CD19-CD28: An affinity-optimized CD28 agonist for combination with glofitamab (CD20-TCB) as off-the-shelf immunotherapy.

Effective T cell responses not only require the engagement of T cell receptors (TCRs, "signal 1"), but also the availability of costimulatory signals ("signal 2"). T cell bispecific antibodies (TCBs) deliver a robust signal 1 by engaging the TCR signaling component CD3ε, while simultaneously binding to tumor antigens. The CD20-TCB glofitamab redirects T cells to CD20-expressing malignant B cells. While glofitamab exhibits strong single agent efficacy, adding costimulatory signaling may enhance the depth and durability of T cell-mediated tumor cell killing. We developed a bispecific CD19-targeted CD28 agonist (RG6333, CD19-CD28) to enhance the efficacy of glofitamab and similar TCBs by delivering signal 2 to tumor-infiltrating T cells. CD19-CD28 distinguishes itself from the superagonistic antibody TGN1412, as its activity requires the simultaneous presence of a TCR signal and CD19 target binding. This is achieved through its engineered format incorporating a mutated Fc region with abolished FcγR and C1q binding, CD28 monovalency, and a moderate CD28 binding affinity. In combination with glofitamab, CD19-CD28 strongly increased T cell effector functions in ex vivo assays using lymphoma patient-derived PBMC and spleen samples, and enhanced glofitamab-mediated regression of aggressive lymphomas in humanized mice. Notably, the triple combination of glofitamab with CD19-CD28 with the costimulatory 4-1BB agonist CD19-4-1BBL, offered substantially improved long-term tumor control over glofitamab monotherapy and respective duplet combinations. Our findings highlight CD19-CD28 as a safe and highly efficacious off-the-shelf combination partner for glofitamab, similar TCBs, and other costimulatory agonists. CD19-CD28 is currently in a Phase 1 clinical trial in combination with glofitamab.

Biodistribution of Drug/ADA Complexes: The Impact of Immune Complex Formation on Antibody Distribution.

The clinical use of therapeutic monoclonal antibodies (mAbs) for the treatment of cancer, inflammation, and other indications has been successfully established. A critical aspect of drug-antibody pharmacokinetics is immunogenicity, which triggers an immune response via an anti-drug antibody (ADA) and forms drug/ADA immune complexes (ICs). As a consequence, there may be a reduced efficacy upon neutralization by ADA or an accelerated drug clearance. It is therefore important to understand immunogenicity in biological therapies. A drug-like immunoglobulin G (IgG) was radiolabeled with tritium, and ICs were formed using polyclonal ADA, directed against the complementary-determining region of the drug-IgG, to investigate in vivo biodistribution in rodents. It was demonstrated that 65% of the radioactive IC dose was excreted within the first 24 h, compared with only 6% in the control group who received non-complexed 3H-drug. Autoradiographic imaging at the early time point indicated a deposition of immune complexes in the liver, lung, and spleen indicated by an increased radioactivity signal. A biodistribution study confirmed the results and revealed further insights regarding excretion and plasma profiles. It is assumed that the immune complexes are readily taken up by the reticuloendothelial system. The ICs are degraded proteolytically, and the released radioactively labeled amino acids are redistributed throughout the body. These are mainly renally excreted as indicated by urine measurements or incorporated into protein synthesis. These biodistribution studies using tritium-labeled immune complexes described in this article underline the importance of understanding the immunogenicity induced by therapeutic proteins and the resulting influence on biological behavior.

ROCKETS - a novel one-for-all toolbox for light sheet microscopy in drug discovery.

