Antibody Fusion Protein Research Articles

Effect of Tripterygium glycosides combined with Recombinant human tumor necrosis factor receptor II antibody fusion protein for injection on serum vascular endothelial growth factor, rheumatoid factor, receptor activator of nuclear factor κ B ligand levels in patients with rheumatoid arthritis

Objective To investigate the effect of tripterygium glycosides combined with recombinant human tumor necrosis factor receptor Ⅱ antibody fusion protein for injection on serum vascular endothelial growth factor(VEGF), rheumatoid factor(RF), receptor activator of nuclear factor κ B ligand(RANKL) levels in patients with rheumatoid arthritis.To provide reference for rational clinical application. Methods From December 2014 to January 2016, 132 patients with rheumatoid arthritis were divided into observation group and control group by random number table method, , with 66 cases in each group, The control group was treated with tripterygium glycosides alone (10 mg each time, 3 times daily, orally) for 3 months.The observation group was treated with a combination of tripterygium glycosides and recombinant human tumor necrosis factor receptor Ⅱ (0.4 mg/kg, once weekly, hypodermic injection)for 3 months.The clinical efficacy, and serum VEGF, RF and RANKL levels were compared between 2 groups. Results The effective rate of the observation group was 92.4% (61/66), which was significantly higher than that in the control group (80.3%, 53/66). There was a significant difference between the two groups (χ2=4.117, P 0.05). After treatment, the levels of serum VEGF, RF and RANKL in the observation group and the control group were (20.8±11.5) ng/L and (27.3±13.1)ng/L, (258.4±54.5)U/L and (298.1±49.5)U/L, (0.083±0.021) pmol/L and (0.197±0.064), respectively.There were significant differences between the two groups (t=3.029, 4.381, 13.750, all P<0.05). The incidence rate of adverse reactions in the control group was 10.5% (7/66), which was significantly higher than that in the observation group (1.5%, 1/66). There was a significant difference between the two groups(χ2=4.790, P<0.05). The one-year recurrence rate was 25.0% (13/52) in the control group and that was 6.7% (4/60) in the observation group, respectively, and there was a significant difference between the two groups(χ2=7.272, P<0.05). Conclusion Tripterygium glycosidescombined with recombinant human tumor necrosis factor receptor Ⅱ antibody fusion protein for injection is effective in the treatment of rheumatoid arthritis, which reduces the levels of serum VEGF, RF and RANKL, and has a low incidence of adverse reactions and recurrence. Key words: Tripterygium glycosides; Recombinant human tumor necrosis factor receptor Ⅱ antibody fusion protein for injection; Rheumatoid arthritis; Vascular endothelial growth factor; Rheumatoid factor; Receptor activator of nuclear factor κB ligand

MRNA: A Novel Avenue to Antibody Therapy?

First attempts to use exogenous mRNA for protein expression invivo were made more than 25 years ago. However, widespread appreciation of invitro transcribed mRNA as a powerful technology for supplying therapeutic proteins to the body has evolved only during the past few years. Various approaches to turning mRNA into a potent therapeutic have been developed. All of them share utilization of specifically designed, rather than endogenous, sequences and thorough purification protocols. Apart from this, there are two fundamental philosophies, one promoting the use of chemically modified nucleotides, the other advocating restriction to unmodified building blocks. Meanwhile, both strategies have received broad support by successful mRNA-based protein treatments in animal models. For such invivo use, specifically optimized mRNA had to be combined with potent formulations to enable efficient invivo delivery. The present review analyzes the applicability of mRNA technology to antibody therapy in all main fields: antitoxins, infectious diseases, and oncology.

TNFRSF receptor-specific antibody fusion proteins with targeting controlled Fc\u03b3R-independent agonistic activity

Antibodies specific for TNFRSF receptors that bind soluble ligands without getting properly activated generally act as strong agonists upon FcγR binding. Systematic analyses revealed that the FcγR dependency of such antibodies to act as potent agonists is largely independent from isotype, FcγR type, and of the epitope recognized. This suggests that the sole cellular attachment, achieved by Fc domain-FcγR interaction, dominantly determines the agonistic activity of antibodies recognizing TNFRSF receptors poorly responsive to soluble ligands. In accordance with this hypothesis, we demonstrated that antibody fusion proteins harboring domains allowing FcγR-independent cell surface anchoring also act as strong agonist provided they have access to their target. This finding defines a general possibility to generate anti-TNFRSF receptor antibodies with FcγR-independent agonism. Moreover, anti-TNFRSF receptor antibody fusion proteins with an anchoring domain promise superior applicability to conventional systemically active agonists when an anchoring target with localized disease associated expression can be addressed.

