Antibody-based Cancer Immunotherapy Research Articles

Abstract A173: ASP2074, a novel tetraspanin 8 x CD3 bispecific antibody, demonstrates selectivity and antitumor activity in preclinical cancer models

Abstract ASP2074 is a novel bispecific antibody that targets the tumor-associated antigen tetraspanin 8 (TSPAN8) and CD3, which is expressed on the plasma membrane of all T cells. By engaging with both TSPAN8 and CD3, ASP2074 enables the retargeting of cytotoxic T cells specifically against TSPAN8-expressing tumor cells in a human leukocyte antigen-independent manner. TSPAN8 is a member of the tetraspanin superfamily of proteins that plays a crucial role in intracellular signal transduction and regulates various cellular functions, including leukocyte trafficking, angiogenesis, and wound repair. TSPAN8 is highly expressed in various types of cancer, such as colorectal cancer, gastric cancer, pancreatic cancer, esophageal adenocarcinoma, and gastroesophageal junction (GEJ) adenocarcinoma. Its overexpression has been associated with tumor cell growth, angiogenesis, invasion, metastasis, and poor prognosis, making it an attractive target for antibody-based cancer immunotherapy. ASP2074 is currently undergoing phase 1 clinical trials for the treatment of patients with colorectal cancer, esophageal adenocarcinoma, pancreatic cancer, gastric cancer, and GEJ adenocarcinoma. In this study, we examined the selectivity and antitumor activity of ASP2074 in vitro and in vivo. Methods: Retrogenix Cell Microarray Technology was used to evaluate the selective binding of ASP2074 to TSPAN8 and CD3 in 6018 full-length human plasma membrane proteins, secreted and cell surface-tethered human secreted proteins, and 397 human heterodimers containing homologs and isoforms of tetraspanin. Cytotoxicity and T-cell activation of ASP2074 were examined using TSPAN8-expressing tumor cells in combination with human peripheral blood mononuclear cells (PBMCs) as effector cells in a redirected T-cell cytotoxicity assay. Furthermore, the antitumor effect of ASP2074 was examined in a human PBMC-engrafted NOG mouse model bearing human TSPAN8-expressing colorectal cancer cells. Results: Our results showed that ASP2074 selectively bound to TSPAN8 and CD3 without significant binding to other human plasma membrane proteins, secreted proteins, and heterodimers. ASP2074 exhibited cytotoxicity against TSPAN8-expressing cancer cells and increased interferon-gamma production and expression of T-cell activation markers. The cytotoxic activities were dependent on the TSPAN8 expression levels in tumor cells. In the NOG mouse model, ASP2074 exhibited an antitumor effect on human TSPAN8-expressing colorectal cancer cells in vivo. Conclusion: Our findings suggest that ASP2074 has potent antitumor activity through cytotoxicity against TSPAN8-expressing tumor cells by T-cell activation via selective binding to TSPAN8 and CD3. These preclinical results support the continued development of ASP2074 as a promising immunotherapy for the treatment of TSPAN8-expressing cancers. Citation Format: Yoshiyuki Tenda, Yuichi Hanada, Hiroaki Tanaka, Masashi Shimazaki, Kenna Shirasuna. ASP2074, a novel tetraspanin 8 x CD3 bispecific antibody, demonstrates selectivity and antitumor activity in preclinical cancer models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A173.

Bispecific antibodies targeting CTLA-4: game-changer troopers in cancer immunotherapy.

Antibody-based cancer immunotherapy has become a powerful asset in the arsenal against malignancies. In this regard, bispecific antibodies (BsAbs) are a ground-breaking novel approach in the therapy of cancers. Recently, BsAbs have represented a significant advancement in improving clinical outcomes. BsAbs are designed to target two different antigens specifically. Over a hundred various BsAb forms currently exist, and more are constantly being manufactured. An antagonistic regulator of T cell activation is cytotoxic T lymphocyte-associated protein 4 (CTLA-4) or CD152, a second counter-receptor for the B7 family of co-stimulatory molecules was introduced in 1996 by Professor James P. Allison and colleagues. Contrary to the explosive success of dual immune checkpoint blockade for treating cancers, a major hurdle still yet persist is that immune-related adverse events (irAEs) observed by combining immune checkpoint inhibitors (ICIs) or monoclonal antibodies such as ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1). A promising strategy to overcome this hurdle is using BsAbs. This article will summarize BsAbs targeting CTLA-4, their applications in cancer immunotherapy, and relevant clinical trial advances. We will also discuss the pre-clinical rationale for using these BsAbs, and provide the current landscape of the field.