Advancing novel immunotherapy strategies requires refined tools in preclinical research to thoroughly assess drug targets, biodistribution, safety, and efficacy. Light sheet fluorescence microscopy (LSFM) offers unprecedented fast volumetric ex vivo imaging of large tissue samples in high resolution. Yet, to date laborious and unstandardized tissue processing procedures have limited throughput and broader applications in immunological research. Therefore, we developed a simple and harmonized protocol for processing, clearing and imaging of all mouse organs and even entire mouse bodies. Applying this Rapid Optical Clearing Kit for Enhanced Tissue Scanning (ROCKETS) in combination with LSFM allowed us to comprehensively study the in vivo biodistribution of an antibody targeting Epithelial Cell Adhesion Molecule (EpCAM) in 3D. Quantitative high-resolution scans of whole organs did not only reveal known EpCAM expression patterns but, importantly, uncovered several new EpCAM-binding sites. We identified gustatory papillae of the tongue, choroid plexi in the brain and duodenal papillae as previously unanticipated locations of high EpCAM expression. Subsequently, we confirmed high EpCAM expression also in human tongue and duodenal specimens. Choroid plexi and duodenal papillae may be considered as particularly sensitive sites due to their importance for liquor production or as critical junctions draining bile and digestive pancreatic enzymes into the small bowel, respectively. These newly gained insights appear highly relevant for clinical translation of EpCAM-addressing immunotherapies. Thus, ROCKETS in combination with LSFM may help to set new standards for preclinical evaluation of immunotherapeutic strategies. In conclusion, we propose ROCKETS as an ideal platform for a broader application of LSFM in immunological research optimally suited for quantitative co-localization studies of immunotherapeutic drugs and defined cell populations in the microanatomical context of organs or even whole mice.

Clearance of therapeutic antibody glycoforms after subcutaneous and intravenous injection in a porcine model.

A relatively low clearance is one of the prominent favorable features of immunoglobulin G1-based therapeutic monoclonal antibodies (mAbs). Various studies have observed differential clearance of mAb glycoforms, including oligomannose glycoforms, which are considered a critical quality attribute because they show higher clearance than complex type glycoforms. Glycoform clearance, however, has not previously been studied after subcutaneous injection or in a porcine model system. Here, we performed glycoform-resolved pharmacokinetic (PK) analysis of two mAbs in Göttingen minipigs. We found glycoform effects on clearance to be largely the same for subcutaneous and intravenous injection and in line with observations in other species. Oligomannose glycoforms were cleared up to 25% faster and monoantennary glycoforms up to 8% faster than agalactosylated complex glycoforms. Sialylated glycoforms were cleared at approximately the same rate as fully galactosylated glycoforms. Importantly, we report here an impact of galactosylation on the PK of a mAb for the first time. Whether increased galactosylation led to slower or faster clearance seemed to depend on the overall glycosylation profile. When clearance of galactosylated glycoforms was slower, the mAb showed higher galactosylation in serum at maximum concentration after subcutaneous injection compared to both intravenous injection and the injected material. Whether this higher galactosylation after subcutaneous injection has consequences for therapeutic efficacy remains to be investigated. In conclusion, preferential clearance of antibody glycoforms can be simulated in the minipig model with intravenous as well as subcutaneous injections. Furthermore, we observed a glycoform bias in the absorption from skin into circulation after subcutaneous injection based on galactosylation.Abbreviations: AUC - area under the curve; CL/F - apparent clearance as a function of bioavailability following SC administration; Cmax - maximum serum concentration; CQA critical quality attribute; FcγR - Fc gamma receptor; IgG - immunoglobulin G; IV - intravenous; LC-MS - liquid chromatography - mass spectrometry; mAb - therapeutic monoclonal antibody; PK - pharmacokinetics; SC - subcutaneous; TMDD - target-mediated drug disposition.

Translating pharmacology models effectively to predict therapeutic benefit.

Many in vitro and in vivo models are used in pharmacological research to evaluate the role of targeted proteins in a disease. Understanding the translational relevance and limitation of these models for analyzing a drug's disposition, pharmacokinetic/pharmacodynamic (PK/PD) profile, mechanism, and efficacy, is essential when selecting the most appropriate model of the disease of interest and predicting clinically efficacious doses of the investigational drug. Selected animal models used in ophthalmology, infectious diseases, oncology, autoimmune diseases, and neuroscience are reviewed here. Each area has specific challenges around translatability and determination of an efficacious dose: new patient-specific dosing methods may help overcome these limitations.

The impact of immunogenicity on therapeutic antibody pharmacokinetics: A preclinical evaluation of the effect of immune complex formation and antibody effector function on clearance.

