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In analyses of protein families that may serve as drug targets, membrane-associated G-protein-coupled receptors (GPCRs) dominate, followed by ion channels, transporters, and—to a lesser extent—membrane-bound enzymes. However, various challenges put such membrane proteins among key groups of underutilized opportunities for the application of therapeutic antibodies. Antibodies hold the promise of exquisite specificity, as they are able to target even specific conformations of a particular membrane protein, as well as adaptability through engineering into various antibody formats. However, the ease of raising and isolating specific, effective antibodies targeting membrane proteins depends on many factors. In particular, the generation of specific antibodies is easier when targeting larger, simpler, extracellular domains with greater uniqueness of amino acid sequence. The rareness of such ideal conditions is illustrated by the limited number of approved biologics for targeting GPCRs and other complex membrane proteins. Challenges in developing antibodies to complex membrane proteins such as GPCRs, ion channels, transporters, and membrane-bound enzymes can be addressed by the design of the antigen, antibody-generation strategies, lead optimization technologies, and antibody modalities. A better understanding of the membrane proteins being targeted would facilitate mechanism-based drug discovery. This review describes the advantages and challenges of targeting complex membrane proteins with antibodies and discusses the preparation of membrane protein antigens and antibody generation, illustrated by select examples of success.

High tumor regulatory T (Treg) cell infiltration is associated with poor prognosis of many cancers. CD25 is highly expressed on tumor Treg cells and is a potential target for Treg deletion. Previously characterized anti-CD25 antibodies appear to have limited efficacy in tumor inhibition. Here we identified two human anti-CD25 antibodies, BA9 and BT942, which did not prevent the activation of IL-2R signaling pathway by IL-2. BT942 had weaker binding and cytotoxic activity to human CD25-expressing cell lines than BA9. But both demonstrated significant tumor growth inhibition in early and late-stage animal cancer models. BT942 resulted in a higher expansion of CD8+T cells and CD4+T cells in tumor microenvironment in mouse MC38 model compared to BA9. BT942 also demonstrated significant higher tumor growth inhibition and higher expansion of CD8+T cells and CD4+T cells in combination with an anti-PD1 antibody. Pharmacokinetic study of BT942 in cynomolgus monkeys demonstrated a half-life of 206.97 ± 19.03 h. Structural analysis by cryo-EM revealed that BT942 recognizes an epitope on opposite side of the CD25-IL-2 binding site, consistent with no IL-2 signaling blockade in vitro. BT942 appears to be an excellent candidate for cancer immunotherapy.

The majority of depleting monoclonal antibody (mAb) drugs elicit responses via Fc-FcγR and Fc-C1q interactions. Optimal C1q interaction is achieved through hexameric Fc:Fc interactions at the target cell surface. Herein is described an approach to exploit the tailpiece of the naturally multimeric IgM to augment hexamerisation of IgG. Fusion of the C-terminal tailpiece of IgM promoted spontaneous hIgG hexamer formation, resulting in enhanced C1q recruitment and complement-dependent cytotoxicity (CDC) but with off-target complement activation and reduced in-vivo efficacy. Mutation of the penultimate tailpiece cysteine to serine (C575S) ablated spontaneous hexamer formation, but facilitated reversible hexamer formation after concentration in solution. C575S mutant tailpiece antibodies displayed increased complement activity only after target binding, in-line with the concept of ‘on-target hexamerisation’, whilst retaining efficient in-vivo efficacy and augmented target cell killing in the lymph node. Hence, C575S-tailpiece technology represents an alternative format for promoting on-target hexamerisation and enhanced CDC., Sopp et al describe an approach, which exploits the tailpiece of the naturally multimeric IgM to augment hexamerisation of IgG. Their approach provides a newly engineered format of antibodies for promoting hexamerisation and enhanced complement-dependent cytotoxicity only when cell surface bound.