Antibody-based cancer immunotherapy by targeting regulatory T cells.

Regulatory T cells (Tregs) are among the most abundant suppressive cells, which infiltrate and accumulate in the tumor microenvironment, leading to tumor escape by inducing anergy and immunosuppression. Their presence has been correlated with tumor progression, invasiveness and metastasis. Targeting tumor-associated Tregs is an effective addition to current immunotherapy approaches, but it may also trigger autoimmune diseases. The major limitation of current therapies targeting Tregs in the tumor microenvironment is the lack of selective targets. Tumor-infiltrating Tregs express high levels of cell surface molecules associated with T-cell activation, such as CTLA4, PD-1, LAG3, TIGIT, ICOS, and TNF receptor superfamily members including 4-1BB, OX40, and GITR. Targeting these molecules often attribute to concurrent depletion of antitumor effector T-cell populations. Therefore, novel approaches need to improve the specificity of targeting Tregs in the tumor microenvironment without affecting peripheral Tregs and effector T cells. In this review, we discuss the immunosuppressive mechanisms of tumor-infiltrating Tregs and the status of antibody-based immunotherapies targeting Tregs.

Abstract 2962: ASP2138, a novel 2+1 format, claudin 18.2 x CD3 bispecific antibody, demonstrates selectivity and activity in preclinical cancer models

Abstract Claudin 18.2 (CLDN18.2) is a member of the claudin family of proteins that are involved in the formation of tight junctions in epithelia and endothelia. In normal tissue, CLDN18.2 is expressed exclusively on gastric epithelial cells. CLDN18.2 expression is maintained during malignant transformation of gastric epithelia. In addition, it is aberrantly expressed in other tumor types, including esophageal, pancreatic, and lung adenocarcinomas. Thus, CLDN18.2 is considered to be an attractive tumor target antigen for antibody-based cancer immunotherapy. ASP2138 is an immunoglobulin G (IgG)-based, asymmetric 2+1 format, T-cell bispecific antibody comprising bivalent humanized anti-CLDN18.2 antigen-binding fragments and a monovalent anti-CD3 single-chain variable fragment. CD3 is a surface protein complex expressed on all T-cell populations and its clustering causes T-cell activation. Therefore, CD3 bispecific antibodies allow for simultaneous engagement with both CD3 and a tumor-associated antigen (TAA), resulting in retargeting of cytotoxic T cells specifically against TAA-positive tumor cells in a human leukocyte antigen-independent manner. ASP2138 is in development for the treatment of patients with CLDN18.2-positive gastric or gastroesophageal junction adenocarcinoma or pancreatic adenocarcinoma, and is being evaluated in an ongoing phase 1/1b clinical trial (NCT05365581). In this study, ASP2138 demonstrated selective binding to CLDN18.2 and CD3 without binding to other homologs and isoforms of CLDN18.2. Cytotoxicity and T-cell activation of ASP2138 were assessed using CLDN18.2-expressing tumor cells in combination with human peripheral blood mononuclear cells (PBMCs) as effector cells in a redirected T-cell cytotoxicity assay in vitro. ASP2138 showed cytotoxicity against CLDN18.2-expressing gastric or pancreatic cancer cells and increased interferon-gamma production and expression of T-cell activation markers. The cytotoxic activities were dependent on the effector cell to target cell ratio and CLDN18.2 expression on tumor cells. ASP2138 exhibited an antitumor effect on human CLDN18.2-expressing gastric cancer in a human PBMC-engrafted NOG mouse model in vivo. In summary, ASP2138 is expected to show a clinical effect through cytotoxicity against CLDN18.2-expressing tumor cells by T-cell activation that results from selective binding to CLDN18.2 and CD3. Citation Format: Taisuke Nakazawa, Hiroaki Tanaka, Aya Kikuchi, Rumana Rashid, Kendra N. Avery, Jing Qi, Alex Nisthal, Masashi Shimazaki, Kenna Shirasuna. ASP2138, a novel 2+1 format, claudin 18.2 x CD3 bispecific antibody, demonstrates selectivity and activity in preclinical cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2962.