The occurrence of an immune response against therapeutic proteins poses a major risk for the development of biologics and for successful treatment of patients. Generation of anti-drug antibodies (ADAs) can lead to formation of immune complexes (ICs), consisting of drug and ADAs, with potential impact on safety, efficacy and exposure. Here, we focus on the effects of IC formation, i.e., specific IC sizes, ADA and drug properties, on drug pharmacokinetics. Pre-formed IC preparations of an IgG1 drug (with wild type or with an ablated effector function at the Fc domain) and different ADA surrogates (directed against the complementarity-determining regions or Fc domain of the drug) were administered to rats and collected serum was analyzed to determine the total drug concentration. A combination of size-exclusion chromatography and ELISA enabled a size-specific evaluation of IC profiles in serum and their changes over time. Within five minutes, total drug concentration decreased by ~20-60% when the drug was complexed. Independent of the ADA surrogate and drug variant used, increasing IC size led to increased clearance. Comparing ICs formed with the same ADA surrogate but different IgG1 variants, we observed that complexed drug with a wildtype Fc domain showed faster clearance compared to immune effector function modified drug. Data generated in this study indicated that clearance of drug due to ADA generation is driven by size and structure of the formed ICs, but also by the immune effector functions of the Fc domains of IgGs.Abbreviations Ab: antibody, ADA: anti-drug antibody, AUC: area under the curve, Bi: biotin, CDR: complementary-determining region, cmax: maximal concentration, Dig: digoxigenin, ELISA: enzyme-linked immunosorbent assay, Fc: fragment crystallizable, FcRn: neonatal Fc receptor, HMW: high molecular weight, IC: immune complex, IC-QC: immune complex quality control, IgG: immunoglobulin G, mAb: monoclonal antibody, mADA: monoclonal ADA, pAb: polyclonal antibody, pADA: polyclonal ADA, PD: pharmacodynamics; PK: pharmacokinetic, QC: quality control, SEC: size-exclusion chromatography, WT: wildtype.

Fibroblast Activation Protein α-Targeted CD40 Agonism Abrogates Systemic Toxicity and Enables Administration of High Doses to Induce Effective Antitumor Immunity.

PURPOSE: CD40 agonists hold great promise for cancer immunotherapy (CIT) as they enhance dendritic cell (DC) activation and concomitant tumor-specific T-cell priming. However, the broad expression of CD40 accounts for sink and side effects, hampering the efficacy of anti-CD40 antibodies. We hypothesized that these limitations can be overcome by selectively targeting CD40 agonism to the tumor. Therefore, we developed a bispecific FAP-CD40 antibody, which induces CD40 stimulation solely in presence of fibroblast activation protein α (FAP), a protease specifically expressed in the tumor stroma. EXPERIMENTAL DESIGN: FAP-CD40's in vitro activity and FAP specificity were validated by antigen-presenting cell (APC) activation and T-cell priming assays. In addition, FAP-CD40 was tested in subcutaneous MC38-FAP and KPC-4662-huCEA murine tumor models. RESULTS: FAP-CD40 triggered a potent, strictly FAP-dependent CD40 stimulation in vitro. In vivo, FAP-CD40 strongly enhanced T-cell inflammation and growth inhibition of KPC-4662-huCEA tumors. Unlike nontargeted CD40 agonists, FAP-CD40 mediated complete regression of MC38-FAP tumors, entailing long-term protection. A high dose of FAP-CD40 was indispensable for these effects. While nontargeted CD40 agonists induced substantial side effects, highly dosed FAP-CD40 was well tolerated. FAP-CD40 preferentially accumulated in the tumor, inducing predominantly intratumoral immune activation, whereas nontargeted CD40 agonists displayed strong systemic but limited intratumoral effects. CONCLUSIONS: FAP-CD40 abrogates the systemic toxicity associated with nontargeted CD40 agonists. This enables administration of high doses, essential for overcoming CD40 sink effects and inducing antitumor immunity. Consequently, FAP-targeted CD40 agonism represents a promising strategy to exploit the full potential of CD40 signaling for CIT.

In Vivo Reoxidation Kinetics of Free Thiols in Multiple Domains of IgG1 Antibodies in Rats.

