Target Cell Lysis Research Articles

Abstract 1645: Advancing the development of CD16A-based bispecific antibodies in humanized CD16A mouse models

Abstract Antibody-dependent cell cytotoxicity (ADCC) is an important mechanism in immunotherapy. CD16A (FcγRIIIA), a low-affinity receptor for the IgG Fc domain, facilitates ADCC and is responsible for triggering the lysis of target cells by natural killer (NK) cells. Human CD16A is closely related to mouse Fcgr4 and is expressed in NK cells and macrophages, but is absent in granulocytes. Alternatively, mouse Fcgr4 is expressed in granulocytes and macrophages and is absent in NK cells. CD16A-based bispecific antibodies targeting NK cells have been studied for cancer therapy. To evaluate the anti-tumor efficacy of CD16A-based bispecific antibody in mouse tumor models, we have successfully generated a novel humanized CD16A knock-in mouse strain (B-hCD16A Mice). Considering the differential expression patterns between human CD16A and mouse Fcgr4, the regulatory region and whole genome sequence of mouse Fcgr4 were replaced by the human CD16A gene. Human CD16A and mouse Fcgr4 protein expression was confirmed in heterozygous B-hCD16A mice by FACS and RT-PCR. Human CD16A protein was detected in NK cells and macrophages of B-hCD16A mice, but not granulocytes. When compared with wild type mice, there were no abnormalities in the populations of immune cells in B-hCD16A mice. CD16A-based bispecific antibody usually binds to CD16A on NK cells, whereas the other arm of the antibody binds to tumor antigen on cancer cells. Initial assessment of the tumorigenicity of MC38 cells were evaluated in B-hCD16A mice, and results indicated that these cancer cells were able to establish tumors in vivo. These data suggest that the B-hCD16A mouse model is an effective tool for future in vivo efficacy evaluation of therapeutic CD16A-based bispecific antibodies prior to clinical development. Citation Format: Jiahui Chang, Tao Li, Limei Ma, Xiaoxia Sun, Yuelei Shen. Advancing the development of CD16A-based bispecific antibodies in humanized CD16A mouse models [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 1645.

Abstract 2864: A CLEC12A immunocytokine with target cell-restricted IL-15 activity shows a favorable toxicity profile and high potency in AML

Abstract IL-15, often referred to as the “anti-cancer cytokine”, potently stimulates proliferation and activation of NK and T cells, but unlike its close relative IL-2, does neither promote activation-induced death of lymphocytes nor activity of immunoinhibitory Tregs. So far, short half-life, poor accumulation at the tumor site and severe toxicity upon systemic application limit IL-15 efficacy in patients. Fusion of IL-15 to antibodies directed to tumor antigens (classical immunocytokines, ICs) improves on accumulation at the tumor site and pharmacokinetics. However, since the activity of the cytokine moiety within classical ICs does not depend on antigen binding, the application of clinically effective doses is still prevented by toxicity due to unspecific immune activation. To overcome this problem, we took advantage of the unique mechanism of action of IL-15 which stimulates IL-15Rβ/γ on NK and T cells as a membrane-bound complex with IL-15Rα on monocytes and DCs (trans-presentation). We used an Fc-optimized antibody directed to CLEC12A, a surface antigen abundantly expressed on AML cells and leukemic stem cells but not on healthy stem cells. This antibody termed 33C2-SDIE was fused to an IL-15E46K mutant with abolished binding to IL-15Rα, allowing to substitute physiological trans-presentation of IL-15 by binding of the construct to its tumor-expressed target. Antigen-specific binding of the resulting modified immunocytokine (MIC12) was confirmed using multiple CLEC12A-expressing cell lines and primary AML samples from patients. Functional analysis of activation, cytokine release, and target cell lysis demonstrated that MIC proteins, in contrast to classical IC, stimulate cytotoxic lymphocytes in a highly target cell-restricted manner, allowing for the desired reduction of unspecific immune activation. At the same time, significantly superior NK cell reactivity against AML cells as compared to even the Fc-optimized antibody was observed. Both, IL-15 signaling and engagement of Fc-receptors by the optimized Fc-domain were found to be essential for optimal activity of our MIC constructs. Importantly, only treatment with MIC12 was capable to induce NK cell proliferation, which is required to overcome unfavorable target to effector ratios that prevail in overt cancer disease. In summary, our novel CLEC12A-targeting immunocytokine allows for tumor-restricted stimulation of cytotoxic lymphocytes and reduced toxicity while displaying superior anti-leukemic activity and constitutes a promising compound for the treatment of AML. Citation Format: Boris Klimovich, Leonard Anton, Yangmi Lim, Jonghwa Won, Anna Chashchina, Martin Pflügler, Latifa Zekri, Gundram Jung, Helmut R. Salih. A CLEC12A immunocytokine with target cell-restricted IL-15 activity shows a favorable toxicity profile and high potency in AML [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 2864.