New Insights on the Role of Anti-PD-L1 and Anti-CTLA-4 mAbs on Different Lymphocytes Subpopulations in TNBC.

Antibody-based cancer immunotherapy includes monoclonals against immune checkpoints (ICs), to modulate specific T cell responses against cancer. NK cells are a newly emerging target for immune checkpoint receptor inhibition in cancer immunotherapy, as ICs are also expressed on NK cells in various cancers. The latter cells are becoming attractive targets for cancer immunotherapy, as they are effector cells similar to CTLs, exerting natural cytotoxicity against primary tumor cells and metastasis, and they are able to distinguish tumor cells from healthy ones, leading to more specific anti-tumor cytotoxicity and reduced off-target effects. Thus, we decided to test the effects on isolated NK cells and T cell subpopulations of novel immunomodulatory mAbs, recently generated in our lab, in comparison with those in clinical use, such as ipilimumab and atezolizumab. Interestingly, we found that the novel anti-CTLA-4 (ID-1) and anti-PD-L1 (PD-L1_1) antibodies are able to induce NK cell activation and exert anti-tumor effects on TNBC cells co-cultured with NK cells more efficiently than the clinically validated ones, either when used as single agents or in combinatorial treatments. On the other hand, ipilimumab was found to be more effective in activating T cells with respect to ID-1. These findings indicate that antibodies targeting different epitopes can have differential effects on different lymphocytes subpopulations and that novel combinations of mAbs could be suitable for therapeutic approaches aimed at activating not only T cells but also NK cells, especially for tumors lacking MHC.

Holdase/Foldase Mimetic Nanochaperone Improves Antibody-Based Cancer Immunotherapy.

Despite unprecedented successes of antibody-based cancer immunotherapy, the serious side effects and rapid clearance following systemic administration remain big challenges to realize its full potential. At the same time, combination immunotherapy using multiple antibodies has shown particularly promising in cancer treatment. It is noticed that the working mechanisms of natural holdase and foldase chaperone are desirable to overcome the limitations of therapeutic antibodies. Holdase chaperone stabilizes unfolded client and prevents it from activation and degradation, while foldase chaperone assists unfolded client to its native state to function. Here a holdase/foldase mimetic nanochaperone (H/F-nChap) to co-delivery two types of monoclonal antibodies (mAbs), αCD16 and αPDL1, and resiquimod (R848) is developed, which significantly improves cancer immunotherapy. The H/F-nChap presents holdase activity in blood and normal tissues that hides and protects mAbs from unnecessary targeted activation and degradation, thereby prolonging blood circulation and reducing immunotoxicity in vivo. Furthermore, H/F-nChap switches to foldase activity in the tumor microenvironment that exposes mAbs and releases R848 to enhance the engagement between NK cells and tumor cells and promote immune activation, respectively. The H/F-nChap represents a strategy for safe and spatiotemporal delivery of multiple mAbs, providing a promising platform for improved cancer immunotherapy.

Human Antibody Domains and Fragments Targeting Neutrophil Elastase as Candidate Therapeutics for Cancer and Inflammation-Related Diseases.

Neutrophil elastase (NE) is a serine protease released during neutrophil maturation. High levels of NE are related to lung tissue damage and poor prognosis in cancer; thus, NE is a potential target for therapeutic immunotherapy for multiple lung diseases and cancers. Here, we isolate and characterize two high-affinity, specific, and noncompetitive anti-NE antibodies Fab 1C10 and VH 1D1.43 from two large phage-displayed human Fab and VH libraries. After fusion with human IgG1 Fc, both of them (VH-Fc 1D1.43 and IgG1 1C10) inhibit NE enzymatic activity with VH-Fc 1D1.43 showing comparable inhibitory effects to that of the small molecule NE inhibitor SPCK and IgG1 1C10 exhibiting even higher (2.6-fold) activity than SPCK. Their epitopes, as mapped by peptide arrays combined with structural modeling, indicate different mechanisms for blocking NE activity. Both VH-Fc and IgG1 antibodies block NE uptake by cancer cells and fibroblast differentiation. VH-Fc 1D1.43 and IgG1 1C10 are promising for the antibody-based immunotherapy of cancer and inflammatory diseases.

Folate Receptor Beta as a Direct and Indirect Target for Antibody-Based Cancer Immunotherapy.