While free thiols in monoclonal antibodies (mAbs) have been extensively characterized by in vitro studies to probe its effect on antibody function and stability, their in vivo biotransformation has not been comprehensively studied. In this study, a panel of five recombinant IgG1 mAbs with elevated free thiols in the VH, VL, and CH2 domains were intravenously administered into Wistar rats. In vivo biotransformation of thirty-five free thiol sites in total (7 disulfide pairs in VL, CL, VH, CH1, HH, CH2, CH3 domains across the 5 mAbs) were monitored using a denaturing differential isotopic tagging procedure on immunopurified timepoints followed by LC-MS of tryptic digests. The free thiol levels in two VH domain and one CH2 domain disulfide sites decreased in vivo following first order kinetics. Free thiol levels of the remaining 32 sites were remarkably stable in vivo. Further analytical characterization highlighted a positive association between a free thiol's solvent accessibility and a free thiol's reoxidation propensity. The data and discussion presented here shed valuable insights into the in vivo fate of free thiols in several recombinant IgG1s and its implications for free thiols as a product quality attribute in therapeutic mAb products.

Glycoform-resolved pharmacokinetic studies in a rat model employing glycoengineered variants of a therapeutic monoclonal antibody.

Good pharmacokinetic (PK) behavior is a key prerequisite for sufficient efficacy of therapeutic monoclonal antibodies (mAbs). Fc glycosylation is a critical quality attribute (CQA) of mAbs, due to its impact on stability and effector functions. However, the effects of various IgG Fc glycoforms on antibody PK remain unclear. We used a combination of glycoengineering and glycoform-resolved PK measurements by mass spectrometry (MS) to assess glycoform effects on PK. Four differently glycoengineered mAbs, each still containing multiple glycoforms, were separately injected into rats. Rat models have been shown to be predictive of human PK. At different time points, blood was taken, from which the mAbs were purified and analyzed with a liquid chromatography-MS-based bottom-up glycoproteomics approach. This allowed us to follow changes in the glycosylation profiles of each glycoengineered mAb over time. Enzyme-linked immunosorbent assay measurements provided an absolute concentration in the form of a sum value for all glycoforms. Information from both readouts was then combined to calculate PK parameters per glycoform. Thereby, multiple glycoform kinetics were resolved within one mAb preparation. We confirmed increased clearance of high-mannose (Man5) and hybrid-type (Man5G0) glycoforms. Specifically, Man5 showed a 1.8 to 2.6-fold higher clearance than agalactosylated, complex glycans (G0F). Unexpectedly, clearance was even higher (4.7-fold) for the hybrid-type glycan Man5G0. In contrast, clearance of agalactosylated, monoantennary glycoforms (G0F-N) was only slightly increased over G0F (1.2 to 1.4-fold). Thus, monoantennary, hybrid-type and high-mannose glycoforms should be distinguished in CQA assessments. Strikingly, α2,3-linked sialylation did not affect clearance, contradicting the involvement of the asialoglycoprotein receptor in mAb clearance.

Comparative assessment of immune complex-mediated hypersensitivity reactions with biotherapeutics in the non-human primate: Critical parameters, safety and lessons for future studies.

With the emergence of novel biotherapeutic formats and immunostimulatory biotherapeutics in cancer immunotherapy, an understanding of immune-complex (IC) mediated hypersensitivity reactions in toxicology studies - and their differentiation from pharmacology - remains key to the preclinical evaluation of these drugs. In this review we provide an in-depth evaluation and comparison of case examples where IC-mediated hypersensitivity reactions were observed in cynomolgus monkeys. We provide details of the parameters evaluated in each study to substantiate and guide the interpretation of these findings. Five study cases (1 therapeutic protein, 4 monoclonal antibodies) are discussed for which effects ranged from minor to fatal. Common characteristics are the high incidence of clinical signs, detectable antidrug antibodies, and accelerated drug clearance up to virtual loss of exposure. In our experience, measurement of cytokine levels in vivo and detection of complement (split products) were supportive markers in situations where coagulopathy was suspected to play a role in the observed effects. Recommendations are outlined to prepare for root-cause analysis of suspected hypersensitivity reactions. Overall, a thorough analysis of the findings has helped to start clinical trials despite major findings. The hypersensitivity reactions with our human(ized) immunoglobulins have not proven to be predictive for humans.