Single-domain antibody fusion proteins can target and shuttle functional proteins into macrophage mannose receptor expressing macrophages

The tumor microenvironment of numerous prevalent cancer types is abundantly infiltrated with tumor-associated macrophages (TAMs). Macrophage mannose receptor (MMR or CD206) expressing TAMs have been shown to be key promoters of tumor progression and major opponents of successful cancer therapy. Therefore, depleting MMR+ TAMs is an interesting approach to synergize with current antitumor therapies. We studied the potential of single-domain antibodies (sdAbs) specific for MMR to target proteins to MMR+ TAMs. Anti-MMR sdAbs were genetically coupled to a reporter protein, mWasabi (wasabi green, WG), generating sdAb “drug” fusion proteins (SFPs), referred to as WG-SFPs. The resulting WG-SFPs were highly efficient in targeting MMR+ macrophages both in vitro and in vivo. As we showed that second mitochondria-derived activator of caspase (SMAC) mimetics modulate MMR+ macrophages, we further coupled the anti-MMR sdAb to an active form of SMAC, referred to as tSMAC. The resulting tSMAC-SFPs were able to bind and upregulate caspase3/7 activity in MMR+ macrophages in vitro. In conclusion, we report the proof-of-concept of an elegant approach to conjugate anti-MMR sdAbs to proteins, which opens new avenues for targeted manipulation of MMR+ tumor-promoting TAMs.

Abstract A014: Phase I clinical trial with PD-1/MUC1 CAR-pNK92 immunotherapy

Abstract Mucin1 (MUC1) proteins represent a family of high molecular weight trans-membrane glycoproteins that protect epithelial cells and mediate signal transduction by communication with extracellular stimuli. On the other hand, MUC1 has been observed to be overexpressed and glycosylated in adenocarcinoma, making it an ideal molecular target for cancer treatment. MUC1 based antibody and specific chimeric antigen receptors (CARs) modified T/NK cells exhibit strong antibody-dependent or direct-cell cytotoxicity to MUC1 positive tumor cells. However, treatment failure with MUC1-targeted therapy was usually caused by tumor microenvironment (TME)-driven immune suppression. PD-L1 has been identified as a negative checkpoint molecule that promotes immune evasion of tumor cells. The interaction of PD-1 and PD-L1 inhibits the function of tumor-infiltrating lymphocytes (TILs) or infused T/NK cells while activating the negative immune-regulatory cells in TME, such as regulatory T-cells and MDSCs. To overcome these barriers, we engineered clinically applicable NK-92 cells by lentiviral gene transfer to express chimeric antigen receptors comprising an anti-MUC1 scFv antibody fusion protein with CD28-CD137 as a signaling moiety and truncated PD-1 peptide. NK92 cells expressing anti-MUC1 CAR specifically and efficiently lysed MUC1 positive tumor cells in vitro and in vivo. The safety of NK-92 cell administration has been demonstrated by numerous phase I clinical trials. In our study, 13 patients with different kinds of tumors (lung cancer, pancreatic cancer, colon cancer and ovarian cancer) with positive PD-L1 and MUC-1 expression were enrolled. CAR-NK cells were infused (1×109 cells/time) into these patients. Of 13 patients, 3 patients were withdrawn, 9 patients (69.2%) showed stable disease and 1 patient showed progressive disease. The cytokine level and hematologic changes were monitored to evaluate the safety. Severe cytokine storm and/or bone marrow suppression were not observed. From this phase I clinical trial we found that CAR-NK therapy has a broad prospect of application as a new cellular immunotherapy due to its stable clinical efficacy, mild side effects and ease of preparation. Citation Format: Qiao Li, Yi Wang, Ming Lin, Leiming Xia, Yangyi Bao, Xiang Sun, Lin Yang. Phase I clinical trial with PD-1/MUC1 CAR-pNK92 immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A014.