Folate receptor beta (FRβ) is a folate binding receptor expressed on myeloid lineage hematopoietic cells. FRβ is commonly expressed at high levels on malignant blasts in patients with acute myeloid leukemia (AML), as well as on M2 polarized tumor-associated macrophages (TAMs) in the tumor microenvironment of many solid tumors. Therefore, FRβ is a potential target for both direct and indirect cancer therapy. We demonstrate that FRβ is expressed in both AML cell lines and patient-derived AML samples and that a high-affinity monoclonal antibody against FRβ (m909) has the ability to cause dose- and expression-dependent ADCC against these cells in vitro. Importantly, we find that administration of m909 has a significant impact on tumor growth in a humanized mouse model of AML. Surprisingly, m909 functions in vivo with and without the infusion of human NK cells as mediators of ADCC, suggesting potential involvement of mouse macrophages as effector cells. We also found that TAMs from primary ovarian ascites samples expressed appreciable levels of FRβ and that m909 has the ability to cause ADCC in these samples. These results indicate that the targeting of FRβ using m909 has the potential to limit the outgrowth of AML in vitro and in vivo. Additionally, m909 causes cytotoxicity to TAMs in the tumor microenvironment of ovarian cancer warranting further investigation of m909 and its derivatives as therapeutic agents in patients with FRβ-expressing cancers.

Bringing Together the Power of T Cell Receptor Mimic and Bispecific Antibodies for Cancer Immunotherapy: Still a Long Way to Go.

Antibody-based cancer immunotherapy has revolutionized oncology. The first successful therapeutic antibodies relied on eliciting immune-mediated cytotoxicity (rituximab) or modulation of intracellular signaling (trastuzumab). Further attempts to enhance the antitumor effects led to the development of immunoconjugates with radioactive or cytotoxic compounds (tositumomab, brentuximab vedotin). Another line of research led to the bispecific antibodies that can enhance the formation of immunological synapse between cancer and cytotoxic T cells (blinatumomab). Despite the constant advances in design and production, the application of monoclonal antibodies in cancer treatment remains limited by the presence of specific cell surface markers. A rational approach to target intracellular cancer antigens was proposed almost two decades ago by the development of anti-peptide human leukocyte antigen (HLA) complex (T cell receptor-like/mimic) antibodies. They could recognize specifically cancer neoantigens expressed in the context of specific HLA molecules theoretically providing unprecedented specificity. Furthermore, they can be developed in a semigeneric format, that is, to target common neoantigens expressed in the context of common HLA molecules. It is rationale to expect that the development of such antibodies in the format of bispecific antibody constructs can bring together the power of specific antibody-based recognition and that of T cell-mediated lysis. There are already some preliminary reports that suggest such constructs would be an achievable goal. In this brief review, we discuss some of the successful preclinical developments in the field and the major challenges that are yet to be addressed.

Immunomodulating nano-adaptors potentiate antibody-based cancer immunotherapy

Modulating effector immune cells via monoclonal antibodies (mAbs) and facilitating the co-engagement of T cells and tumor cells via chimeric antigen receptor- T cells or bispecific T cell-engaging antibodies are two typical cancer immunotherapy approaches. We speculated that immobilizing two types of mAbs against effector cells and tumor cells on a single nanoparticle could integrate the functions of these two approaches, as the engineered formulation (immunomodulating nano-adaptor, imNA) could potentially associate with both cells and bridge them together like an ‘adaptor’ while maintaining the immunomodulatory properties of the parental mAbs. However, existing mAbs-immobilization strategies mainly rely on a chemical reaction, a process that is rough and difficult to control. Here, we build up a versatile antibody immobilization platform by conjugating anti-IgG (Fc specific) antibody (αFc) onto the nanoparticle surface (αFc-NP), and confirm that αFc-NP could conveniently and efficiently immobilize two types of mAbs through Fc-specific noncovalent interactions to form imNAs. Finally, we validate the superiority of imNAs over the mixture of parental mAbs in T cell-, natural killer cell- and macrophage-mediated antitumor immune responses in multiple murine tumor models.