Characterization of a re-engineered, mesothelin-targeted Pseudomonas exotoxin fusion protein for lung cancer therapy.

Mesothelin overexpression in lung adenocarcinomas correlates with the presence of activating KRAS mutations and poor prognosis. Hence SS1P, a mesothelin-targeted immunotoxin, could offer valuable treatment options for these patients, but its use in solid tumor therapy is hampered by high immunogenicity and non-specific toxicity. To overcome both obstacles we developed RG7787, a de-immunized cytotoxic fusion protein comprising a humanized SS1 Fab fragment and a truncated, B-cell epitope silenced, 24 kD fragment of Pseudomonas exotoxin A (PE24). Reactivity of RG7787 with sera from immunotoxin-treated patients was >1000 fold reduced. In vitro RG7787 inhibited cell viability of lung cancer cell lines with picomolar potency. The pharmacokinetic properties of RG7787 in rodents were comparable to SS1P, yet it was tolerated up to 10 fold better without causing severe vascular leak syndrome or hepatotoxicity. A pharmacokinetic/pharmacodynamic model developed based on NCI-H596 xenograft studies showed that for RG7787 and SS1P, their in vitro and in vivo potencies closely correlate. At optimal doses of 2-3 mg/kg RG7787 is more efficacious than SS1P. Even large, well established tumors (600 mm(3)) underwent remission during three treatment cycles with RG7787. Also in two patient-derived lung cancer xenograft models, Lu7336 and Lu7187, RG7787 showed anti-tumor efficacy. In monotherapy two treatment cycles were moderately efficacious in the Lu7336 model but showed good anti-tumor activity in the KRAS mutant Lu7187 model (26% and 80% tumor growth inhibition, respectively). Combination of RG7787 with standard chemotherapies further enhanced efficacy in both models achieving near complete eradication of Lu7187 tumors.

Immunopurification and mass spectrometric quantification of the active form of a chimeric therapeutic antibody in human serum.

In this study, we show that liquid chromatography coupled with tandem mass spectrometry provides a sensitive, specific, and accurate absolute quantification of Erbitux, a human:murine chimeric mAb used for the treatment of colorectal cancer. Micrometric magnetized beads, functionalized with soluble epidermal growth factor receptor (sEGFR), the pharmacological target of Erbitux, were used for specific immunocapture of Erbitux allowing assessment of the antibody's biological potency and sample purification. Following digestion with trypsin, specific peptides from light and heavy chains were monitored in the selected reaction monitoring (SRM) mode. Assay variability below 20% was provided through optimization of the digestion step and rigorous monitoring of the whole analytical process using an appropriate internal standard. The 20 ng/mL lower limit of quantification was similar to that of ELISA methods. These results show that this mass spectrometric approach is a potential alternative for pharmacokinetic evaluation of mAbs during clinical development.

Vaccination trial with HPV16 L1E7 chimeric virus-like particles in women suffering from high grade cervical intraepithelial neoplasia (CIN 2/3).

Persistent infection with human papillomaviruses (HPV) is a prerequisite for the development of cervical cancer. Vaccination with virus-like particles (VLP) has demonstrated efficacy in prophylaxis but lacks therapeutic potential. HPV16 L1E7 chimeric virus-like particles (CVLP) consist of a carboxy-terminally truncated HPV16L1 protein fused to the amino-terminal part of the HPV16 E7 protein and self-assemble by recombinant expression of the fusion protein. The CVLP are able to induce L1- and E7-specific cytotoxic T lymphocytes. We have performed a first clinical trial to gain information about the safety and to generate preliminary data on the therapeutic potential of the CVLP in humans. A randomized, double blind, placebo-controlled clinical trial has been conducted in 39 HPV16 mono-infected high grade cervical intraepithelial neoplasia (CIN) patients (CIN 2/3). Two doses (75 mug or 250 mug) of CVLP were applied. The duration of the study was 24 weeks with 2 optional visits after another 12 and 24 weeks. The vaccine showed a very good safety profile with only minor adverse events attributable to the immunization. Antibodies with high titers against HPV16 L1 and low titers against HPV16 E7 as well as cellular immune responses against both proteins were induced. Responses were equivalent for both vaccine concentrations. A trend for histological improvement to CIN 1 or normal was seen in 39% of the patients receiving the vaccine and only 25% of the placebo recipients. Fifty-six percent of the responders were also HPV16 DNA-negative by the end of the study. Therefore, we demonstrated evidence for safety and a nonsignificant trend for the clinical efficacy of the HPV16 L1E7 CVLP vaccine.