Similar to pediatric acute myeloid leukemia (AML) the subgroup of biphenotypic acute lymphoblastic leukemia (ALL) is a rare complex entity with adverse outcome, characterized by the surface expression of CD33. Despite novel and promising anti-CD19 targeted immunotherapies such as chimeric antigen receptor T cells and bispecific anti-CD19/CD3 antibodies, relapse and resistance remain a major challenge in about 30% to 60% of patients. To investigate the potential role of the fully humanized bispecific antibody CD16 × CD33 (BiKE) in children with CD33+ acute leukemia, we tested whether the reagent was able to boost NK cell effector functions against CD33+ AML and biphenotypic ALL blasts. Stimulation of primary NK cells from healthy volunteers with 16 × 33 BiKE led to increased cytotoxicity, degranulation and cytokine production against CD33+ cell lines. Moreover, BiKE treatment significantly increased degranulation, IFN-γ and TNF-α production against primary ALL and AML targets. Importantly, also NK cells from leukemic patients profited from restoration of effector functions by BiKE treatment, albeit to a lesser extent than NK cells from healthy donors. In particular, those patients with low perforin and granzyme expression showed compromised cytotoxic function even in the presence of BiKE. In patients with intrinsic NK cell deficiency, combination therapy of CD16xCD33 BiKE and allogeneic NK cells might thus be a promising therapeutic approach. Taken together, CD16xCD33 BiKE successfully increased NK cell effector functions against pediatric AML and biphenotypic ALL blasts and constitutes a promising new option for supporting maintenance therapy or “bridging” consolidation chemotherapy before hematopoietic stem cell transplantation. Supplementary Information The online version contains supplementary material available at 10.1007/s00262-021-03008-0.

T-cell-redirecting bispecific antibodies have emerged as a new class of therapeutic agents designed to simultaneously bind to T cells via CD3 and to tumor cells via tumor-cell-specific antigens (TSA), inducing T-cell-mediated killing of tumor cells. The promising preclinical and clinical efficacy of TSAxCD3 antibodies is often accompanied by toxicities such as cytokine release syndrome due to T-cell activation. How the efficacy and toxicity profile of the TSAxCD3 bispecific antibodies depends on the binding affinity to CD3 remains unclear. Here, we evaluate bispecific antibodies that were engineered to have a range of CD3 affinities, while retaining the same binding affinity for the selected tumor antigen. These agents were tested for their ability to kill tumor cells in vitro, and their biodistribution, serum half-life, and anti-tumor activity in vivo. Remarkably, by altering the binding affinity for CD3 alone, we can generate bispecific antibodies that maintain potent killing of TSA + tumor cells but display differential patterns of cytokine release, pharmacokinetics, and biodistribution. Therefore, tuning CD3 affinity is a promising method to improve the therapeutic index of T-cell-engaging bispecific antibodies.

In the absence of a prophylactic vaccine, the use of antiretroviral therapy (ART) as pre-exposure prophylaxis (PrEP) to prevent HIV acquisition by uninfected individuals is a promising approach to slowing the epidemic, but its efficacy is hampered by incomplete patient adherence and ART-resistant variants. Here, we report that competitive inhibition of HIV Env-CCR5 binding via the CCR5-specific antibody Leronlimab protects rhesus macaques against infection following repeated intrarectal challenges of CCR5-tropic SHIVSF162P3. Injection of Leronlimab weekly at 10 mg/kg provides significant but partial protection, while biweekly 50 mg/kg provides complete protection from SHIV acquisition. Tissue biopsies from protected macaques post challenge show complete CCR5 receptor occupancy and an absence of viral nucleic acids. After Leronlimab washout, protected macaques remain aviremic, and adoptive transfer of hematologic cells into naïve macaques does not transmit viral infection. These data identify CCR5 blockade with Leronlimab as a promising approach to HIV prophylaxis and support initiation of clinical trials., CCR5 is a co-receptor for many transmitted HIV strains. Here, the authors show that biweekly injection of the CCR5-specific antibody Leronlimab protects rhesus macaques against infection following repeated intrarectal challenges of a CCR5-tropic SHIV.

