Electrospun patch delivery of anti-TNFα F(ab) for the treatment of inflammatory oral mucosal disease.

Chronic ulcerative oral mucosal inflammatory diseases, including oral lichen planus and recurrent aphthous stomatitis, are painful and highly prevalent, yet lack effective clinical management. In recent years, systemic biologic therapies, including monoclonal antibodies that block the activity of cytokines, have been increasingly used to treat a range of immune-mediated inflammatory conditions such as rheumatoid arthritis and psoriasis. The ability to deliver similar therapeutic agents locally to the oral epithelium could radically alter treatment options for oral mucosal inflammatory diseases, where pro-inflammatory cytokines, in particular tumour-necrosis factor-α (TNFα), are major drivers of pathogenesis. To address this, an electrospun dual-layer mucoadhesive patch comprising medical-grade polymers was investigated for the delivery of F(ab) biologics to the oral mucosa. A fluorescent-labelled F(ab) was incorporated into mucoadhesive membranes using electrospinning with 97% v/v ethanol as a solvent. The F(ab) was detected within the fibres in aggregates when visualised by confocal microscopy. Biotinylated F(ab) was rapidly eluted from the patch (97 ± 5% released within 3 h) without loss of antigen-binding activity. Patches applied to oral epithelium models successfully delivered the F(ab), with fluorescent F(ab) observed within the tissue and 5.1 ± 1.5% cumulative transepithelial permeation reached after 9 h. Neutralising anti-TNFα F(ab) fragments were generated from whole IgG by papain cleavage, as confirmed by SDS-PAGE, then incorporated into patches. F(ab)-containing patches had TNFα neutralising activity, as shown by the suppression of TNFα-mediated CXCL8 release from oral keratinocytes cultured as monolayers. Patches were applied to lipopolysaccharide-stimulated immune-competent oral mucosal ulcer equivalents that contained primary macrophages. Anti-TNFα patch treatment led to reduced levels of active TNFα along with a reduction in the levels of disease-implicated T-cell chemokines (CCL3, CCL5, and CXCL10) to baseline concentrations. This is the first report of an effective device for the delivery of antibody-based biologics to the oral mucosa, enabling the future development of new therapeutic strategies to treat painful conditions.

Design of a Novel Fab-Like Antibody Fragment with Enhanced Stability and Affinity for Clinical use.

With increasing interest in applying recombinant monoclonal antibodies (mAbs) in human medicine, engineered mAb fragments with reduced size and improved stability are in demand to overcome current limitations in clinical use. Herein, a novel Fab-like antibody fragment generated via an in silico-based engineering approach where the CH1 and CL domains of Fab are replaced by the IgG1 CH3 domains is described. This construct, designated as FabCH3, maintains the natural N-terminus and C-terminus of IgG antibody, can be expressed at a high level in bacterial cells and, importantly, exhibits much higher stability and affinity than the parental Fab when tested in a mesothelin-specific Fab m912, as well as a vascular endothelial growth factor A (VEGFA)-specific Fab Ranibizumab (in vivo). The high-resolution crystal structures of m912 FabCH3 and m912 Fab are determined, and the comparative analysis reveals more rigid structures in both constant domains and complementarity-determining regions of FabCH3, explaining its enhanced stability and affinity. Overall, the stabilized FabCH3 described in this report provides a versatile platform for engineering Fab-like antibody fragments with higher stability and antigen-binding affinity that can be used as a distinct class of antibody therapeutics.

Crotalidae Polyvalent Immune Fab and Cost-Effective Management of Hospital Admissions for Snakebites.