Epithelioid granulomatous lesions express abundant programmed death ligand-1 (PD-L1): a discussion of adverse events in anti-PD-1 antibody-based cancer immunotherapy

ABSTRACT The immune system is often called a double-edged sword, due to the inextricable link between cancer immunity and allergy/autoimmunity. Intriguingly, a growing number of cases have been reported in which PD-1 blockade triggers the exacerbation of tuberculosis (TB), an organ-invasive granulomatous disease caused by bacterial infection. As a result, the exacerbation of TB is now considered a severe adverse effect of nivolumab and pembrolizumab. In this letter, we report the strong expression of PD-L1 in epithelioid granulomatous lesions in tuberculosis, sarcoidosis, Crohn’s disease, and foreign body granuloma. In addition, we discussed the exacerbation of tuberculosis after anti-PD-1 antibody-based cancer immunotherapy.

Antibody and antibody fragments for cancer immunotherapy

Antibody has become the most rapidly expanding class of pharmaceuticals for treating a wide variety of human diseases including cancers. Especially, with the fast development of cancer immunotherapy, antibody drugs have become the most promising therapeutic for curing cancers. Immune-mediated cell killing by antibodies including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) as well as regulation of T cell function through immune checkpoint blockade. Due to the absence of Fc fragment, antibody fragments including single-chain variable fragments (scFvs) and single-domain antibodies (sdAds) are mainly applied in chimeric antigen receptors (CAR) T cell therapy for redirecting T cells to tumors and T cell activation by immune checkpoint blockade. In this review, the cancer immunity is first discussed. Then the principal mechanisms of antibody-based immunotherapy will be reviewed. Next, the antibody and antibody fragments applied for cancer immunotherapy will be summarized. Bispecific and multispecific antibodies and a combination of cancer immunotherapy with other tumor treatments will also be mentioned. Finally, an outlook and perspective of antibody-based cancer immunotherapy will be given. This review would provide a comprehensive guidance for the researchers who are interested in and intended to involve in the antibodies- or antibody fragments-based tumor immunity.

TLR7/8 Agonist-Loaded Nanoparticles Augment NK Cell-Mediated Antibody-Based Cancer Immunotherapy.

Activated natural killer (NK) cells can kill malignant tumor cells via granule exocytosis and secretion of IFN-γ, a key regulator of the TH1 response. Thus, mobilization of NK cells can augment cancer immunotherapy, particularly when mediated through antibody-dependent cellular cytotoxicity (ADCC). Stimulation of toll-like receptor (TLR)7/8 activity in dendritic cells promotes pro-inflammatory cytokine secretion and costimulatory molecule upregulation, both of which can potentiate NK cell activation. However, currently available TLR7/8 agonists exhibit unfavorable pharmacokinetics, limiting their in vivo efficacy. To enable efficient delivery to antigen-presenting cells, we encapsulated a novel imidazoquinoline-based TLR7/8 agonist in pH-responsive polymeric NPs. Enhanced costimulatory molecule expression on dendritic cells and a stronger pro-inflammatory cytokine response were observed with a NP-encapsulated agonist, compared to that with the soluble form. Treatment with NP-encapsulated agonists resulted in stronger in vivo cytotoxicity and prolonged activation of NK cells compared to that with a soluble agonist. In addition, TLR7/8 agonist-loaded NPs potentiated stronger NK cell degranulation, which resulted in enhanced in vitro and in vivo ADCC mediated by the epidermal growth factor receptor-targeting antibody cetuximab. TLR7/8 agonist-loaded NP treatment significantly enhanced the antitumor efficacy of cetuximab and an anti-HER2/neu antibody in mouse tumor models. Collectively, our data show that a pH-responsive NP-encapsulating TLR7/8 agonist could be used as a potent immunostimulatory adjuvant for antibody-based cancer immunotherapy by promoting NK cell activation.

Downregulation of PD-L1 via amide analogues of brefelamide: Alternatives to antibody-based cancer immunotherapy.