Therapeutic antibodies are decorated with complex-type N-glycans that significantly affect their biodistribution and bioactivity. The N-glycan structures on antibodies are incompletely processed in wild-type CHO cells due to their limited glycosylation capacity. To improve N-glycan processing, glycosyltransferase genes have been traditionally overexpressed in CHO cells to engineer the cellular N-glycosylation pathway by using random integration, which is often associated with large clonal variations in gene expression levels. In order to minimize the clonal variations, we used recombinase-mediated-cassette-exchange (RMCE) technology to overexpress a panel of 42 human glycosyltransferase genes to screen their impact on antibody N-linked glycosylation. The bottlenecks in the N-glycosylation pathway were identified and then released by overexpressing single or multiple critical genes. Overexpressing B4GalT1 gene alone in the CHO cells produced antibodies with more than 80% galactosylated bi-antennary N-glycans. Combinatorial overexpression of B4GalT1 and ST6Gal1 produced antibodies containing more than 70% sialylated bi-antennary N-glycans. In addition, antibodies with various tri-antennary N-glycans were obtained for the first time by overexpressing MGAT5 alone or in combination with B4GalT1 and ST6Gal1. The various N-glycan structures and the method for producing them in this work provide opportunities to study the glycan structure-and-function and develop novel recombinant antibodies for addressing different therapeutic applications.

Myostatin, a member of the transforming growth factor-b superfamily, is an attractive target for muscle disease therapy because of its role as a negative regulator of muscle growth and strength. Here, we describe a novel antibody therapeutic approach that maximizes the potential of myostatin-targeted therapy. We generated an antibody, GYM329, that specifically binds the latent form of myostatin and inhibits its activation. Additionally, via “sweeping antibody technology”, GYM329 reduces or “sweeps” myostatin in the muscle and plasma. Compared with conventional anti-myostatin agents, GYM329 and its surrogate antibody exhibit superior muscle strength-improvement effects in three different mouse disease models. We also demonstrate that the superior efficacy of GYM329 is due to its myostatin specificity and sweeping capability. Furthermore, we show that a GYM329 surrogate increases muscle mass in normal cynomolgus monkeys without any obvious toxicity. Our findings indicate the potential of GYM329 to improve muscle strength in patients with muscular disorders.

In this review, we have summarized the current landscape of therapeutic antibody optimization for successful development. By engineering antibodies with display technology, computer-aided design and site mutagenesis, various properties of the therapeutic antibody candidates can be improved with the purpose of enhancing their safety, efficacy and developability. These properties include antigen binding affinity and specificity, biological efficacy, pharmacokinetics and pharmacodynamics, immunogenicity and physicochemical developability features. A best-in-class strategy may require the optimization of all these properties to generate a good therapeutic antibody.

Contains fulltext : 229558.pdf (Publisher’s version ) (Closed access) Malaria eradication is a global priority but requires innovative strategies. Humoral immune responses attack different parasite stages, and antibody-based therapy may prevent malaria infection or transmission. Here, we discuss targets of monoclonal antibodies in mosquito sexual stages of Plasmodium.

Highlights • There are totally five kinds of traditional mechanisms of antibody-dependent enhancement. Mostly, the effect is reported to related with FcγR. • Antibody-dependent enhancement has been discovered in both SARS-CoV and MERS-CoV but the mechanism wasn’t thoroughly clear. Different researches may provide different opinions. • Many scientist have mentioned the potential existence of antibody-dependent enhancement in the 2019 novel coronavirus. • Latest study of both convalescent plasma transmission and application of inactivated vaccine haven’t report case of antibody-dependent enhancement., Antibody-dependent enhancement (ADE) exists in several kinds of virus. It has a negative influence on antibody therapy for viral infection. This effect was first identified in dengue virus and then was also described for coronavirus. The rapid spread of the newly emerged 2019 novel coronavirus (COVID-19) has sickened over 3.8 million people across the globe up to now. The novel coronavirus causes great challenge and arouses a wave of panic. In this review, antibody-dependent enhancements in dengue virus and two kinds of coronavirus are summarized. Solutions for the effects are mentioned, too. We also speculate that antibody-dependent enhancement may exist in COVID-19.

SARS-CoV-2 is a newly emergent coronavirus that causes COVID-19, which has adversely impacted human health and has led to a pandemic. There is an unmet need to develop therapies against SARS-CoV-2 due to its severity and lack of treatment options. A promising approach to combat COVID-19 is through the neutralization of SARS-CoV-2 by therapeutic antibodies. Previously, we described a strategy to rapidly identify and generate llama nanobodies (VHH) from naïve and synthetic humanized VHH phage libraries that specifically bind the S1 SARS-CoV-2 spike protein, and block the interaction to the ACE2 human receptor. In this study, we then used computer-aided designed and constructed multi-specific VHH antibodies fused to human IgG1 Fc domains based on the epitope predictions for leading VHHs. The resulting tri-specific VHH-Fc antibodies show more potent S1 binding, S1/ACE2 blocking, and SARS-CoV-2 pseudovirus neutralization than the bi-specific VHH-Fcs or combination of individual monoclonal VHH-Fcs. Furthermore, protein stability analysis of the VHH-Fcs show favorable developability features, which enable them to be quickly and successfully developed into therapeutics against COVID-19.