BACKGROUND: Venomous snakebites are a common clinical scenario in the Southeastern United States. CroFab® (Crotalidae Polyvalent Immune Fab (Ovine), BTG, Wales, UK) antivenom is indicated in cases involving pit vipers and is known to be expensive. The treatment protocol for snakebites is based on clinically subjective measures triggering the application, or escalation of, antivenom administration. The purpose of this study is to characterize the use of CroFab at our institution and to evaluate the impact of its use regarding cost and overall outcomes. We suspect that it is often used but potentially less often needed. We hypothesized that CroFab use was associated with increased length of stay (LOS) without an observed difference in patient outcomes. MATERIALS AND METHODS: A retrospective chart review of snakebite patients at our level-1 trauma center from 2000 to 2016 was performed. Snakebite location, snake species, number of vials of CroFab administered, hospital LOS, intensive care unit (ICU) LOS, and complications were identified for each patient. Patients were divided into CroFab (C) and no CroFab (NC) groups. RESULTS: One hundred ninety patients with venomous snakebites were included. 53.7% of patients received CroFab. There was no difference in the complication rate of C versus NC groups, (P = .1118). CroFab use was associated with longer hospital LOS (P < .0001) and ICU LOS (P < .0001). DISCUSSION: CroFab use was associated with increased LOS in our patient population. There was no difference in observed outcomes between the C and NC groups. These findings imply that CroFab is potentially over-used in our patient population.

Pharmacokinetics of Monoclonal Antibody and Antibody Fragments in the Mouse Eye Following Systemic Administration.

The ocular pharmacokinetics (PK) of antibody-based therapies are infrequently studied in mice due to the technical difficulties in working with the small murine eye. This study is the first of its kind to quantitatively measure the PK of variously sized proteins in the plasma, cornea/ICB, vitreous humor, retina, and posterior cup (including choroid) of the mouse and to evaluate the relationship between molecular weight (MW) and antibody biodistribution coefficient (BC) to the eye. Proteins analyzed include trastuzumab (150 kDa), trastuzumab-vc-MMAE (T-vc-MMAE, 155 kDa), F(ab)2 (100 kDa), Fab (50 kDa), and scFv (27 kDa). As expected, ocular PK mirrored the systemic PK as plasma was the driving force for ocular exposure. For trastuzumab, T-vc-MMAE, F(ab)2, Fab, and scFv, respectively, the BCs in the cornea/ICB were 0.610%, 0.475%, 1.74%, 3.39%, and 13.7%; the BCs in the vitreous humor were 0.0198%, 0.0427%, 0.0934%, 0.234%, and 5.56%; the BCs for the retina were 0.539%, 0.230%, 0.704%, 2.44%, and 20.4%; the BCs for the posterior cup were 0.557%, 0.650%, 1.47%, 4.06%, and 13.9%. The relationship between BC and MW was best characterized by a log-log regression in which BC decreased as MW increased, with every doubling in MW leading to a decrease in BC by a factor of 3.44 × , 6.76 × , 4.74 × , and 3.43 × in cornea/ICB, vitreous humor, retina, and posterior cup, respectively. In analyzing the disposition of protein therapeutics to the eye, these findings enhance our understanding of the potential for ocular toxicity of systemically administered protein therapeutics and may aid in the discovery of systemically administered protein therapeutics for ocular disorders.

The Deleterious Effects of Shiga Toxin Type 2 Are Neutralized In Vitro by FabF8:Stx2 Recombinant Monoclonal Antibody.

Hemolytic Uremic Syndrome (HUS) associated with Shiga-toxigenic Escherichia coli (STEC) infections is the principal cause of acute renal injury in pediatric age groups. Shiga toxin type 2 (Stx2) has in vitro cytotoxic effects on kidney cells, including human glomerular endothelial (HGEC) and Vero cells. Neither a licensed vaccine nor effective therapy for HUS is available for humans. Recombinant antibodies against Stx2, produced in bacteria, appeared as the utmost tool to prevent HUS. Therefore, in this work, a recombinant FabF8:Stx2 was selected from a human Fab antibody library by phage display, characterized, and analyzed for its ability to neutralize the Stx activity from different STEC-Stx2 and Stx1/Stx2 producing strains in a gold standard Vero cell assay, and the Stx2 cytotoxic effects on primary cultures of HGEC. This recombinant Fab showed a dissociation constant of 13.8 nM and a half maximum effective concentration (EC50) of 160 ng/mL to Stx2. Additionally, FabF8:Stx2 neutralized, in different percentages, the cytotoxic effects of Stx2 and Stx1/2 from different STEC strains on Vero cells. Moreover, it significantly prevented the deleterious effects of Stx2 in a dose-dependent manner (up to 83%) in HGEC and protected this cell up to 90% from apoptosis and necrosis. Therefore, this novel and simple anti-Stx2 biomolecule will allow further investigation as a new therapeutic option that could improve STEC and HUS patient outcomes.