Antibody-based Delivery of TNF to the Tumor Neovasculature Potentiates the Therapeutic Activity of a Peptide Anticancer Vaccine.

There is a growing interest in the use of tumor antigens for therapeutic vaccination strategies. Unfortunately, in most cases, the use of peptide vaccines in patients does not mediate shrinkage of solid tumor masses.Experimental Design: Here, we studied the opportunity to boost peptide vaccination with F8-TNF, an antibody fusion protein that selectively delivers TNF to the tumor extracellular matrix. AH1, a model antigen to investigate CD8+ T-cell immunity in BALB/c mice, was used as vaccine. Peptide antigens alone exhibited only a modest tumor growth inhibition. However, anticancer activity could be substantially increased by combination with F8-TNF. Analysis of T cells in tumors and in draining lymph nodes revealed a dramatic expansion of AH1-specific CD8+ T cells, which were strongly positive for PD-1, LAG-3, and TIM-3. The synergistic anticancer activity, observed in the combined use of peptide vaccination and F8-TNF, was largely due to the ability of the fusion protein to induce a rapid hemorrhagic necrosis in the tumor mass, thus leaving few residual tumor cells. While the cell surface phenotype of tumor-infiltrating CD8+ T cells did not substantially change upon treatment, the proportion of AH1-specific T cells was strongly increased in the combination therapy group, reaching more than 50% of the CD8+ T cells within the tumor mass. Because both peptide vaccination strategies and tumor-homing TNF fusion proteins are currently being studied in clinical trials, our study provides a rationale for the combination of these 2 regimens for the treatment of patients with cancer.

ProBDNF inhibits the proliferation and migration of OLN‑93 oligodendrocytes.

In contrast with mature brain-derived neurotrophic factor (mBDNF), proBDNF induces cell apoptosis. However, the function of proBDNF in oligodendrocytes remains unclear. In the present study, the OLN-93 oligodendroglia cell line was utilized as an in vitro model to analyse the functions of proBDNF in oligodendroglia. p75NTR, sortilin and proBDNF were expressed in cultured OLN-93 cells. It was indicated that proBDNF inhibited OLN-93 cell proliferation in a dose-dependent manner as determined using the MTT assay and BrdU staining. Furthermore, proBDNF suppressed the migration of OLN-93 cells as demonstrated using the scratch assay. proBDNF also decreased cell viability and promoted apoptosis as indicated by activated cysteine-aspartic acid protease-3 (caspase-3) immunocytochemistry. Notably, anti-proBDNF treatment neutralized the effect of proBDNF and resulted in increased cell proliferation and migration and decreased apoptosis. However, these effects were not observed in the presence of recombinant p75NTR extracellular domain-human FC fusion protein and p75NTR antibody, indicating that proBDNF imparts its inhibitory effects on oligodendrocytes through the p75NTR signal pathway.

Antibody fusion proteins with human ribonucleases 1 to 8.

ImmunoRNases combine tumor targeting by antibodies with the cytotoxic action of ribonucleases from the RNase A superfamily. This study investigated for the first time all catalytic active human RNase A family members (1 to 8) as effector components of antibody fusion proteins. ImmunoRNase fusion proteins were constructed using the CD30-specific bivalent recombinant scFv-Fc antibody SH313-B5. Production of the resulting entirely human immunoRNases 1 to 8 was done in mammalian cells by secretion of active forms. The immunoRNases mediated CD30-specific cell binding and showed ribonucleolytic activity. Interestingly, immunoRNases 1 and 2 were active in the presence of up to 5-/20-fold molar excess of the pancreatic RNase inhibitor (RI), which is supposed to efficiently inhibit all human RNase A activity. ImmunoRNases 3, 4, 6 and 7 were only inhibited by several fold molar excess of RI, whereas immunoRNases 5 and 8 were already completely inactive at equimolar RI concentrations. Compared to free RNases, activity and RI sensitivity were not significantly changed by antibody fusion or dimerisation. ImmunoRNase3 and 5 mediated tumor growth inhibition at low nanomolar concentrations. Anti-tumor activity was antigen-specific and did not show any correlation with ribonucleolytic activity or RI sensitivity.