Monoclonal antibody (mAb) 10E8 recognizes a highly conserved epitope on HIV and is capable of neutralizing > 95% of circulating viral isolates making it one of the most promising Abs against HIV. Solution instability and biochemical heterogeneity of 10E8 has hampered its development for clinical use. We identify the source of 10E8 heterogeneity being linked to cis/trans isomerization at two prolines within the YPP motif in the CRD3 loop that exists as two predominant conformers that interconvert on a slow timescale. The YtransP conformation conformer can bind the HIV gp41 epitope, while the YcisP is not binding competent and shows a higher aggregation propensity. The high barrier of isomerization and propensity to adopt non-binding competent proline conformers provides novel insight into the slow binding kinetics, low potency, and poor solubility of 10E8. This study highlights how proline isomerization should be considered a critical quality attribute for biotherapeutics with paratopes containing potential cis proline amide bonds.

COVID-19 presents varied clinical features, ranging from asymptomatic, mild, to acute respiratory distress syndrome (ARDS). People of any age can be infected by the novel coronavirus but some people are at more risk than others of becoming seriously ill with COVID-19. Older people, and people with pre-existing medical conditions (such as asthma, diabetes, and heart disease) appear to be more vulnerable developing severe or even lethal illness. Children can be infected with SARS-CoV-2, and become ill with COVID-19. However, they appear to be less susceptible to infection than adults and their symptoms are generally milder. Immunoglobulin D (IgD) has remained an enigmatic antibody class since its discovery more than 50 years ago. The function of secreted IgD has been a longstanding puzzle in immunology. In this pandemic, it is the proper time to rediscover its role and find additional therapy to vaccine. In this article I will review IgD discovery, its role in immunity, its concentration in some groups and their susceptibility to infection with COVID 19.

Broadly neutralising antibodies (bNAbs) against human immunodeficiency virus type 1 (HIV-1), such as CAP256-VRC26 are being developed for HIV prevention and treatment. These Abs carry a unique but crucial post-translational modification (PTM), namely O-sulfated tyrosine in the heavy chain complementarity determining region (CDR) H3 loop. Several studies have demonstrated that plants are suitable hosts for the generation of highly active anti-HIV-1 antibodies with the potential to engineer PTMs. Here we report the expression and characterisation of CAP256-VRC26 bNAbs with posttranslational modifications (PTM). Two variants, CAP256-VRC26 (08 and 09) were expressed in glycoengineered Nicotiana benthamiana plants. By in planta co-expression of tyrosyl protein sulfotransferase 1, we installed O-sulfated tyrosine in CDR H3 of both bNAbs. These exhibited similar structural folding to the mammalian cell produced bNAbs, but non-sulfated versions showed loss of neutralisation breadth and potency. In contrast, tyrosine sulfated versions displayed equivalent neutralising activity to mammalian produced antibodies retaining exceptional potency against some subtype C viruses. Together, the data demonstrate the enormous potential of plant-based systems for multiple posttranslational engineering and production of fully active bNAbs for application in passive immunisation or as an alternative for current HIV/AIDS antiretroviral therapy regimens.