Perspectives on metals-based radioimmunotherapy (RIT): moving forward.

Radioimmunotherapy (RIT) is FDA-approved for the clinical management of liquid malignancies, however, its use for solid malignancies remains a challenge. The putative benefit of RIT lies in selective targeting of antigens expressed on the tumor surface using monoclonal antibodies, to systemically deliver cytotoxic radionuclides. The past several decades yielded dramatic improvements in the quality, quantity, recent commercial availability of alpha-, beta- and Auger Electron-emitting therapeutic radiometals. Investigators have created new or improved existing bifunctional chelators. These bifunctional chelators bind radiometals and can be coupled to antigen-specific antibodies. In this review, we discuss approaches to develop radiometal-based RITs, including the selection of radiometals, chelators and antibody platforms (i.e. full-length, F(ab')2, Fab, minibodies, diabodies, scFv-Fc and nanobodies). We cite examples of the performance of RIT in the clinic, describe challenges to its implementation, and offer insights to address gaps toward translation.

Intravenous Vipera berus Venom-Specific Fab Fragments and Intramuscular Vipera ammodytes Venom-Specific F(ab')2 Fragments in Vipera ammodytes-Envenomed Patients.

Vipera ammodytes (V. ammodytes) is the most venomous European viper. The aim of this study was to compare the clinical efficacy and pharmacokinetic values of intravenous Vipera berus venom-specific (paraspecific) Fab fragments (ViperaTAb) and intramuscular V. ammodytes venom-specific F(ab')2 fragments (European viper venom antiserum, also called "Zagreb" antivenom) in V.ammodytes-envenomed patients. This was a prospective study of V.ammodytes-envenomed patients that were treated intravenously with ViperaTAb or intramuscularly with European viper venom antiserum that was feasible only due to the unique situation of an antivenom shortage. The highest venom concentration, survival, length of hospital stay and adverse reactions did not differ between the groups. Patients treated with intravenous Fab fragments were sicker, with significantly more rhabdomyolysis and neurotoxicity. The kinetics of Fab fragments after one or more intravenous applications matched better with the venom concentration in the early phase of envenomation compared to F(ab')2 fragments that were given intramuscularly only on admission. F(ab')2 fragments given intramuscularly had 25-fold longer apparent total body clearance and 14-fold longer elimination half-time compared to Fab fragments given intravenously (2 weeks vs. 24 h, respectively). In V.ammodytes-envenomed patients, the intramuscular use of specific F(ab')2 fragments resulted in a slow rise of antivenom serum concentration that demanded their early administration but without the need for additional doses for complete resolution of all clinical signs of envenomation. Intravenous use of paraspecific Fab fragments resulted in the immediate rise of antivenom serum concentration that enabled their use according to the clinical progress, but multiple doses might be needed for efficient therapy of thrombocytopenia due to venom recurrence, while the progression of rhabdomyolysis and neurotoxic effects of the venom could not be prevented.

Engineered Antibody Fragment against the Urokinase Plasminogen Activator for Fast Delineation of Triple-Negative Breast Cancer by Positron Emission Tomography.