Preventive effect of tumor necrosis factor inhibitors versus nonsteroidal anti-inflammatory drugs on uveitis in patients with ankylosing spondylitis.

To compare the preventive effect of tumor necrosis factor (TNF) inhibitors (anti-TNF antibody and soluble TNF receptor fusion protein (TNFR)) and nonsteroidal anti-inflammatory drugs (NSAIDs) on uveitis in patients with ankylosing spondylitis (AS). This retrospective cohort study included all AS patients (n = 1055) who have been treated with either TNF inhibitor or NSAIDs at the Seoul National University Hospital from 2004 to 2016. Treatment episodes of each patient were assigned to anti-TNF antibody (n = 517), TNFR (n = 341), and NSAID (n = 704) groups. The incidence of uveitis in each group was compared using a Cox proportional hazard model. The incidence rates of uveitis before and after initiation of TNF inhibitors were also assessed. A propensity score-matched (PSM) comparison was performed for a sensitivity analysis. Uveitis was significantly less common in the anti-TNF antibody group than the NSAID group (adjusted hazard ratio (HR) 0.53; 95% confidence interval (CI) 0.29-0.96) while it was higher in the TNFR group (adjusted HR 2.25, 95% CI 1.43-3.53). Anti-TNF antibody further reduced the incidence of uveitis when prescribed with NSAIDs (combination therapy) (adjusted HR 0.39; 95% CI 0.19-0.79). Combination therapy was preventive in AS patients with a history of uveitis (adjusted HR 0.31; 95% CI 0.12-0.81), but not in those without history of uveitis. The incidence rate of uveitis fell from 6.36 to 2.60 per 100 person-years when anti-TNF antibody was added to NSAIDs. Anti-TNF antibody plus NSAIDs reduces the risk of uveitis to a greater extent than NSAIDs alone in AS patients with a history of uveitis. Combination therapy may be an effective secondary prevention measure.

Investigation of PD-1H in DEN-induced mouse liver cancer model.

Immune therapy has recently become a novel strategy for treating liver cancer, making it of critical importance to identify novel targets for treatment. Programmed death-1 homology (PD-1H) is one newly discovered negative co-stimulating molecule, and plays important regulatory roles in suppressing T cell activation. However, the expression or function of PD-1H in liver tumors has not been reported. Liver cancer tissues were collected from The Cancer Genome Atlas (TCGA) (http://tcga-data.nci-nih.gov). This study then utilized diethylnitrosamine (DEN) induced liver cancer mice, on which PD-1H monoclonal antibody and PD-1H extra-cellular Fc domain fusion protein were injected intraperitoneal. General status, gross morphology of liver tissues was examined, followed by hematoxylin-eosin (HE) staining and plotting survival curve. Among TCGA samples, PD-1H expression was significantly elevated. Induced liver cancer mice showed depressed mental status, early onset of hepatitis and liver cirrhosis. Five mice dead in model group (mortality=33.33%). No natural death occurred in control group. Injection of PD-1H-Fc-Ig fusion and PD-1H monoclonal antibody improved the condition to certain extents, with morality at about 20%. Comparing to DEN group, combined treatment group showed significantly fewer tumor lesion on liver surface, with increased body weight and lower liver-body weight ratio. HE staining showed significantly elevated ratio of normal cells in combined treatment group, although large amounts of cancer cells still existed. Blocking of PD-1H signal pathway could suppress liver cancer cell growth, decrease mouse mortality, indicating promising application of PD-1H in tumor immune therapy.