Abstract 3506: CLN-619, a clinical-stage MICA/MICB-specific IgG1 antibody, restores the MICA/MICB-NKG2D axis to promote NK-mediated tumor cell lysis

Abstract Natural killer (NK) cells play a central role in the immune surveillance of cancer. NK cells express multiple inhibitory and activating surface receptors, including the activating receptor, NKG2D. Engagement of NKG2D by the stress-induced ligands MICA/MICB stimulates NK cell activation and subsequent target cell lysis. While MICA/MICB expression is restricted in normal tissues, expression is strongly induced upon viral infection, genotoxic stress or malignant transformation. In cancer patients, the tumor microenvironment (TME) facilitates evasion of immune surveillance by NK cells. One mechanism by which tumor cells can escape NKG2D-mediated lysis is via proteolytic release of MICA/MICB from the cell surface due to proteases present in the TME. High concentrations of shed MICA have been observed in serum from patients across multiple tumor types and correlate with poor survival. In mouse models, expression of MICA on tumor cells delayed tumor growth, while the absence of NKG2D accelerated tumor growth. Given the established role of the MICA/MICB-NKG2D axis in the immune surveillance of tumor cells, a therapeutic agent that effectively restores this axis is a compelling approach to harness the immune system for treating cancer. CLN-619 is a humanized anti-MICA/MICB monoclonal IgG1 antibody being developed for the treatment of multiple cancer indications. The antibody was selected based on its high-affinity binding to MICA/MICB, potent in vitro activity and robust anti-tumor activity in xenograft models. MICA/MICB is highly polymorphic, with >150 MICA and 47 MICB alleles in humans, and expression level, binding affinity to NKG2D, and degree of MICA/MICB shedding is thought to be allele-dependent. CLN-619 demonstrated high affinity binding to all common allelic variants of MICA, including the most prevalent MICA allele, MICA*008, and the canonical allelic variant of MICB. CLN-619 was able to prevent proteolytic cleavage of MICA/MICB as evidenced by the reduction of shed MICA/MICB in cell culture supernatants and the corresponding increase in surface MICA/MICB on cancer cells. In addition to the ability to restore the MICA/MICB-NKG2D axis, CLN-619 enhanced the binding of MICA to NKG2D on NK cells. This was enabled by both FcγR engagement on NK cells as well as an intrinsic enhancement of MICA binding to NKG2D. Finally, CLN-619 was shown to elicit potent ADCC of MICA/MICB expressing tumor cells. Consistent with the multimodal mechanisms of action described above, CLN-619 treatment resulted in robust anti-tumor activity at low doses in mice bearing MICA/MICB-expressing human tumor xenografts. Given the pan-cancer expression of MICA/MICB in both solid and hematological malignancies, CLN-619 is expected to have broad anti-tumor activity. CLN-619 is currently being investigated in a Phase 1 clinical trial for the treatment of patients with advanced solid tumors. Citation Format: Kerry A. Whalen, Naveen K. Mehta, Safak Yalcin, Kristan Meetze, Neil W. Gibson, Jennifer S. Michaelson, Patrick A. Baeuerle. CLN-619, a clinical-stage MICA/MICB-specific IgG1 antibody, restores the MICA/MICB-NKG2D axis to promote NK-mediated tumor cell lysis [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 3506.

Abstract 2859: Platelet-derived TGFβ undermines treatment efficacy of t cell recruiting bispecific antibodies

Abstract Immunotherapeutic strategies such as T cell-recruiting bispecific antibodies (bsAb), chimeric antigen receptor (CAR) T cells, and immune checkpoint inhibition have revolutionized oncological treatment. However, many patients do not respond to treatment with the so far available therapeutics, others for limited time only. Various tumor immune evasion mechanisms have been reported to counteract efficiency of T cell engaging therapeutics. Thrombocytes have recently been described to affect cancer biology by mediating epithelial-mesenchymal transition, tumor invasion, metastasis and notably also immune evasion. Here we report that, upon treatment of prostate cancer patients within a clinical trial evaluating a novel PSMAxCD3 bsAb (NCT04104607), we observed profound treatment-associated platelet activation, mirrored by a decrease of total platelet count. Upon modelling the treatment setting, we found that platelet activation significantly reduced bsAb-mediated CD4+ and CD8+ T cell reactivity as revealed by impaired T cell activation, reduced secretion of perforin and ultimately inhibition of target cell lysis. The immunosuppressive effect mediated by platelets occurred in a TGFβ-dependent manner and was not restricted to the PSMAxCD3 bsAb, but was also observed with various other CD3-directed bispecific constructs including the clinically approved CD19xCD13 bsAb Blinatumomab. BsAb-mediated T cell reactivity could generally be restored by inhibiting platelets using dabigatran, but also specifically by blocking the TGFβ axis. Together, our findings unravel that platelets undermine the efficacy of T cell-recruiting bsAb and identify modulation of platelet function and TGFβ as means to reinforce the effectiveness of bsAb treatment. Citation Format: Martina S. Lutz, Boris Klimovich, Stefanie Maurer, Jonas S. Heitmann, Melanie Maerklin, Latifa Zekri, Gundram Jung, Helmut R. Salih, Clemens Hinterleitner. Platelet-derived TGFβ undermines treatment efficacy of t cell recruiting bispecific antibodies [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 2859.

MTORC1 Inhibition Protects Human Regulatory T Cells From Granzyme-B-Induced Apoptosis.