The urokinase plasminogen activator (uPA) and its cofactors are important regulators of tumor initiation and progression (including metastasis), and its overexpression is associated with unfavorable situations in cancer patients. We have previously used positron emission tomography (PET) imaging with a radiolabeled monoclonal antibody against the uPA (named ATN-291) to detect the uPA signaling activity in various cancer types; however, good tumor contrast can only be observed 24 h postinjection. To shorten the antibody circulation time and decrease interactions of ATN-291 with the mononuclear phagocyte system (MPS), our goal in this study is to develop an engineered antibody fragment (F(ab')2) from the parent antibody. By pepsin digestion and chromatography purification, ATN-291 F(ab')2 was obtained and characterized. Subsequently, it was conjugated with NOTA-Bn-NCS or fluorescein isothiocyanate (FITC) for PET imaging and fluorescence-mediated cellular analysis (i.e., flow cytometry or fluorescence microscopy). We confirmed that ATN-291 F(ab')2 still maintained a good targeting efficacy for the uPA in MDA-MB-231 cells (uPA+) and it had a faster blood clearance speed compared with ATN-291, while its interaction with MPS has been significantly decreased. In rodent tumor xenografts, radiolabeled ATN-291 F(ab')2 had a selective and persistent uptake in MDA-MB-231 tumors, with an early tumor-to-blood ratio of 1.3 ± 0.8 (n = 4) at 2 h postinjection from PET imaging. During our observation, radiolabeled ATN-291 F(ab')2 was excreted from both renal and hepatobiliary pathways. Radiolabeled ATN-291 F(ab')2 was also used for detecting uPA fluctuation during the tumor treatment in test animals. We concluded that radiolabeled ATN-291 F(ab')2 could be used as fast as PET cancer diagnostics with versatile applicability.

Use of both Fab and F(ab')2 fragment antivenom in a pediatric patient for treatment of a North American Crotalidae envenomation.

Crotalidae envenomation has been managed successfully in emergency departments across the world with antivenom. Over the years, antivenom has evolved and newer agents have been studied with the possibility of eliminating maintenance antivenom therapy. Here we report a patient who had worsening platelet and fibrinogen concentrations, as well as complaints of swelling and pain at the site of a rattlesnake envenomation following an initial dose of F(ab')2AV (Crotalidae immune F(ab')2 (equine) [ANAVIP®]) Crotalidae antivenom. The patient was subsequently transferred to a tertiary children's hospital for a higher level of care and received FabAV (Crotalidae polyvalent immune Fab (ovine) [CroFab®]) Crotalidae antivenom. The details of this patient's treatment course highlight the possibility that patients who receive F(ab')2AV, may require additional antivenom treatment. Furthermore, it appears that based on our single patient experience, giving FabAV after F(ab')2AV is safe and effective.

Intracerebral transfection of anti-rabies virus antibodies is an effective therapy for rabies.

Rabies is a neurological disease with 100% lethality. Some of the rare human patients who survived after multiple drug treatment had severe sequelae. The present study showed that after 48 h of RABV inoculation, mice injected intracerebrally with anti-RABV F (ab')2 plus Bioporter® showed 70% survival compared to the control group, suggesting that transfection of anti-RABV antibodies to the brain may prevent or delay the spread of RABV at an early stage of infection. This result may provide important protocol results in intracellular antibody delivery to prevent the fatal outcome of the disease.

Symptomatic digoxin toxicity in a patient on haemodialysis.

We present a man undergoing regular haemodialysis sessions, who presented with non-specific symptoms of nausea, vomiting and light-headedness. He was found to have significantly raised serum digoxin concentrations, as well as a heart rate of 30 beats per minutes. An ECG showed complete heart block. He has a history of non-ischaemic dilated cardiomyopathy with resistant supraventricular and ventricular tachycardias and was on concomitant beta-blockade and digoxin. On questioning, he reported a gradual decline in his residual urine output over the past 6 months. He was reviewed by the cardiology team and required both pharmacological therapy for reversal of digoxin toxicity and temporary pacing in view of significant bradyarrhythmias. The beta-blockade and digoxin were discontinued. He was kept on continuous monitoring at the Cardiac Critical Care Unit. His symptoms resolved spontaneously once digoxin-specific antibody fragments were administered and temporary pacing successfully performed.

Characterization of an HLA-restricted and human cytomegalovirus-specific antibody repertoire with therapeutic potential.

