies for therapeutic applications.

A novel bispecific antibody, S-Fab, induces potent cancer cell killing.

Bispecific antibodies that engage immune cells to kill cancer cells have been actively studied in cancer immunotherapy. In this study, we present a novel bispecific format, S-Fab, fabricated by linking a single-domain anti-carcinoembryonic antigen VHH to a conventional anti-CD3 Fab. In contrast to most bispecific antibodies, the S-Fab bispecific antibody can be efficiently expressed and purified from bacteria. The purified S-Fab is stable in serum and is able to recruit T cells to drive potent cancer cell killing. In xenograft models, the S-Fab antibody suppresses tumor growth in the presence of human immune cells. Our study suggested that the bispecific S-Fab format can be applied to a wide range of immunotherapies.

Multiformat T-cell-engaging bispecific antibodies targeting human breast cancers.

Four different formats of bispecific antibodies (bsAbs) were generated that consist of anti-Her2 IgG or Fab site-specifically conjugated to anti-CD3 Fab using the genetically encoded noncanonical amino acid. These bsAbs varied in valency or in the presence or absence of an Fc domain. Different valencies did not significantly affect antitumor efficacy, whereas the presence of an Fc domain enhanced cytotoxic activity, but triggered antigen-independent T-cell activation. We show that the bsAbs can efficiently redirect T cells to kill all Her2 expressing cancer cells, including Her2 1+ cancers, both in vitro and in rodent xenograft models. This work increases our understanding of the structural features that affect bsAb activity, and underscores the potential of bsAbs as a promising therapeutic option for breast cancer patients with low or heterogeneous Her2 expression.

Delayed Anti-CD3 Therapy Results in Depletion of Alloreactive T Cells and the Dominance of Foxp3+CD4+ Graft Infiltrating Cells

The engineered Fc-nonbinding (crystallizable fragment-nonbinding) CD3 antibody has lower mitogenicity and a precise therapeutic window for disease remission in patients with type 1 diabetes. Before anti-CD3 can be considered for use in transplantation, the most effective timing of treatment relative to transplantation needs to be elucidated. In this study anti-CD3F(ab′)2 fragments or saline were administered intravenously for 5 consecutive days (early: d1–3 or delayed: d3–7) to mice transplanted with a cardiac allograft (H2b-to-H2k; d0). Survival of allografts was prolonged in mice treated with the early protocol (MST = 48 days), but most were rejected by d100. In contrast, in mice treated with the delayed protocol allografts continued to survive long term. The delayed protocol significantly inhibited donor alloreactivity at d30 as compared to the early protocol. A marked increase in Foxp3+ T cells (50.3 ± 1.6%) infiltrating the allografts in mice treated with the delayed protocol was observed (p < 0.0001 vs. early (24.9 ± 2.1%)) at d10; a finding that was maintained in the accepted cardiac allografts at d100. We conclude that the timing of treatment with anti-CD3 therapy is critical for inducing long-term graft survival. Delaying administration effectively inhibits the alloreactivity and promotes the dominance of intragraft Foxp3+ T cells allowing long-term graft acceptance.