Regulatory T cells (Tregs) have shown great promise as a means of cellular therapy in a multitude of allo- and auto-immune diseases—due in part to their immunosuppressive potency. Nevertheless, the clinical efficacy of human Tregs in patients has been limited by their poor in vivo homeostasis. To avert apoptosis, Tregs require stable antigenic (CD3ζ/T-cell-receptor-mediated), co-stimulatory (CD28-driven), and cytokine (IL-2-dependent) signaling. Notably, this sequence of signals supports an activated Treg phenotype that includes a high expression of granzymes, particularly granzyme B (GrB). Previously, we have shown that aside from the functional effects of GrB in lysing target cells to modulate allo-immunity, GrB can leak out of the intracellular lysosomal granules of host Tregs, initiating pro-apoptotic pathways. Here, we assessed the role of inhibiting mechanistic target of rapamycin complex 1 (mTORC1), a recently favored drug target in the transplant field, in regulating human Treg apoptosis via GrB. Using ex vivo models of human Treg culture and a humanized mouse model of human skin allotransplantation, we found that by inhibiting mTORC1 using rapamycin, intracytoplasmic expression and functionality of GrB diminished in host Tregs; lowering human Treg apoptosis by in part decreasing the phosphorylation of S6K and c-Jun. These findings support the already clinically validated effects of mTORC1 inhibition in patients, most notably their stabilization of Treg bioactivity and in vivo homeostasis.

Bispecific mAb2 Antibodies Targeting CD59 Enhance the Complement-Dependent Cytotoxicity Mediated by Rituximab.

Inhibition of complement activation via the overexpression of complement-regulatory proteins (CRPs), most notably CD46, CD55 and CD59, is an efficient mechanism of disguise of cancer cells from a host immune system. This phenomenon extends to counteract the potency of therapeutic antibodies that could lyse target cells by eliciting complement cascade. The manifold functions and ubiquitous expression of CRPs preclude their systemic specific inhibition. We selected CD59-specific Fc fragments with a novel antigen binding site (Fcabs) from yeast display libraries using recombinant antigens expressed in bacterial or mammalian cells. To produce a bispecific antibody, we endowed rituximab, a clinically applied anti-CD20 antibody, used for therapy of various lymphoid malignancies, with an anti-CD59 Fcab. This bispecific antibody was able to induce more potent complement-dependent cytotoxicity for CD20 and CD59 expressing Raji cell line measured with lactate dehydrogenase-release assay, but had no effect on the cells with lower levels of the primary CD20 antigen or CD20-negative cells. Such molecules are promising candidates for future therapeutic development as they elicit a higher specific cytotoxicity at a lower concentration and hence cause a lower exhaustion of complement components.

In Silico Designed Gain-of-Function Variants of Complement C2 Support Cytocidal Activity of Anticancer Monoclonal Antibodies.

Simple SummaryThe complement system can be exploited by anticancer antibody-based therapeutics. Activation of the classical complement pathway by the heavy chain of antibodies eventually leads to the lysis of target cells. However, overexpression of complement inhibitors by tumor cells limits the therapeutic efficacy. We designed and produced recombinant gain-of-function variants of complement C2 protein that counteract the activity of complement inhibitors. The dominant character of designed mutations in C2 allows supplementation of human serum with recombinant proteins for enhancing the cytocidal activity of complement-activating antibodies. In vitro functional assays demonstrate that this strategy is compatible with several clinically approved antibodies.The molecular target for the classical complement pathway (CP) is defined by surface-bound immunoglobulins. Therefore, numerous anticancer monoclonal antibodies (mAbs) exploit the CP as their effector mechanism. Conversely, the alternative complement pathway (AP) is spontaneously induced on the host and microbial surfaces, but complement inhibitors on host cells prevent its downstream processing. Gain-of-function (GoF) mutations in the AP components that oppose physiological regulation directly predispose carriers to autoimmune/inflammatory diseases. Based on the homology between AP and CP components, we modified the CP component C2 so that it emulates the known pathogenic mutations in the AP component, factor B. By using tumor cell lines and patient-derived leukemic cells along with a set of clinically approved immunotherapeutics, we showed that the supplementation of serum with recombinant GoF C2 variants not only enhances the cytocidal effect of type I anti-CD20 mAbs rituximab and ofatumumab, but also lowers the threshold of mAbs necessary for the efficient lysis of tumor cells and efficiently exploits the leftovers of the drug accumulated in patients’ sera after the previous infusion. Moreover, we demonstrate that GoF C2 acts in concert with other therapeutic mAbs, such as type II anti-CD20, anti-CD22, and anti-CD38 specimens, for overcoming cancer cells resistance to complement attack.

Enhanced Calcium Signal Induces NK Cell Degranulation but Inhibits Its Cytotoxic Activity.

Although the mechanism of NK cell activation is still unclear, the strict calcium dependence remains the hallmark for lytic granule secretion. A plethora of studies claiming that impaired Ca2+ signaling leads to severely defective cytotoxic granule exocytosis accompanied by weak target cell lysis has been published. However, there has been little discussion about the effect of induced calcium signal on NK cell cytotoxicity. In our study, we observed that small-molecule inhibitor UNC1999, which suppresses global H3K27 trimethylation (H3K27me3) of human NK cells, induced a PKD2-dependent calcium signal. Enhanced calcium entry led to unbalanced vesicle release, which resulted into fewer target cells acquiring lytic granules and subsequently being killed. Further analyses revealed that the ability of conjugate formation, lytic synapse formation, and granule polarization were normal in NK cells treated with UNC1999. Cumulatively, these data indicated that induced calcium signal exclusively enhances unbalanced degranulation that further inhibits their cytotoxic activity in human NK cells.