Efficacy of oncolytic herpesvirus NV1020 can be enhanced by combination with chemotherapeutics in colon carcinoma cells.

NV1020, an oncolytic herpes simplex virus type 1, can destroy colon cancer cells by selectively replicating within these cells, while sparing normal cells. NV1020 is currently under investigation in a clinical phase I/II trial as an agent for the treatment of colon cancer liver metastases, in combination with conventional chemotherapeutic agents such as 5-fluorouracil (5-FU), SN38 (the active metabolite of irinotecan), and oxaliplatin. To study the synergy of NV1020 and chemotherapy, cytotoxicity and viral replication were evaluated in vitro by treating various human and murine colon carcinoma cell lines, using a colorimetric viability assay, a clonogenic assay, and a plaque-forming assay. In vivo experiments, using a subcutaneous syngeneic CT-26 tumor model in BALB/c mice, were performed to determine the efficacy of combination therapy. In vitro studies showed that the efficacy of NV1020 on human colon carcinoma cell lines HT-29, WiDr, and HCT-116 was additively or synergistically enhanced in combination with 5-FU, SN38, or oxaliplatin. The sequence of application was not important and effects were still apparent after a 21-day incubation period. Three intra-tumoral treatments with NV1020 (1 x 10(7) plaque-forming units), followed by three subcutaneous treatments with 5-FU (50 mg/kg), resulted in substantially higher inhibition of tumor growth and prolongation of survival compared with monotherapies (NV1020/5-FU vs. NV1020, p = 0.027). On WiDr cells, reduced replication of NV1020, in combination with 5-FU, indicated that additive and synergistic effects of combination therapy must be independent from viral replication. These results suggest that NV1020, in combination with chemotherapy, is a promising therapy for treating patients with metastatic colorectal cancer of the liver. We hypothesize that infection of cells with NV1020 sensitizes the infected cells for the cytotoxic effect of the chemotherapeutics.

Immunization with hepatitis C virus-like particles protects mice from recombinant hepatitis C virus-vaccinia infection.

We have recently demonstrated that immunization with hepatitis C virus-like particles (HCV-LPs) generated in insect cells can elicit both humoral and cellular immune responses in BALB/c mice. Here, we evaluate the immunogenicity of HCV-LPs in HLA2.1 transgenic (AAD) mice in comparison to DNA immunization. HCV-LP immunization elicited a significantly stronger humoral immune response than DNA immunization. HCV-LP-immunized mice also developed stronger HCV-specific cellular immune responses than DNA-immunized mice as determined by using quantitative enzyme-linked immunospot (ELISpot) assay and intracellular cytokine staining. In BALB/c mice, immunization with HCV-LPs resulted in a >5 log10 reduction in vaccinia titer when challenged with a recombinant vaccinia expressing the HCV structural proteins (vvHCV.S), as compared to 1 log10 decrease in DNA immunization. In HLA2.1 transgenic mice, a 1-2 log10 reduction resulted from HCV-LP immunization, whereas no reduction was seen from DNA immunization. Adoptive transfer of lymphocytes from HCV-LP-immunized mice to naive mice provided protection against vvHCV.S challenge, and this transferred immunity can be abrogated by either CD4 or CD8 depletion. Our results suggest that HCV-LPs can induce humoral and cellular immune responses that are protective in a surrogate HCV challenge model and that a strong cellular immunity provided by both CD4 and CD8 effector lymphocytes may be important for protection from HCV infection.