The therapeutic blockade of immune checkpoint has emerged as an effective treatment option for a broad range of tumors. However, the objective tumor response is still limited to a small number of cases and tumor types. The full utility of monoclonal antibody (mAb)-based treatment is hindered by several inherent limitations. Thus, there is an urgent requirement to explore alternative modalities targeting the same pathways. In the present study, two amide analogues of brefelamide, TPFS-201 and TPFS-202, were identified as small molecular immune checkpoint inhibitors, as they downregulated PD-L1 expression in tumor cells. PD-L1 was suppressed in cancer cells treated with TPFD compounds at both mRNA and protein levels, as detected by reverse transcription quantitative PCR and flow cytometric analysis, respectively. Reporter assays using a PD-L1 promoter luciferase construct confirmed the transcriptional inhibition of PD-L1 by TPFS compunds. TPFS compound-mediated PD-L1 downregulation in cancer cells consequently restored T cell activity, as identified by the reduction of apoptosis and an increase in interleukin-2 promoter activity in Jurkat T cells, which were co-cultured with TPFS compound-treated A549 cells. TPFS compound-mediated PD-L1 inhibition was partially abolished by the disruption of the putative transcriptional co-activator with PDZ (TAZ)/TEA domain (TEAD)-binding motif in the PD-L1 promoter. The inhibitory effect of TPFS compounds on PD-L1 was markedly inhibited in mouse cell lines, which is consistent with previous research demonstrating that PD-L1 regulation by TAZ is not conserved in mice due to distinct promoter sequences flanking the TAZ/TEAD-binding motif. Together, the data of the current study indicated the potential utility of the brefelamide amide analogues as small molecule immune checkpoint inhibitors, thereby providing therapeutic alternatives, which could be used as monotherapy or in combination with mAbs-based treatment.

Machine learning-aided quantification of antibody-based cancer immunotherapy by natural killer cells in microfluidic droplets

Natural killer (NK) cells have emerged as an effective alternative option to T cell-based immunotherapies, particularly against liquid (hematologic) tumors. However, the effectiveness of NK cell therapy has been less than optimal for solid tumors, partly due to the heterogeneity in target interaction leading to variable anti-tumor cytotoxicity. This paper describes a microfluidic droplet-based cytotoxicity assay for quantitative comparison of immunotherapeutic NK-92 cell interaction with various types of target cells. Machine learning algorithms were developed to assess the dynamics of individual effector-target cell pair conjugation and target death in droplets in a semi-automated manner. Our results showed that while short contacts were sufficient to induce potent killing of hematological cancer cells, long-lasting stable conjugation with NK-92 cells was unable to kill HER2+ solid tumor cells (SKOV3, SKBR3) significantly. NK-92 cells that were engineered to express FcγRIII (CD16) mediated antibody-dependent cellular cytotoxicity (ADCC) selectively against HER2+ cells upon addition of Herceptin (trastuzumab). The requirement of CD16, Herceptin and specific pre-incubation temperature served as three inputs to generate a molecular logic function with HER2+ cell death as the output. Mass proteomic analysis of the two effector cell lines suggested differential changes in adhesion, exocytosis, metabolism, transport and activation of upstream regulators and cytotoxicity mediators, which can be utilized to regulate specific functionalities of NK-92 cells in future. These results suggest that this semi-automated single cell assay can reveal the variability and functional potency of NK cells and may be used to optimize immunotherapeutic efficacy for preclinical analyses.

Insluin and epithelial growth factor (EGF) promote programmed death ligand 1(PD-L1) production and transport in colon cancer stem cells

BackgroundProgrammed cell death ligand 1 (PD-L1) is an important immune-inhibitory protein expressed on cancer cells to mediate cancer escape through interaction with PD-1 expressed on activated T lymphocytes (T cells). Previously, we reported that colon and breast cancer stem cells (CSCs) expressed much higher levels of PD-L1 than their parental cells, suggesting they will be more resistant to immune attack.MethodsWe investigated the underlining mechanism of PD-L1 increase in colon CSCs, with a special focus on the effect of insulin and epithelial growth factor (EGF), the two fundamental components to sustain the metabolism and stemness in the culture of CSCs.ResultsWe found that insulin increased the total and surface PD-L1 levels through PI3K/Akt/mTOR pathway as the increase could be inhibited by the dual inhibitor of the pathway, BEZ235. EGF didn’t affect the total PD-L1 levels of CSCs but increased the cell surface protein levels by flow cytometry analysis, indicating EGF promotes the transport of PD-L1 to the cell surface. Blocking cell surface PD-L1 with a specific antibody resulted in a significant reduction of tumour sphere formation but didn’t interfere with the sphere growth, suggesting that cell surface PD-L1 may act as an adhering molecule for CSCs.ConclusionsApart from the essential roles in metabolism and stemness, insulin and EGF involve in up-regulation of PD-L1 expression in colon CSCs, therefore the inhibition of insulin and EGF/EGFR pathways can be considered for cancer immunotherapy or combined with PD-1/PD-L1 antibody-based cancer immunotherapy to eliminate CSCs.