Summary Introduction So far, anti-PD-1/anti-PD-L1 antibody therapy is reportedly in treating gastric cancer or gastro-oesophageal junction cancer (GC/GEJC) in a number of clinical trials. Based on this, we conducted current meta-analysis to assess the safety and efficacy of anti-PD-1/anti-PD-L1 antibody for previously treated advanced GC/GEJC patients. Methods We searched five electronic databases for eligible records. Outcomes were presented and analyzed by objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and adverse effects (AEs). Results Nine records involving 1388 participants were selected in our study. The pooled ORR, DCR, OS rate (6 month), PFS rate (6 month), OS rate (12 month) and PFS rate (12 month) were 10% (95% confidence interval [CI]: 6%–14%), 32% (95%CI: 25%–38%), 52% (95%CI: 44%–61%), 18% (95%CI: 13%–24%), 40% (95%CI: 31%–48%) and 8% (95%CI: 5%–10%), respectively. Meanwhile, grade ≥ 3 AEs rate was 12% (95% CI: 10%-15%). Programmed death ligand 1 (PD-L1) positive cases had higher rate of ORR (odds ratio [OR]: 3.75, 95%CI: 2.09–6.74, P = 0.58) compared with negative cases. Conclusion The results indicated that anti-PD-1/anti-PD-L1 antibody therapy has an effectual anti-tumor activity and controllable AEs in advanced GC/GEJC patients. Furthermore, overexpression of PD-L1 in advanced GC/GEJC patients had better ORR from anti-PD-1/anti-PD-L1 antibody therapy (PROSPERO registration number: CRD42018116480).

In clinical trials with early Alzheimer’s patients, administration of anti-amyloid antibodies reduced amyloid deposits, suggesting that immunotherapies may be promising disease-modifying interventions against Alzheimer’s disease (AD). Specific forms of amyloid beta (Aβ) peptides, for example post-translationally modified Aβ peptides with a pyroglutamate at the N-terminus (pGlu3, pE3), are attractive antibody targets, due to pGlu3-Aβ’s neo-epitope character and its propensity to form neurotoxic oligomeric aggregates. We have generated a novel anti-pGlu3-Aβ antibody, PBD-C06, which is based on a murine precursor antibody that binds with high specificity to pGlu3-Aβ monomers, oligomers and fibrils, including mixed aggregates of unmodified Aβ and pGlu3-Aβ peptides. PBD-C06 was generated by first grafting the murine antigen binding sequences onto suitable human variable light and heavy chains. Subsequently, the humanized antibody was de-immunized and site-specific mutations were introduced to restore original target binding, to eliminate complement activation and to improve protein stability. PBD-C06 binds with the same specificity and avidity as its murine precursor antibody and elimination of C1q binding did not compromise Fcγ-receptor binding or in vitro phagocytosis. Thus, PBD-C06 was specifically designed to target neurotoxic aggregates and to avoid complement-mediated inflammatory responses, in order to lower the risk for vasogenic edemas in the clinic.

Fabs offer an attractive platform for monoclonal antibody discovery/engineering, but library construction can be cumbersome. We report a simple method – Golden Gate assembly with a bi-directional promoter (GBid) – for constructing phage display Fab libraries. In GBid, the constant domains of the Fabs are located in the backbone of the phagemid vector and the library insert comprises only the variable regions of the antibodies and a central bi-directional promoter. This vector design reduces the process of Fab library construction to “scFv-like” simplicity and the double promoter ensures robust expression of both constituent chains. To maximize the library size, the 3 fragments comprising the insert – two variable chains and one bi-directional promoter – are assembled via a 3-fragment overlap extension PCR and the insert is incorporated into the vector via a high-efficiency one-fragment, one-pot Golden Gate assembly. The reaction setup requires minimal preparatory work and enzyme quantities, making GBid highly scalable. Using GBid, we constructed a chimeric chicken-human Fab phage display library comprising 1010 variants targeting the multi-transmembrane protein human CD20 (hCD20). Selection/counter-selection on transfected whole cells yielded hCD20-specific antibodies in four rounds of panning. The simplicity and scalability of GBid makes it a powerful tool for the discovery/engineering of Fabs and IgGs.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action for many therapeutic antibodies. A therapeutic immunoglobulin (Ig) G1 monoclonal antibody lost more than half of its ADCC activity after heat stress at 40 °C for 4 months. Size-exclusion and ion-exchange chromatography were used to fractionate various size and charge variants from the stressed IgG1. Physicochemical characterization of these fractions revealed that a rarely seen crystallizable fragment (Fc) modification, N325 deamidation, exhibited a positive correlation with the loss of ADCC activity. A further surface plasmon resonance study showed that this modification disrupted the binding between the IgG1 Fc and Fcγ receptor IIIa, resulting in decreased ADCC activity of the IgG1 antibody. Mutants of N325/D and N325/Q were made to confirm the effect of N325 deamidation on ADCC. We hypothesize that N325 deamidation altered the local three-dimensional structure, which might interfere with the binding and interaction with the effector cell. Because of its impact on biological activity, N325 deamidation is a critical quality attribute for products whose mechanism of action includes ADCC. A thorough understanding of the criticality of N325 deamidation and appropriate monitoring can help ensure the safety and efficacy of IgG1 or Fc-fusion products.