With an infection rate of 60-90%, the human cytomegalovirus (HCMV) is very common among adults but normally causes no symptoms. When T cell-mediated immunity is compromised, HCMV reactivation can lead to increased morbidity and mortality. HCMV antigens are processed and presented as peptides on the cell surface via HLA I complexes to the T cell receptor (TCR) of T cells. The generation of antibodies against HCMV peptides presented on HLA complexes (TCR-like antibodies) has been described, but is without therapeutic applications to date due to the polygenic and polymorphic nature of HLA genes. We set out to obtain antibodies specific for HLA/HCMV-peptides, covering the majority of HLA alleles present in European populations. Using phage display technology, we selected 10 Fabs, able to bind to HCMV-peptides presented in the 6 different HLA class I alleles A*0101, A*0201, A*2402, B*0702, B*0801 and B*3501. We demonstrate specific binding of all selected Fabs to HLA-typed lymphoblastoid cell lines (EBV-transformed B cells) and lymphocytes loaded with HCMV-peptides. After infection with HCMV, 4/10 tetramerized Fabs restricted to the alleles HLA-A*0101, HLA-A*0201 and HLA-B*0702 showed binding to infected primary fibroblasts. When linked to the pseudomonas exotoxin A, these Fab antibodies induce highly specific cytotoxicity in HLA matched cell lines loaded with HCMV peptides. TCR-like antibody repertoires therefore represent a promising new treatment modality for viral infections and may also have applications in the treatment of cancers.

Visual Function Decline Resulting from Geographic Atrophy: Results from the Chroma and Spectri Phase 3 Trials.

PURPOSE: To assess visual function outcomes to 48 weeks in patients with bilateral geographic atrophy (GA) secondary to age-related macular degeneration included in 2 interventional clinical trials: relationship to baseline lesion size, outcomes by baseline lesion characteristic subgroups, and correlation of visual function outcomes with GA area. DESIGN: The Chroma and Spectri studies (ClinicalTrials.gov identifiers, NCT02247479 and NCT02247531, respectively) were identically designed phase 3, double-masked, multicenter, randomized, sham injection-controlled clinical trials that evaluated intravitreal lampalizumab in GA. PARTICIPANTS: Eligible patients were 50 years of age or older with well-demarcated bilateral GA (lesion size, 1-7 disc areas) without evidence of or previous treatment for choroidal neovascularization in either eye and best-corrected visual acuity (BCVA) letter score of 49 letters or more (≥1 GA lesion within 250 µm of foveal center if BCVA ≥79 letters). METHODS: Patients (pooled n = 1881) were randomized 2:1:2:1 to lampalizumab every 4 weeks, sham every 4 weeks, lampalizumab every 6 weeks, or sham every 6 weeks. Sham arms were pooled for analysis. MAIN OUTCOME MEASURES: Functional end points included change in BCVA from baseline to week 48, low-luminance visual acuity, mesopic microperimetry (number of absolute scotomatous points, mean macular sensitivity), binocular and monocular maximum reading speed, and 2 validated patient-reported outcome measures: Functional Reading Independence Index and 25-item National Eye Institute Visual Function Questionnaire. RESULTS: Enlargement of GA area, approximately 2 mm2/year on average across all treatment groups in each study, was accompanied by overall deterioration in all functional end points. No statistically significant differences were found between lampalizumab or sham arms for changes from baseline in functional assessment scores. Of visual function tests, only microperimetry outcomes were correlated moderately with GA lesion area when assessed cross-sectionally at baseline and week 48. CONCLUSIONS: Chroma and Spectri provide a unique data set of functional end points in GA that are relevant for future clinical trials. Patients with bilateral GA experienced a consistent decline in visual function over 48 weeks, but measures of visual function were not correlated strongly with GA lesion area. It is not possible to predict visual function outcomes from GA lesion size.

Antibody Fragment F(ab')2 Targeting Caveolae-Associated Protein PV1 for Selective Kidney Targeting and Retention.