SKIK-zipbody-alkaline phosphatase, a novel antibody fusion protein expressed in Escherichia coli cytoplasm

Antibody-enzyme fusion proteins have been used for various immunological detection techniques, such as ELISA, Western blotting and so on. The use of genetically-fused antibody-enzyme complexes has advantages over conventional chemical conjugation methods, as they require no complex chemical reactions and allow for the strict control of the number of enzymes fused with antibodies, resulting in a more stable performance of the bifunctional protein. Here, we describe efficient cytoplasmic soluble expression of an antigen-binding fragment (Fab) fused with Escherichia coli alkaline phosphatase (AP), N-terminal Ser-Lys-Ile-Lys (SKIK) tag that can improve the synthesis of the tagged protein, as well as leucine zipper (LZ) to enhance the association of the light chain and the heavy chain of Fab. Our results demonstrated that the SKIK-Fab-LZ-AP fusion was well expressed in E.coli oxidative cytoplasm in soluble form having both antigen-binding and AP activity, and was purified to homogeneity by two step column chromatography, suggesting that the combination of the SKIK tag and AP fusion can greatly increase the productivity and solubility of the Fab-enzyme fusion in an E.coli cytoplasmic expression system.

Antitumor effect of recombinant Mycobacterium smegmatis expressing MAGEA3 and SSX2 fusion proteins.

Mycobacterium smegmatis (M. smegmatis), which is a nonpathogenic and fast-growing mycobacterium, is a potential vaccine vector capable of expressing heterologous antigens. Spontaneous humoral and cellular immune responses have been demonstrated against cancer/testis antigens (CTA), including melanoma-associated antigen A (MAGEA) and SSX. In the present study, recombinant plasmids expressing MAGEA3 and SSX2 were constructed. The recombinant plasmids were transferred into M. smegmatis to generate the novel antitumor DNA vaccine. As MAGEA3 and SSX2 were in different ligation sequences, the two DNA vaccines were recombinant M. smegmatis MAGEA3-SSX2 (rM.S-MS) and recombinant M. smegmatis SSX2-MAGEA3 (rM.S-SM), respectively. The expression levels of Fusion proteins were assessed by western blotting. BALB/c mice were immunized with rM.S and western blot analysis was used to determine whether antibodies against MAGEA3 or SSX2 were produced in immunized mice. EC9706 cells were inoculated into BALB/c nude mice and the mice were maintained until an obvious visible tumor appeared on the back. Subsequently, the blood from the rM.S immunized BALB/c mice was injected into the BALB/c nude mice via the tail vein. In order to evaluate the antitumor effect of the vaccines, tumor volume and weight were measured 5 to 21 days after injection. Mice were euthanized on day 21 of tumor growth, and the tumor was dissected and weighed. The two fusion proteins were expressed in the rM.S and the specific fusion protein antibodies were expressed in the blood of immunized BALB/c mice. The tumor volumes and weight in the recombinant M. smegmatis MAGEA3 (rM.S-M) and recombinant M. smegmatis SSX2 (rM.S-S) groups were significantly reduced compared with the control group. Furthermore, the decrease in tumor volumes and weight in the rM.S-MS and rM.S-SM groups was more severe than in the rM.S-M or rM.S-S groups. There was no significant difference in the antitumor effect of the rM.S-MS and rM.S-SM groups. The present findings suggest that this rM.S may be a potential candidate therapeutic vaccine for the treatment of cancer.

Expression of 5T4 extracellular domain fusion protein and preparation of anti-5T4 monoclonal antibody with high affinity and internalization efficiency

5T4, a membrane protein, is overexpressed in many tumor tissues but rarely expressed in normal tissues. Here, CHO-5T4+ cells were generated and served as the antigen to immunize mice. Hybridoma techniques were employed to produce monoclonal antibodies (mAbs). The recombinant protein of human IgG Fc-fused extracellular domain of 5T4 (5T4 ECD-Fc) was obtained from transient expression in HEK293F cells. The fusion protein 5T4 ECD-Fc and CHO-5T4+ cells were respectively utilized to screen anti-5T4 antibodies that could bind to the native antigen. In preliminary screening, three hundred and fifty mAbs were obtained. Via surface plasmon resonance and flow cytometry screening, seven anti-5T4 mAbs stood out. Among them, H6 showed a high affinity (KD = 1.6 × 10−11 M) and internalization percentage (36% for 1 h and 80% for 4 h). The molecular weight and isoelectric point of H6 were determined by LC-MS and iCIEF. Moreover, the specific reactivity of H6 was demonstrated by western blotting, flow cytometry, and immunohistochemistry, respectively. In conclusion, we produced human recombinant protein of 5T4 extracellular domain and developed high-affinity internalizing monoclonal antibodies which may be applied in the 5T4-targeting ADC therapy and basic research.