Monoclonal antibodies (mAbs) are one of the most widely used drug platforms for infectious diseases or cancer therapeutics because they selectively target pathogens, infectious cells, cancerous cells, and even immune cells. In this way, they mediate the elimination of target molecules and cells with fewer side effects than other therapeutic modalities. In particular, cancer therapeutic mAbs can recognize cell-surface proteins on target cells and then kill the targeted cells by multiple mechanisms that are dependent upon a fragment crystallizable (Fc) domain interacting with effector Fc gamma receptors, including antibody-dependent cell-mediated cytotoxicity and antibody-dependent cell-mediated phagocytosis. Extensive engineering efforts have been made toward tuning Fc functions by either reinforcing (e.g. for targeted therapy) or disabling (e.g. for immune checkpoint blockade therapy) effector functions and prolonging the serum half-lives of antibodies, as necessary. In this report, we review Fc engineering efforts to improve therapeutic potency, and propose future antibody engineering directions that can fulfill unmet medical needs., Monoclonal antibodies: Engineering immunity Fine-tuning the function of monoclonal antibodies (mAbs) holds promise for developing new therapeutic agents. Antibodies bind to pathogens or cancer cells, flagging them with Fc (fragment crystallizable) domain for destruction by the immune system. mAbs attached only to specific target cells enable lower side effect than other conventional drugs. Sang Taek Jung at Korea University and Tae Hyun Kang at Kookmin University, both in Seoul, reviewed recent developments in engineering therapeutic potency of mAbs. They report that mAbs can be engineered to activate effective immune cell types to treat a particular disease. Engineering can also increase mAbs’ persistence in the blood, enabling less frequent administration. Antibodies engineered to bind to two different antigens at once can also improve therapeutic efficacy. Applying these techniques could help developing new treatments against cancer, and infectious and autoimmune diseases.

The epidemiological impact of viral diseases, combined with the emergence and reemergence of some viruses, and the difficulties in identifying effective therapies, have encouraged several studies to develop new therapeutic strategies for viral infections. In this context, the use of immunotherapy for the treatment of viral diseases is increasing. One of the strategies of immunotherapy is the use of antibodies, particularly the monoclonal antibodies (mAbs) and multi-specific antibodies, which bind directly to the viral antigen and bring about activation of the immune system. With current advancements in science and technology, several such antibodies are being tested, and some are already approved and are undergoing clinical trials. The present work aims to review the status of mAb development for the treatment of viral diseases.

Streptococcal peptide of virulence (SpoV) is a Streptococcus pyogenes (group A streptococcus (GAS))-specific peptide that is important for GAS survival in murine blood, and the expression of the virulence factors streptolysin O (slo) and streptolysin S (sagA). We used a spoV mutant in isolate MGAS315 to assess the contribution of the SpoV peptide to virulence by using a murine model of invasive disease and an ex vivo human model (Lancefield assay). We then used antibodies to SpoV in both models to evaluate their ability to decrease morbidity and mortality. Results showed that SpoV is essential for GAS virulence, and targeting the peptide has therapeutic potential.