The histone demethylase Kdm6b regulates the maturation and cytotoxicity of TCRαβ+CD8αα+ intestinal intraepithelial lymphocytes

Intestinal intraepithelial lymphocytes (IELs) are distributed along the length of the intestine and are considered the frontline of immune surveillance. The precise molecular mechanisms, especially epigenetic regulation, of their development and function are poorly understood. The trimethylation of histone 3 at lysine 27 (H3K27Me3) is a kind of histone modifications and associated with gene repression. Kdm6b is an epigenetic enzyme responsible for the demethylation of H3K27Me3 and thus promotes gene expression. Here we identified Kdm6b as an important intracellular regulator of small intestinal IELs. Mice genetically deficient for Kdm6b showed greatly reduced numbers of TCRαβ+CD8αα+ IELs. In the absence of Kdm6b, TCRαβ+CD8αα+ IELs exhibited increased apoptosis, disturbed maturation and a compromised capability to lyse target cells. Both IL-15 and Kdm6b-mediated demethylation of histone 3 at lysine 27 are responsible for the maturation of TCRαβ+CD8αα+ IELs through upregulating the expression of Gzmb and Fasl. In addition, Kdm6b also regulates the expression of the gut-homing molecule CCR9 by controlling H3K27Me3 level at its promoter. However, Kdm6b is dispensable for the reactivity of thymic precursors of TCRαβ+CD8αα+ IELs (IELPs) to IL-15 and TGF-β. In conclusion, we showed that Kdm6b plays critical roles in the maturation and cytotoxic function of small intestinal TCRαβ+CD8αα+ IELs.

Filamin A Is Required for NK Cell Cytotoxicity at the Expense of Cytokine Production via Synaptic Filamentous Actin Modulation.

Natural killer (NK) cells are innate cytotoxic lymphocytes that efficiently eliminate malignant and virus-infected cells without prior activation via the directed and focused release of lytic granule contents for target cell lysis. This cytolytic process is tightly regulated at discrete checkpoint stages to ensure the selective killing of diseased target cells and is highly dependent on the coordinated regulation of cytoskeletal components. The actin-binding protein filamin crosslinks cortical actin filaments into orthogonal networks and links actin filament webs to cellular membranes to modulate cell migration, adhesion, and signaling. However, its role in the regulation of NK cell functions remains poorly understood. Here, we show that filamin A (FLNa), a filamin isoform with preferential expression in leukocytes, is recruited to the NK cell lytic synapse and is required for NK cell cytotoxicity through the modulation of conjugate formation with target cells, synaptic filamentous actin (F-actin) accumulation, and cytotoxic degranulation, but not granule polarization. Interestingly, we also find that the loss of FLNa augments the target cell-induced expression of IFN-γ and TNF-α by NK cells, correlating with enhanced activation signals such as Ca2+ mobilization, ERK, and NF-κB, and a delayed down-modulation of the NKG2D receptor. Thus, our results identify FLNa as a new regulator of NK cell effector functions during their decision to kill target cells through a balanced regulation of NK cell cytotoxicity vs cytokine production. Moreover, this study implicates the cross-linking/bundling of F-actin mediated by FLNa as a necessary process coordinating optimal NK effector functions.

Abstract PR01: Oncolytic adenoviruses expressing metabolic targets can improve viro-immunotherapy with bispecific T cell engagers through reducing acidosis in an in vitro model

Abstract Oncolytic viro-immunotherapy holds huge promise for cancer treatment. While oncolytic viruses can robustly trigger immune activation, negative feedback is often upregulated in the tumor microenvironment (TME), an issue that is often neglected in early evaluation in vitro. Lactate accumulation and the resulting acidosis are commonly associated with viral infections and has been reported to be a key factor in shaping the immunosuppressive TME through various mechanisms which favor suppressive Tregs and MDSCs whilst polarizing macrophages towards an immunosuppressive state Given that the success of oncolytic viro-immunotherapy is now widely agreed to rely upon the induction of an immune response either from a transgene or from the release of DAMPs and PAMPs as opposed to direct cell lysis, understanding the impact of the increased acidosis upon immune cells and generating oncolytic viruses capable of avoiding or correcting acidosis is very promising. We found that infection with an oncolytic adenovirus led to a significant increase in lactic acid accumulation and acidosis in keeping with prior reports. An immunotherapeutic that shows promising results is the bispecific antibodies known as bispecific T cell engagers (BiTEs). Although BiTEs have particular success in hematological cancers, they have often shown negligible effects in solid tumor models. Here we show that BiTEs can robustly activate T cells resulting in target cell lysis; however, both T cell activation and BiTE-directed cytotoxicity are reduced when lactate concentrations increase or when acid is added to the media in vitro. Using media from virally infected cells, we show that acidosis following infection of tumor cells with an oncolytic adenovirus also negatively impacted T cell stimulation with BiTEs. Building on previously published results which show that inhibiting glycolysis can improve oncolytic viral infection, we have created oncolytic adenoviruses which inhibit acidosis at various stages through the expression of transgenes targeting this pathway. These next-generation oncolytic viruses were able to replicate whilst inhibiting glycolysis, or acidosis, and were able to halt the production of excess lactic acid following infection of tumor cells, ultimately allowing a rescuing of BiTE-mediated T cell activation. These findings provide a rationale for arming oncolytic viruses with anti-acidity agents, alongside those with T cell-activating abilities, which may offer a novel approach to overcome metabolic barriers of the tumor microenvironment and combat immune evasion. Citation Format: Arthur Dyer, Sally Frost, Flurin Caviezel, Len Seymour. Oncolytic adenoviruses expressing metabolic targets can improve viro-immunotherapy with bispecific T cell engagers through reducing acidosis in an in vitro model [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr PR01.

Complement System in Alzheimer's Disease.