Targeted strategies to deliver and retain drugs to kidneys are needed to improve drug accumulation and efficacy in a myriad of kidney diseases. These drug delivery systems show potential for improving the therapeutic windows of drugs acting in the kidney. Biodistribution of antibody-based therapeutics in vivo is governed by several factors including binding affinity, size, and valency. Investigations of how the biophysical and biochemical properties of biologics enable them to overcome biological barriers and reach kidneys are therefore of interest. Although renal accumulation of antibody fragments in cancer diagnostics and treatment has been observed, reports on effective delivery of antibody fragments to the kidneys remain scarce. Previously, we demonstrated that targeting plasmalemma vesicle-associated protein (PV1), a caveolae-associated protein, can promote accumulation of antibodies in both the lungs and the kidneys. Here, by fine-tuning the binding affinity of an antibody toward PV1, we observe that the anti-PV1 antibody with reduced binding affinity lost the capability for kidney targeting while retaining the lung targeting activity, suggesting that binding affinity is a critical factor for kidney targeting of the anti-PV1 antibody. We next use the antibody fragment F(ab')2 targeting PV1 to assess the dual effects of rapid kidney filtration and PV1 targeting on kidney-selective targeting. Ex vivo fluorescence imaging results demonstrated that after rapidly accumulating in kidneys at 4 h, PV1-targeted F(ab')2 was continually retained in the kidney at 24 h, whereas the isotype control F(ab')2 underwent urinary elimination with significantly reduced signaling in the kidney. Confocal imaging studies confirmed the localization of PV1-targeted F(ab')2 in the kidney. In addition, the monovalent antibody fragment (Fab-C4) lost the capability for kidney homing, indicating that the binding avidity of anti-PV1 F(ab')2 is important for kidney targeting. Our findings suggest that PV1-targeted F(ab')2 might be useful as a drug carrier for renal targeting and highlight the importance of affinity optimization for tissue targeting antibodies.

Ocular Half-Life of Intravitreal Biologics in Humans and Other Species: Meta-Analysis and Model-Based Prediction.

Therapeutic antibodies administered intravitreally are the current standard of care to treat retinal diseases. The ocular half-life (t1/2) is a key determinant of the duration of target suppression. To support the development of novel, longer-acting drugs, a reliable determination of t1/2 is needed together with an improved understanding of the factors that influence it. A model-based meta-analysis was conducted in humans and nonclinical species (rat, rabbit, monkey, and pig) to determine consensus values for the ocular t1/2 of IgG antibodies and Fab fragments. Results from multiple literature and in-house pharmacokinetic studies are presented within a mechanistic framework that assumes diffusion-controlled drug elimination from the vitreous. Our analysis shows, both theoretically and experimentally, that the ocular t1/2 increases in direct proportion to the product of the hydrodynamic radius of the macromolecule (3.0 nm for Fab and 5.0 nm for IgG) and the square of the radius of the vitreous globe, which varies approximately 24-fold from the rat to the human. Interspecies differences in the proportionality factors are observed and discussed in mechanistic terms. In addition, mathematical formulae are presented that allow prediction of the ocular t1/2 for molecules of interest. The utility of these formulae is successfully demonstrated in case studies of aflibercept, brolucizumab, and PEGylated Fabs, where the predicted ocular t1/2 values are found to be in reasonable agreement with the experimental data available for these molecules.

Presumed Copperhead Snakebite and Antivenom Administration in the Third Trimester.

Snake envenomation during pregnancy is an uncommon emergency with several potential complications associated with the poisoning and its treatment. This case discusses a 27-y-old gravida 3, para 1102 (3 total pregnancies, 1 term birth, 1 premature birth, 0 abortions, 2 living births, twins) at 36 wk gestation who was bitten by a presumed Agkistrodon contortrix (copperhead snake). She had worsening pain and swelling in the right lower limb. Crotalidae polyvalent immune Fab was administered. The patient felt significantly better with improvement in swelling. She had a reactive nonstress test and reassuring coagulation studies. She gave birth to a healthy female infant 12 d later. This case supports the use of Crotalidae polyvalent immune Fab for venomous snakebites in pregnant patients to prevent possible maternal and fetal morbidity and mortality.

Infliximab-Tumor Necrosis Factor Complexes Elicit Formation of Anti-Drug Antibodies.