Potentiation of PD-L1 blockade with a potency-matched dual cytokine-antibody fusion protein leads to cancer eradication in BALB/c-derived tumors but not in other mouse strains.

We have recently described a novel therapeutic antibody product (IL2-F8-TNFmut), featuring the simultaneous fusion of murine IL2 and of a TNF mutant with scFv(F8), an antibody specific to the alternatively-spliced extra domain A of fibronectin (EDA). Here, we report on the in vivo characterization of the anti-cancer activity of IL2-F8-TNFmut in four immunocompetent murine models of cancer, CT26, WEHI-164, F9 teratocarcinoma and Lewis lung carcinoma (LLC), using the product alone or in combination with a monoclonal antibody specific to murine PD-L1. All four models exhibited a strong expression of EDA-fibronectin, which was confined to vascular structures for F9 tumors, while the other three malignancies exhibited a more stromal pattern of staining. A complete and long-lasting tumor eradication of CT26 and WEHI-164 tumors was observed in BALB/c mice when IL2-F8-TNFmut was used in combination with PD-L1 blockade. The combination treatment led to improved tumor growth inhibition in 129/SvEv mice bearing murine teratocarcinoma or in C57BL/6 mice bearing murine LLC, but those cancer cures were difficult to achieve in those models. A microscopic analysis of tumor sections, obtained 24h after pharmacological treatment, revealed that the PD-L1 antibody had homogenously reached tumor cells in vivo and that the combination of PD-L1 blockade with IL2-F8-TNFmut stimulated an influx of NK cells and of T cells into the neoplastic mass. These data indicate that potency-matched dual-cytokine fusion proteins may be ideally suited to potentiate the therapeutic activity of immune check-point inhibitors.

Abstract 957: Design of CD19-4-1BBL, a novel CD19-targeted 4-1BB ligand for combination therapy with CD20 T-cell bispecific antibodies and CD20 antibodies

Abstract Co-stimulation through 4-1BB has shown promising anti-tumor activity in preclinical models, but the development of 4-1BB agonistic antibodies in the clinic has been hampered by Fc-mediated liver toxicity. Here, we describe a novel CD19-targeted 4-1BB ligand designed to deliver a safe but potent 4-1BB agonist to effector T and NK cells with the goal to improve treatment of B cell malignancies. The antibody fusion protein is composed of split trimeric 4-1BB ligands and a tumor antigen targeting moiety recognizing CD19 fused to a silent Fc part (CD19-4-1BBL). The construct is devoid of FcgR-mediated crosslinking responsible for Fc-mediated toxicity and reintroduces 4-1BB hyperclustering upon binding to CD19 on B cells exclusively. In mice and cynomolgus monkeys, CD19-4-1BBL shows IgG-like pharmacokinetic properties. When cross-linked via CD19 on Non-Hodgkin Lymphoma cell lines or normal B cells, CD19-4-1BBL is biologically active in co-stimulating T cells. As 4-1BB is an inducible protein on activated T cells and NK cells, we combined CD19-4-1BBL with a T cell bispecific Ab targeting CD20 (CD20-TCB) to provide initial T cell activation while engaging with CD19+CD20+ tumor cells. In vitro, pre-treatment with CD20-TCB mediates the clustering of CD19-4-1BBL molecules to the interaction synapse of T cells and tumor cells. Live imaging revealed that the addition of CD19-4-1BBL induces significantly prolonged T cell-tumor contact (&amp;gt;30 min), leading to fast and efficient killing of tumor cells. In vivo in WSU-DLCL2-bearing human stem cell engrafted NSG mice (HSC-NSG) mice, CD20-TCB treatment quickly up-regulates 4-1BB on activated T cells, resulting in tumor growth inhibition. The combination of CD19-4-1BBL and CD20-TCB (used at a suboptimal dose) synergizes to eradicate the tumor completely. The combination induces strong T cell infiltration into the tumor, accompanied by an elevated CD8/Treg ratio, as compared to the monotherapies. Similarly, the combination also induces complete remission in a “difficult-to-treat” Nalm6 tumor model associated with low and patchy CD20 expression, mimicking patterns seen in the ABC subtype of DLBCL. Finally, the combination of CD19-4-1BBL and the ADCC-enhanced Type II CD20 antibody obinutuzumab induces complete tumor remissions in WSU-DLCL2 tumor-bearing HSC-NSG mice, confirming effective 4-1BB co-stimulation on NK cells. Taken together, tumor-targeted cross-linking of 4-1BB mediated by CD19-4-1BBL provides safe and efficient co-stimulation of T cells that are pre-activated by a TCB, or of NK cells pre-activated by an ADCC mediating antibody. This novel and effective combination immunotherapy warrants clinical investigation and offers possible “chemo-free” treatment of B cell malignancies. Citation Format: Wei Xu, Johannes Sam, Mario Perro, John Challier, Christina Claus, Stanford Chen, Claudia Ferrara Koller, Michael Mølhøj, Stella Tournaviti, Marina Bacac, Tom Moore, Christian Klein, Pablo Umana. Design of CD19-4-1BBL, a novel CD19-targeted 4-1BB ligand for combination therapy with CD20 T-cell bispecific antibodies and CD20 antibodies [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 957.