Alzheimer’s disease is a type of dementia characterized by problems with short-term memory, cognition, and difficulties with activities of daily living. It is a progressive, neurodegenerative disorder. The complement system is an ancient part of the innate immune system and comprises of more than thirty serum and membrane-bound proteins. This system has three different activating pathways and culminates into the formation of a membrane attack complex that ultimately causes target cell lysis (usually pathogens) The complement system is involved in several important functions in the central nervous system (CNS) that include neurogenesis, synaptic pruning, apoptosis, and neuronal plasticity. Here, we discuss how the complement system is involved in the effective functioning of CNS, while also contributing to chronic neuroinflammation leading to neurodegenerative disorders such as Alzheimer’s disease. We also discuss potential targets in the complement system for stopping its harmful effects via neuroinflammation and provide perspective for the direction of future research in this field.

Abstract IAP0301: Bispecific T cell engagers (TCEs) for treatment of solid tumors: Challenges and opportunities

Abstract T cell-engaging bispecific antibody constructs (TCEs) have the potential to engage all cytotoxic T cells for redirected cancer cell lysis, including CD8, CD4, gamma/delta, NKT and regulatory T cells. TCEs transiently connect T cells with target cells leading to formation of a cytolytic synapse. Once activated by TCEs, T cells can adopt a serial lysis mode and increase troop size by their local proliferation. In numerous animal models, TCEs can eradicate large tumors as single-agent therapy. TCEs show a remarkable single-agent activity in hematological diseases, including ALL, NHL (DLBCL and FL) and AML. A CD19/CD3-bispecific TCE called blinatumomab (Blincyto) has been approved in 2014, and six other TCEs targeting CD20, BCMA and CD123 are in late-stage development. With complete response rates >50% and durability of responses, TCEs are in par with autologous CD19 CAR-T cells in NHL but are easier to manufacture and allow for a faster treatment start. Treatment of solid tumor indications with TCEs is not as successful as treatment of blood-borne cancers. This may be due to issues with tumor penetration, the hostile tumor microenvironment and the scarcity of T cells in cold tumors. A variety of TCEs explore their single-agent activity against solid tumors by targeting surface antigen like PSMA, GPC3, GD2, or HER-2. Enhancements in the form of combination therapy with checkpoint inhibitors, or 4-1BB and CD28 agonists are being explored as ways of augmenting solid tumor activity of TCEs. The first TCE showing robust anti-tumor activity in a solid tumor indication is targeting a gp100 peptide/MHC (developed by Immunocore). It is made from a soluble TCR fragment of very high affinity for the gp100 peptide HLA*0201 complex fused to an anti-CD3 scFv fragment. Although only few partial responses were observed in uveal melanoma patients, the single-agent treatment showed a very robust improvement in overall survival with a hazard ratio of 0.51 compared to investigators’ choice. This data will inspire the development of more TCEs that are specific for peptide/MHC complexes. This class of targets can also be addresses by TCR-mimetic antibodies. TCEs using such antibodies showed high anti-tumor activity in mouse models against MAGE-A4, p53 mutant or tyrosinase peptide/MHC complexes, all targets that are otherwise not amenable to classical TCEs. Another emerging opportunity for treating cancers are conditional TCEs. These are infused as inactive precursors. Inside tumors, the locally produced proteases convert the precursors into highly active TCEs. One example is the so called ProTriTACs, where a half-life extending albumin-binding domain masks the CD3-binding domain. The mask confers a 500-fold reduction in the cytolytic activity of the TCE. Inside tumors, the mask is cleaved off and a TCE is activated that has a very short serum half-life once it is released from the tumor. Conditional TCEs promise not only a wider therapeutic index but can also leverage as targets tumor-associated antigens that are widely expressed on tumors but also on healthy normal tissues, such as EGFR or EpCAM/TROP1. On-target toxicity of TCEs relates to the quality of target antigens. Finding more tumor-specific surface antigens, appropriate pMHC targets (e.g., mutant oncogene epitopes) and conditional T cell engagers are ways to address this challenge. Off-target toxicity of TCEs relates to T cell activation because of redirected target cell lysis. In the worst case, a cytokine release syndrome is induced. In the best case, a low-level cytokine release is contributing to the efficacy of TCEs. CRS can be managed by steroids, step dosing, anti-IL6 antibody and/or dasatinib. Another promising means is subcutaneous administration of TCEs. A dozen TCE programs currently explore this route, which allows reduction of Cmax and of the initial cytokine release. TCEs will soon become a major weapon in the arsenal of cancer therapy. They will not only work in blood-borne cancers but will soon conquer select solid tumor indications. Peptide/MHC-targeting and conditional TCEs are very promising approaches to achieve solid tumor activity of TCEs. Citation Format: Patrick A. Baeuerle. Bispecific T cell engagers (TCEs) for treatment of solid tumors: Challenges and opportunities [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr IAP0301.

Resveratrol Enhances the Cytotoxic Activity of Lymphocytes from Menopausal Women.

Nutraceuticals and functional foods are the main sources of antioxidants and have positive effects on health through regulation of the redox balance. Accordingly, they represent a useful nutritional source for the prevention of noncommunicable diseases (NCDs). Menopausal women have an increased risk of developing NCDs due to hormonal dysregulation and the ongoing aging process. Accordingly, a healthy lifestyle and good nutritional habits are of utmost importance in this population. Resveratrol (RSV) is a natural polyphenol, and it is used as a nutraceutical given its estrogenic, anti-inflammatory, and antioxidant properties. The aim of this study was to analyze the effects of RSV on the lymphocyte cytotoxicity in menopausal women. Lymphocytes from 13 healthy menopausal women (56.18 ± 4.24 years) were isolated, and then cocultured with hTERT-HME1, a breast cell line with a precancerous phenotype. The results showed that, when treated with RSV, lymphocytes significantly increased the TNF- production (p < 0.001), the formation of immune synapses (p = 0.009), and the target cell lysis (p = 0.002). No effects were detected in the lymphocyte total antioxidant capacity. In conclusion, RSV might enhance the immune surveillance in menopausal women by increasing the cytotoxic activity of lymphocytes.