BACKGROUND & AIMS: Some patients develop anti-drug antibodies (ADAs), which reduce the efficacy of infliximab, a monoclonal antibody against tumor necrosis factor (TNF), in the treatment of immune-mediated diseases, including inflammatory bowel diseases. ADAs arise inconsistently, and it is not clear what factors determine their formation. We investigated features of the immune system, the infliximab antibody, and its complex with TNF that might contribute to ADA generation. METHODS: C57BL/6 mice were given injections of infliximab and recombinant human TNF or infliximab F(ab')2 fragments. Blood samples were collected every 2-3 days for 2 weeks and weekly thereafter for up to 6 weeks; infliximab-TNF complexes and ADAs were measured by enzyme-linked immunosorbent assay (ELISA). Intestinal biopsy and blood samples were obtained from patients having endoscopy who had received infliximab therapy for inflammatory bowel diseases; infliximab-TNF complexes were measured with ELISA. Infliximab-specific plasma cells were detected in patient tissue samples by using mass cytometry. We studied activation of innate immune cells in peripheral blood mononuclear cells (PBMCs) from healthy donors incubated with infliximab or infliximab-TNF complexes; toll-like receptors (TLRs) were blocked with antibodies, endocytosis was blocked with the inhibitor PitStop2, and cytokine expression was measured by real-time polymerase chain reaction and ELISAs. Uptake of infliximab and infliximab-TNF complexes by THP-1 cells was measured with confocal microscopy. RESULTS: Mice given increasing doses of infliximab produced increasing levels of ADAs. Blood samples from mice given injections of human TNF and infliximab contained infliximab-TNF complexes; complex formation was associated with ADA formation with an area under the curve of 0.944 (95% confidence interval, 0.851-1.000; P = .003). Intestinal tissues from patients, but not blood samples, contained infliximab-TNF complexes and infliximab-specific plasma cells. Incubation of PBMCs with infliximab-TNF complexes resulted in a 4.74-fold increase in level of interleukin (IL) 1ß (IL1B) messenger RNA (P for comparison = .005), increased IL1B protein secretion, and a 2.69-fold increase in the expression of TNF messenger RNA (P for comparison = 0.013) compared with control PBMCs. Infliximab reduced only IL1B and TNF expression. Antibodies against TLR2 or TLR4 did not block the increases in IL1B or TNF expression, but endocytosis was required. THP-1 cells endocytosed higher levels of infliximab-TNF complexes than infliximab alone. CONCLUSIONS: In mice, we found ADA formation to increase with dose of infliximab given and concentration of infliximab-TNF complexes detected in blood. Based on studies of human intestinal tissues and blood samples, we propose that infliximab-TNF complexes formed in the intestine are endocytosed by and activate innate immune cells, which increase expression of IL1B and TNF and production of antibodies against the drug complex. It is therefore important to optimize the infliximab dose to a level that is effective but does not activate an innate immune response against the drug-TNF complex.

Optimal design, anti-tumour efficacy and tolerability of anti-CXCR4 antibody drug conjugates.

Antibody-drug conjugates (ADCs) are promising therapies for haematological cancers. Historically, their therapeutic benefit is due to ADC targeting of lineage-restricted antigens. The C-X-C motif chemokine receptor 4 (CXCR4) is attractive for targeted therapy of haematological cancers, given its expression in multiple tumour types and role in cancer "homing" to bone marrow. However, CXCR4 is also expressed in haematopoietic cells and other normal tissues, raising safety challenges to the development of anti-CXCR4 ADCs for cancer treatment. Here, we designed the first anti-CXCR4 ADC with favourable therapeutic index, effective in xenografts of haematopoietic cancers resistant to standard of care and anti-CXCR4 antibodies. We screened multiple ADC configurations, by varying type of linker-payload, drug-to-antibody ratio (DAR), affinity and Fc format. The optimal ADC bears a non-cleavable linker, auristatin as payload at DAR = 4 and a low affinity antibody with effector-reduced Fc. Contrary to other drugs targeting CXCR4, anti-CXCR4 ADCs effectively eliminated cancer cells as monotherapy, while minimizing leucocytosis. The optimal ADC selectively eliminated CXCR4+ cancer cells in solid tumours, but showed limited toxicity to normal CXCR4+ tissues, sparing haematopoietic stem cells and progenitors. Our work provides proof-of-concept that through empirical ADC design, it is possible to target proteins with broad normal tissue expression.