Abstract 2785: Potency-matched dual cytokine antibody fusion proteins in combination with PD-L1 blockade

Abstract Among the different pharmacological approaches for cancer treatment, antibody-cytokine fusion proteins (also called “immunocytokines”) represent an emerging class of biopharmaceutical products, capable of boosting the immune system to attack tumor cells. These products promote the selective accumulation of pro-inflammatory molecules at the site of disease, mediated by the antibody fragment. These fusion proteins have been designed to reduce the systemic toxicity of traditional cytokines, currently approved for certain malignancies. Immunocytokine products based on interleukin-2 (IL2), interleukin-12 (IL12) or tumor necrosis factor (TNF) have been extensively studied in mouse model of cancer and in patients. When used as single agents, immunocytokines are often not able to cure mice and patients. However it as been shown by our group that the combination of IL2- and TNF- based products was capable to completely eradicate tumors in mouse models and to induce complete responses in patients. The development of combination products at the industrial level leads to a duplication of activities and costs. On the other hand, the opportunity to develop products with two payloads fused into a single molecular entity may facilitate development. This approach is limited by the fact that different cytokines often show different maximal tolerated doses (MTDs), and their relative potency need to be tuned. For example, TNF is administered at a dose, which is ten times lower compered to the one of IL2. We have recently described a novel class of immunocytokine products, termed “potency-matched dual cytokine antibody fusion proteins” consisting of a tumor-targeting antibody (for example F8, specific to the alternatively spliced EDA domain of fibronectin), simultaneously fused to both IL2 and TNF. To match biological activity of the two payloads a single-point mutation was inserted in the TNF sequence and the resulting product, named IL2-F8-TNFmut was able to selectively localize to lesions with an excellent tumor-to-organ ratios and it was found to completely eradicate soft-tissue sarcomas in immunocompetent mice, which did not respond to standard chemotherapy. We now report that complete responses were observed in a variety of mouse models of cancer and that the therapeutic efficacy of IL2-F8-TNFmut was further improved when used in combination with an anti-PD-L1 antibody. Potency-matched dual cytokine antibody fusion proteins may find clinical applications for various types of malignancies, used either as single agents or in combination with immune-checkpoint inhibitors. Citation Format: Roberto De Luca, Dario Neri. Potency-matched dual cytokine antibody fusion proteins in combination with PD-L1 blockade [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 2785.

Domain II of Pseudomonas Exotoxin Is Critical for Efficacy of Bolus Doses in a Xenograft Model of Acute Lymphoblastic Leukemia.