SLAMF7 selectively favors degranulation to promote cytotoxicity in human NK cells.

NK cells play an important role in immunity by recognizing and eliminating cells undergoing infection or malignant transformation. This role is dependent on the ability of NK cells to lyse targets cells in a perforin-dependent mechanism and by secreting inflammatory cytokines. Both effector functions are controlled by several cell surface receptors. The Signaling Lymphocyte Activation Molecule (SLAM) family of receptors plays an essential role in regulating NK cell activation. Several studies have demonstrated that SLAMF7 regulates NK cell activation. However, the molecular and cellular mechanisms by which SLAMF7 influences NK effector functions are unknown. Here, we present evidence that physiological ligation of SLAMF7 in human NK cells enhances the lysis of target cells expressing SLAMF7. This effect was dependent on the ability of SLAMF7 to promote NK cell degranulation rather than cytotoxic granule polarization or cell adhesion. Moreover, SLAMF7-dependent NK cell degranulation was predominantly dependent on PLC-γ when compared to PI3K. These data provide novel information on the cellular mechanism by which SLAMF7 regulates human NK cell activation. Finally, this study supports a model for NK cell activation where activated receptors contribute by regulating specific discrete cellular events rather than multiple cellular processes.

Bispecific Vγ9Vδ2-T and Type 1 NKT Cell Engager Lava-051 As First-in-Class Clinical Candidate to Target CD1d Expressing CLL, MM and AML

Bispecific Vγ9Vδ2-T and Type 1 NKT Cell Engager Lava-051 As First-in-Class Clinical Candidate to Target CD1d Expressing CLL, MM and AML

A High Sensitivity aCD22 CAR Combined with aCD19 CAR to Generate Dual Targeting CAR T Cells for the Treatment of r/r B-ALL

Background:Anti-CD19 CAR treatment has shown a remarkable response in r/r B-ALL patients, however, a major cause for relapse following CAR administration is the loss of the CD19 antigen. CD22 is a promising alternative target for B-ALL malignancies, although generating an effective CAR to CD22 is challenging due to the size, density and rigidity of this ligand. Furthermore, in the B-ALL setting the CD22 expression levels are known to down regulate in response to selective CAR pressure. This resulted in patients relapsing with lower CD22 density post-treatment (2,839 epitopes/cell) presumably due to the target density falling below the sensitivity threshold for the aCD22 CAR 1. In this study we aim to improve CAR efficacy for the treatment of r/r B-ALL by firstly developing a highly sensitive aCD22 CAR capable of targeting cells that express less than 1000 CD22 molecules per cell. Secondly, we combined this new aCD22 CAR with an aCD19 CAR to generate a dual targeting CD19 and CD22 product via co-transduction for the treatment of paediatric r/r B-ALL as part of an extension cohort of the CARPALL clinical trial (NCT02443831).Results:We screened 18 anti-CD22 binders with a range of affinities (1-36.5nM) and binding epitopes in CAR format for high functional sensitivity to low density targets. We filtered binders for their ability to maintain cytotoxicity, proliferation and cytokine release with low expressing CD22 targets. We identified the optimal aCD22 binder (9A8) which when combined in a CAR structure containing a 41BBz endodomain was capable of lysing target cells transduced to express less than 255 CD22 mols/cell while not affecting CD22 negative targets. Similar to the previously tested aCD22 binder M971, our new 9A8 binder also targets the membrane proximal Ig domain of the CD22 molecule. However unlike M971, 9A8 utilises a conventional 16 amino acid linker between the heavy and light chain to form the scFv and has a lower propensity to tonically signal compared to the M971 CAR. To generate a dual CD19 and CD22 targeting product, the 9A8 aCD22 CAR was co-transduced with the aCD19 CAT19 CAR vector (AUTO1) utilised in the CARPALL clinical trial for the treatment of paediatric r/r B-ALL. In a NSG animal model engrafted with WT CD19+/CD22+ Nalm-6 cells, the dual targeting co-transduced T cells (AUTO1/22) were significantly better at supressing tumour growth compared to single targeting AUTO1 CAR T cells (Figure 1). Furthermore, CAR T cells recovered from these animals showed an equal persistence of both aCD19 CAR and aCD22 CAR. In an effort to recapitulate the loss of CD19 target observed in aCD19 CAR relapsed patients, we challenged our transduced T cells in a CD19-knockout Nalm-6 NSG model (Figure 1). In this setting, single transduced AUTO1 CAR T cells were unable to control tumour growth above the non-transduced T cell controls. In contrast, the dual targeting co-transduced AUTO1/22 CAR T cells significantly supressed tumour growth and showed enrichment of CAR T cells expressing the aCD22 9A8 CAR in the bone marrow.The dual targeting co-transduced AUTO1/22 is currently being evaluated in an extension cohort of the CARPALL clinical trial (NCT02443831) for the treatment of paediatric r/r B-ALL.Conclusion:We engineered a highly sensitive aCD22 9A8 CAR which when combined with the aCD19 CAT19 CAR allows for improved in vivo function and the targeting of CD19 negative, CD22 positive cells. The use of these dual targeting T cells may reduce the incidence of CD19 negative relapses in the r/r B-ALL setting.1. Fry TJ, Shah NN, Orentas RJ, et al. CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy. Nat. Med. 2018;24(1):20-28. [Display omitted] DisclosuresKokalaki: Autolus Ltd: Current Employment. Grothier: Autolus Ltd: Ended employment in the past 24 months. Manzoor: Autolus Ltd: Ended employment in the past 24 months. Costu: Autolus Ltd: Ended employment in the past 24 months. Srivastava: Autolus Ltd: Current Employment. Jha: Autolus Ltd: Current Employment. Gealy: Autolus Ltd: Current Employment. Stanczuk: Autolus Ltd: Ended employment in the past 24 months. Robson: Autolus Ltd: Current Employment. Taylor: Autolus Ltd: Current Employment. El-Kholy: Autolus Ltd: Current Employment. Baldan: Autolus Ltd: Current Employment. Righi: Autolus Ltd: Current Employment. Sillibourne: Autolus Ltd: Current Employment. Onuoha: Autolus: Ended employment in the past 24 months. Ghorashian: UCLB: Patents & Royalties; Novartis: Honoraria. Cordoba: Autolus Ltd: Current Employment. Amrolia: ADC Therapeutics: Other: Named inventor on a patent which is being transferred to ADCT.; Autolus: Patents & Royalties. Pule: Autolus Ltd: Current Employment.