Abstract 5760: An IgG1 version of the anti-TfR1 antibody ch128.1 shows significant antitumor activity against different xenograft models of multiple myeloma

Abstract The transferrin receptor 1 (TfR1), also identified as CD71, is a type II transmembrane homodimeric protein involved in the cellular uptake of iron and in the regulation of cell growth. TfR1 is a meaningful target for antibody-based cancer immunotherapy due to its high expression levels on the surface of cancer cells and its central role in cancer pathology. We previously developed a mouse/human chimeric IgG3 specific for human TfR1 (ch128.1), which exhibits antitumor activity in SCID-Beige mice bearing disseminated multiple myeloma (MM) KMS-11 cells. Since human IgG1 has been the isotype of choice for therapeutic antibodies targeting malignant cells and has several advantages compared to IgG3, including a more established developability, we constructed an IgG1 version of ch128.1. This novel antibody, ch128.1/IgG1, expressed in murine cells (NS0/1), is properly assembled and secreted and binds antigen (TfR1) similar to its IgG3 counterpart, as demonstrated by ELISA and flow cytometry. Administration of a single dose of the IgG1 completely blocks subcutaneous KMS-11 tumor formation in SCID-Beige mice. Importantly, a single dose of the antibody also shows significant protection, including long-term survival, in this same strain of mice bearing disseminated human MM cells KMS-11 (Asian origin) or MM.1S (African American origin), at different stages of the disease. As expected, the protection was stronger when the mice were treated at an earlier disease stage. Studies using MM.1S cells are of particular relevance given the higher incidence and mortality of MM in African Americans compared to other racial groups. Importantly, we also found that ch128.1/IgG1 confers in vivo protection against MM.1R cells, the MM.1S variant that is dexamethasone resistant. We also developed a ch128.1/IgG1 triple mutant (L234A/L235A/P329S) antibody that does not bind to FcγRs and C1q (the initial protein of the complement pathway), but retains TfR1 binding, as well as binding to FcRn, the neonatal Fc receptor (also known as the Brambell receptor), which is critical in retaining antibody bioavailability in the blood. The triple mutant failed to confer protection in vivo against KMS-11 and MM.1S cells, suggesting a critical role of the antibody Fc fragment in ch128.1/IgG1-mediated antitumor activity, consistent with a potential role of functional effector cells present in this mouse model, such as macrophages. In fact, we found that ch128.1/IgG1 elicits antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) in the presence of murine bone marrow derived macrophages as effectors and KMS-11 cells as targets, similar to its IgG3 counterpart. Taken together, our results suggest that ch128.1/IgG1 has great potential as an effective therapy for incurable human B-cell malignancies such as MM. Citation Format: Tracy R. Daniels-Wells, Pierre V. Candelaria, Lai Sum Leoh, Otoniel Martinez-Maza, Manuel L. Penichet. An IgG1 version of the anti-TfR1 antibody ch128.1 shows significant antitumor activity against different xenograft models of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5760.

Human Cell Line-Derived Monoclonal IgA Antibodies for Cancer Immunotherapy.

IgA antibodies have great potential to improve the functional diversity of current IgG antibody-based cancer immunotherapy options. However, IgA production and purification is not well established, which can at least in part be attributed to the more complex glycosylation as compared to IgG antibodies. IgA antibodies possess up to five N-glycosylation sites within their constant region of the heavy chain as compared to one site for IgG antibodies. The human GlycoExpress expression system was developed to produce biotherapeutics with optimized glycosylation and used here to generate a panel of IgA isotype antibodies directed against targets for solid (TA-mucin 1, Her2, EGFR, Thomsen–Friedenreich) and hematological (CD20) cancer indications. The feasibility of good manufacturing practice was shown by the production of 11 g IgA within 35 days in a one liter perfusion bioreactor, and IgA antibodies in high purity were obtained after purification. The monoclonal IgA antibodies possessed a high sialylation degree, and no non-human glycan structures were detected. Kinetic analysis revealed increased avidity antigen binding for IgA dimers as compared to monomeric antibodies. The IgA antibodies exhibited potent Fab- and Fc-mediated functionalities against cancer cell lines, whereby especially granulocytes are recruited. Therefore, for patients who do not sufficiently benefit from therapeutic IgG antibodies, IgA antibodies may complement current regiment options and represent a promising strategy for cancer immunotherapy. In conclusion, a panel of novel biofunctional IgA antibodies with human glycosylation was successfully generated.