Moxetumomab pasudotox is a fusion protein of a CD22-targeting antibody and Pseudomonas exotoxin. Minutes of exposure to Moxetumomab achieves similar cell killing than hours of exposure to a novel deimmunized variant against some acute lymphoblastic leukemia (ALL). Because blood levels fall quickly, Moxetumomab is more than 1000-fold more active than the deimmunized variant in vivo. We aimed to identify which part of Moxetumomab increases in vivo efficacy and generated five immunotoxins, tested time-dependent activity, and determined the efficacy in a KOPN-8 xenograft model. Full domain II shortened the time cells had to be exposed to die to only a few minutes for some ALL; deimmunized domain III consistently extended the time. Against KOPN-8, full domain II accelerated time to arrest protein synthesis by three-fold and tripled PARP-cleavage. In vivo efficacy was increased by more than 10-fold by domain II and increasing size, and therefore half-life enhanced efficacy two- to four-fold. In summary, in vivo efficacy is determined by the time cells have to be exposed to immunotoxin to die and serum half-life. Thus, domain II is most critical for activity against some ALL treated with bolus doses; however, immunotoxins lacking all but the furin-cleavage site of domain II may be advantageous when treating continuously.

A novel dual-cytokine–antibody fusion protein for the treatment of CD38-positive malignancies

A novel dual-cytokine-antibody fusion protein, consisting of an antibody directed against CD38 [a tumor-associated antigen mainly expressed on the surface of multiple myeloma (MM) cells], simultaneously fused to both tumor necrosis factor ligand superfamily member 10 (TRAIL) and interleukin-2 (IL2), was designed, expressed and purified to homogeneity. The novel fusion protein, termed IL2-αCD38-αCD38-scTRAIL, was able to selectively recognize its cognate antigen expressed on the surface of MM and lymphoma cell lines, as evidenced by flow cytometry analysis. Moreover, the targeted version of TRAIL was able to induce cancer cell death in vitro, both with MM cell lines and with fresh isolates from the bone marrow of MM patients. The experiments provide a rationale for possible future applications of IL2-αCD38-αCD38-scTRAIL for the treatment of patients with MM or other CD38-positive malignancies.

A novel bispecific antibody fusion protein co-targeting EGFR and CD47 with enhanced therapeutic index.

To promote targeting specificity of anti-CD47 agents, we have constructed a novel bispecific antibody fusion protein against EGFR and CD47, which may minimize the "off-target" effects caused by CD47 expression on red blood cells. The novel bispecific antibody fusion protein, denoted as Bi-SP could simultaneously bind to EGFR and CD47 and exhibited potent phagocytosis-stimulation effects in vitro. Bi-SP treatment with a low dose more effectively inhibited tumor growth than either EGFR-targeting antibody, Pan or the SIRPα variant-Fc (SIRPαV-Fc) in the A431 xenograft tumor model. In addition, the treatment with Bi-SP produced less red blood cell (RBC) losses than the SIRPαV-Fc treatment, suggesting its potential use for minimizing RBC toxicity in therapy. Bi-SP with improved therapeutic index has the potential to treat CD47+ and EGFR+ cancers in clinics.

Novel antibody-cytokine fusion proteins featuring granulocyte-colony stimulating factor, interleukin-3 and interleukin-4 as payloads

Neutrophils can strongly influence disease activity in cancer and in chronic inflammation. Here, we report for the first time the construction and characterization of antibody-fusion proteins featuring granulocyte-colony stimulating factor and interleukin-3 as payloads capable of enhancing neutrophil activity and a novel antibody-interleukin-4 fusion protein with neutrophil inhibitory potential. We used the F8 antibody specific to the alternatively-spliced extra domain A (EDA) of fibronectin as a targeting agent, since the cognate antigen is strongly upregulated in diseases characterized by angiogenesis.The fusion proteins GCSF-F8, F8-IL3 and F8-IL4-F8, were cloned, expressed, and their targeting ability assessed, exhibiting preferential tumor uptake with tumor:blood ratios at 24 h after injection of 3.3, 18.2 and 27.3, respectively.In F9 tumor bearing-mice GCSF-F8 and F8-IL3 did not provide a therapeutic benefit, while F8-IL4-F8 showed a potent tumor growth retardation. In the collagen-induced model of arthritis, GCSF-F8 and F8-IL3 induced a worsening of the disease, while F8-IL4-F8 slowed arthritis progression but, surprisingly, exhibited substantial toxicity when used in combination with dexamethasone.Collectively, the results indicate that the novel fusion proteins could be expressed and efficiently delivered to the site of disease. However, they were not superior to other antibody-cytokine fusions previously described by our laboratory.