Preclinical Assessment of CDR101 - a BCMAxCD3xPD-L1 Trispecific Antibody with Superior Anti-Tumor Efficacy

BCMAxCD3 targeting therapies have demonstrated anti-myeloma activity, and high minimal residual disease negativity rates can be achieved with this approach in heavily pre-treated patients with relapsed or refractory multiple myeloma (RRMM). Despite these promising clinical results, patients eventually develop resistant disease and relapse. Thus, there is a high need for novel BCMA therapies that can evade the resistance mechanisms and provide more durable responses. Recently, we reported on the promising activity of the Local Activator and T cell Engager (LocATE) technology, a trispecific molecule that targets CD3, BCMA and PD-L1, redirecting T cells to multiple myeloma (MM) cells while selectively counteracting PD-L1/PD-1 induced immunosuppression at the immune synapse (ASH, 2020). Here we present CDR101, an optimized LocATE candidate with potential for clinical development.First, we analyzed the ability of CDR101 to induce PBMC-mediated cytotoxicity in two MM cell-lines expressing BCMA (U-266 and NCI-H929) and compared it to four BCMAxCD3 bispecific formats currently in clinical development (a half-life extended BCMAxCD3 BiTE, a BCMA-TCB, and two different BCMAxCD3 bispecific monoclonal antibodies) alone or in combination with a separate PD-L1 blocking antibody. CDR101 resulted in at least 10-fold increased T cell-mediated target cell lysis compared to control BCMAxCD3 bispecifics. Strikingly, CDR101 also resulted in increased MM cell killing when compared to free, independent combinations of BCMAxCD3 bispecifics and the PD-L1 inhibitor. These results, together with the observation that MM cells upregulate the expression of PD-L1 in response to treatment with BCMAxCD3 bispecifics, suggest that the superior effect of CDR101 could be attributed to preferential and highly selective inhibition of the PD-1/PD-L1 axis at the cellular interaction within the immune synapse.Next, bone marrow aspirates from newly diagnosed and RRMM patients were treated with increasing concentrations of CDR101 or a BCMAxCD3 bispecific control. After 24h of incubation, percentage of viable CD138-positive cells and activation status of autologous T cells were analyzed by FACS. Overall, CDR101 potently induced lysis of primary MM cells independently of the E:T ratio (range of E:T ratio between 1.3:1 and 33:1). CDR101 achieved higher target cell killing in all samples compared to the bispecific control, with at least 2-fold difference in 3 out of 4 samples at the highest concentration tested. Concomitantly, CDR101 induced a dose-dependent increase of the T cell activation marker CD25, corroborating the ability of CDR101 to counteract PD-L1/PD-1 induced immunosuppression.In vivo anti-tumor activity of CDR101 was evaluated using a human MM (NCI-H929) xenograft model in NPG mice. Treatment with four different doses of CDR101 or BCMAxCD3 bispecific control demonstrated that CDR101 induced stronger and more durable responses compared to the bispecific control leading to complete tumor regression in 55 out of 60 mice at the last day of treatment (day 29) with no relapse until the end of the observation time (day 41).Collectively, CDR101 demonstrated that targeting BCMA with simultaneous blockade of PD-L1 leads to improved myeloma cell killing compared to clinically validated therapies. In contrast to high-affinity PD-L1 immune checkpoint inhibitors, CDR101 selectively inhibits PD-L1 at the immune synapse preventing on-target off-tumor effects. This is expected to translate into a decreased incidence of immune related adverse events (irAEs) and better efficacy arguing for a high clinical potential and swift translation into the clinic. DisclosuresVrohlings: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Jungmichel: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Senn: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Howald: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Schleier: CDR-Life Inc: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Scheifele: CDR-Life Inc: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Wendelspiess: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Richle: CDR-Life Inc: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Merten: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Lenherr-Frey: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Leisner: CDR-Life Inc: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Manz: CDR-Life Inc: Consultancy, Current holder of stock options in a privately-held company; University of Zurich: Patents & Royalties: CD117xCD3 TEA. Borras: CDR-Life Inc: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