Efficacy of an Anti-transferrin Receptor 1 Antibody Against AIDS-related Non-Hodgkin Lymphoma: A Brief Communication.

The transferrin receptor 1 (TfR1), also known as CD71, is a target for antibody-based cancer immunotherapy due to its high expression on the surface of cancer cells and its ability to internalize. We have previously developed a mouse/human chimeric IgG3 specific for human TfR1 genetically fused to avidin, as a vector to deliver biotinylated anticancer agents into malignant cells. However, we found that this fusion protein (ch128.1Av), and to a lesser extent the same antibody without avidin (ch128.1), exhibits direct cytotoxic activity in vitro against certain malignant hematopoietic cells through the induction of TfR1 degradation and lethal iron starvation. Importantly, both ch128.1 and ch128.1Av have also shown significant anticancer activity in 2 xenograft models of the B-cell malignancy multiple myeloma. It is interesting to note that ch128.1 exhibited superior anticancer activity in both models compared with ch128.1Av, even against malignant cells that show no sensitivity to ch128.1 in vitro. In the present study, we evaluated the efficacy of ch128.1 against an AIDS-related human Burkitt lymphoma cell line (2F7) to determine if ch128.1 can eliminate these cells in vitro and in an in vivo model of AIDS-related non-Hodgkin lymphoma (AIDS-NHL). Even though 2F7 cells expressed high TfR1 levels, these cells lacked sensitivity to the cytotoxicity induced by ch128.1 in vitro. However, ch128.1 showed significant anticancer activity against these AIDS-NHL cells in vivo by significantly prolonging the survival of immunodeficient mice bearing 2F7 tumors. Therefore, ch128.1 warrants further study as a candidate for the treatment of AIDS-NHL and other B-cell malignancies.

Abstract P5-04-02: Hyaluronan (HA) depletion sensitizes HAhigh tumors to antibody-dependent cell-mediated cytotoxicity

Abstract Therapeutic efficacy of monoclonal antibodies (MAbs) against solid tumor targets, like trastuzumab (anti-HER2) and cetuximab (anti-EGFR), have been used successfully to treat cancer, despite the many physical barriers impeding their access to the malignant cell surface1. For example, only about 50% of HER23+ patients have a durable response to therapy with trastuzumab2. Extracellular matrix (ECM)-mediated inhibition may be among the mechanisms of resistance to MAb therapy of solid tumors. Aberrant accumulation of hyaluronan (HA), a major component of the ECM in many tumors, is associated with poor prognosis and treatment-resistance in multiple malignancies3-5. We investigated HA-dependent pericellular matrix-mediated inhibition to ADCC in HAhigh human cancer cells in vitro and in vivo. We observed high levels of tumor associated HA (HA3+) in >50% of HER23+ breast adenocarcinoma and ∼40% of EGFR+ head and neck squamous cell carcinoma (HNSCC) primary tumors. Human hyaluronan synthase 2 (HAS2)-overexpressing breast cancer cells formed an HAhigh pericellular matrix, which inhibited both natural killer (NK) cell access to tumor cells and ADCC in vitro. Hyaluronan depletion by PEGPH20, a pegylated recombinant human PH20 hyaluronidase currently in clinical study for pancreatic cancer, increased NK cell access to HAS2-overexpressing breast cancer cells and greatly enhanced trastuzumab- or cetuximab-dependent ADCC. Trastuzumab and NK cell accessibility to HAS2-overexpressing tumors was enhanced following HA-depletion by PEGPH20. In an in vivo ADCC-based efficacy study, PEGPH20 treatment in combination with trastuzumab and NK cells enhanced tumor growth inhibition. This work describes a novel tumor microenvironment (TME)-dependent mechanism of inherent resistance to therapeutic antibody-mediated ADCC in vitro and in vivo, and furthermore shows that ADCC can be enhanced by hyaluronan depletion. These results may help to explain as to why tumors with high levels of HA are more aggressive, and suggest potential benefits of PEGPH20-mediated HA depletion in combination with therapeutic antibodies like trastuzumab or cetuximab in the treatment of HAhigh solid tumors.