CD22 CAR Optimization for Improved in-Human Activity Following Inadequate CD22 CAR Activity in Phase 1 Clinical Trial PLAT-04

Background: CD19 targeting chimeric antigen receptor (CAR) T cells have induced unprecedented remission rates in high-risk precursor B Acute Lymphoblastic Leukemia (ALL); however recurrent disease with CD19 antigen escape variants is not uncommon. Therefore, we developed a novel CD22 targeting CAR, and following preclinical validation, tested it in a first-in-human pediatric and young adult phase 1 clinical trial, PLAT-04 (NCT03244306). Four subjects were treated at 2 dose levels (DL) (1x10 6/kg (DL1) and 3x10 6/kg (DL2)). The CD22 CAR T cell product (SCRI-CAR22v1) was successfully manufactured (n=4) and no dose limiting toxicity (DLTs), cytokine release syndrome (CRS) or neurotoxicity was observed. However, all subjects had minimal CAR T cell expansion, with 3 of 4 subjects demonstrating persistent or progressive disease at day 21 evaluation despite continued CD22 expression on leukemic blasts. Based on the poor in vivo expansion and lack of activity, enrollment was voluntarily halted to interrogate and optimize the CAR construct for enhanced performance.Methods: Human T cells were transduced to express one of two CD22 CAR constructs. We designed SCRI-CAR22v2, a CD22 CAR that utilizes the same scFv as SCRI-CAR22v1 but with a shorter linker between M971 VH and VL and a shorter hinge with differing transmembrane region, and both using CD8 alpha (Figure A). This construct maintained the truncated EGFR extracellular tag (EGFRt) for tracking and potential in vivo suicide mechanism. The two transduced CAR T cell products were compared preclinically by flow cytometry, chromium release assay and in an in vivo murine model to understand differing T cell activity between the CAR constructs. Additionally, SCRI-CAR22v2 is currently under investigation in a dose finding phase 1 clinical trial, PLAT-07 (NCT04571138).Results: Following use of cetuximab-APC and biotinylated anti-human Fab antibody for surface EGFRt and CAR detection, the SCRI-CAR22v1 expresses lower levels of EGFRt but similar CAR levels on the cell surface demonstrated by MFI (Figure B). Biotinylated, soluble CD22 antigen was also used to evaluate CD22 CAR receptor activity and, as measured by MFI, a higher affinity is suggested via SCRI-CAR22v2 as compared to SCRI-CAR22v1 (Figure B). K562 cells expressing low, medium or high CD22 were used to evaluate the impact of surface antigen expression on the CAR activity level. SCRI-CAR22v2 demonstrates improved targeted cell lysis at all 3 antigen quantity levels by chromium release assay (Figure C). In NSG mice inoculated with Raji tumor cells expressing ffluc, SCRI-CAR22v2 demonstrated improved survival compared to SCRI-CAR22v1 (Figure D) and clearance of Raji tumor cells (Figure E). Based on this promising preclinical data, we initiated enrollment onto PLAT-07, a phase 1 dose finding trial (2x10 5cells/kg (DL1), 5x10 5cells/kg (DL2) and 1x10 6cells/kg (DL3)) of SCRI-CAR22v2. To date, 3 subjects have been enrolled and successfully infused at DL1. All had prior CD19-CAR therapy and 2 lacked CD19 leukemic expression at the time of SCRI-CAR22v2 infusion. At the time of cell infusion, one subject had only extramedullary disease, one had MRD of <1% and one subject had a larger disease burden of 30% ALL. None experienced a DLT and all were MRD negative in the bone marrow at day 28 and the subject with EMD demonstrated a complete metabolic response by PET scan. Figure F exhibits the improved expansion and engraftment of the SCRI-CAR22v2 cells as compared to SCRI-CAR22v1 DL1 (n=3) and DL2 (n=1), and higher peak levels of CD22 CAR T cells as compared to SCRI-CAR22v1 DL1 and DL2 (Figure G).Conclusions: Despite encouraging preclinical data, SCRI-CAR22v1 demonstrated poor expansion and engraftment in a Phase 1 trial. Notably, minor CAR alterations lead to encouraging in-human activity in early clinical findings. Our experience suggests a shorter linker and hinge as well as incorporation of an CD8 alpha transmembrane region improves the clinical activity of CD22 targeted CAR T cells in subjects with recurrent disease following CD19 CAR T cells. Further evaluation is needed to elucidate the critical CAR components and/or assays at the preclinical level that can best predict which CAR should be brought to the clinic for further evaluation. [Display omitted] DisclosuresOrentas: Lentigen: Patents & Royalties. Jensen: BMS: Patents & Royalties; Umoja Biopharma: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bluebird Bio: Research Funding. Gardner: Novartis: Consultancy; BMS: Patents & Royalties.

P53.05 Inhibition of Tumor Cell Intrinsic Complement Regulatory Proteins Leads to Decreased Tumor Growth in a Mouse Model of NSCLC

The complement system is part of the innate immune system that interfaces with adaptive immunity. Complement activation triggers a series of proteolytic cascades that converge on C3, leading to the formation of the membrane attack complex (MAC) that causes cell lysis of target cells. Within the complement system, there are multiple regulatory proteins that act to inhibit or decrease complement activation; these proteins include Factor H (fH), CD55, and CD59. Complement activation can mediate tumor progression through production of anaphylatoxins (C3a/C5a) that act to block anti-tumor immunity.