BackgroundRecently, TIGIT+PD1 blockade was shown to confer additive survival benefits in orthotopic glioblastoma and colon cancer mouse models1 2—notably, this included experiments3 that used the 1G9 TIGIT monoclonal antibody (mAb) that has potentially agonistic effects.1 Herein we investigated the TIGIT/CD226/CD155 axis and effects of combining clinically analogous PD-1/PD-L1 mAbs with TIGIT-targeting 1G9 mAb in murine glioblastoma models.MethodsThe overall survival (OS) associated with TIGIT (non-depleting IG9; 200μg every 3days for 4 doses),1 PD-1 (8H3; initial 500μg followed by 250μg every 3days for 7 doses), PD-L1 (6A2; initial 500μg followed by 250μg every 3days for 7 doses), and/or IgG mAbs was assessed in immunocompetent C57BL/6 albino mice intracranially implanted with syngeneic GL261-luc2 or CT2A-luc.4 5 The roles of T cells and NK cells were examined using depletion with CD8a, CD4, or NK1.1 mAbs. Expression of TIGIT/CD226/CD155 and PD-1/PD-L1/PD-L2 by tumor and tumor-infiltrating immune cells was evaluated using flow cytometry and RT-qPCR.ResultsIn vitro, GL261-luc2 and CT2A-luc tumor cells moderately expressed PD-L1, PD-1, and TIGIT; but strongly expressed TIGIT’s inhibitory ligand CD155.(Figure1A-B) Ex vivo, >83% of CD8+ and CD4+TILs in GL261-luc2 co-expressed TIGIT+/CD226+: ≥2x the proportions in CT2A-luc.(Figure 2A) CD155 and PD-L1 were highly co-expressed on tumor-infiltrating macrophages: greater in GL261-luc2 than CT2A-luc tumors.(Figure 2B)In GL261-luc2 mice, anti-TIGIT monotherapy displayed minimal OS improvement; whereas anti-PD-1 and anti-PD-L1 monotherapies demonstrated robust OS responses.(Figure 3A) Adding anti-TIGIT to PD-1/PD-L1 blockade demonstrated synergism with anti-PD-1. Anti-TIGIT plus anti-PD-1 displayed nominally improved OS in CT2A-luc compared to anti-PD-1 monotherapy (p=0.11).(Figure 3B).Given robust T-cell expression of TIGIT and PD-1, we examined how CD4+ or CD8+ depletion affected responses in GL261-luc2 mice: depletion completely abrogated anti-PD-1’s benefits.(Figure 4) Although CD4/CD8 depletion also reduced anti-TIGIT+anti-PD1’s efficacy, the resulting OS matched that of non-depleted anti-PD-1 monotherapy. Additionally, NK cell depletion had no effect on anti-TIGIT+anti-PD1’s efficacy.Abstract 256 Figure 1TIGIT/CD155 axis and PD-1/PD-L1/PD-L2 axis expression in murine glioblastoma model tumor cells. Protein and RNA expression of the TIGIT and PD-1 immune checkpoints — and their ligands CD155 and PDL-1/PD-L2 respectively — on GL261-luc2 and CT2A-luc tumor cells using (A) flow cytometry (blue=samples, red=unstained controls) and (B) RT-qPCR (grey=GL261-luc2, black=CT2A-luc). Pdcd1 encodes PD-1, Cd274 encodes PD-L1, Pvr encodes CD155Abstract 256 Figure 2TIGIT/CD155 axis and PD-1/PD-L1/PD-L2 axis expression in murine glioblastoma model tumor-infiltrating immune cells. (A) Flow cytometry analysis of protein expression for TIGIT (y-axis) and CD226 (x-axis; a competitor of TIGIT) on CD8+, CD4+/FOXP3+ Treg, and CD4+/FOXP3- Teff tumor-infiltrating lymphocytes (TILs) from GL261-luc2 and CT2A-luc tumor-bearing mice. (B) Flow cytometry analysis of protein expression for CD155 (TIGIT’s ligand; bottom) and PD-L1 (PD-1’s ligand; top) on tumor-infiltrating myeloid populations from GL261-luc2 and CT2A-luc tumor-bearing mice. Mean fluorescent intensity (MFI) was compared between cell lines, *indicates statistical significance. Myeloid populations included CD45+/CD11b+/CD11c+/F4-80+ macrophages, CD11b+/CD11c+ and CD11b-/CD11c+ dendritic cells (DCs), CD11b+/CD11c-/Ly6C+/Ly6G- monocytes, CD11b+/CD11c-/Ly6Cmid/Ly6G+ granulocytes, and CD45dim/CX3CR1+ microglia. Tumors were dissociated and leukocytes were enriched for using Percoll gradient. n=5 mice per group.Abstract 256 Figure 3The survival associated with TIGIT-targeting mAb therapy with/without clinically-analogous PD-1/PD-L1 blockade. (A) Kaplan-Meier estimated overall survival (measured from day of intracranial tumor implantation) of GL261-luc2 mice treated with TIGIT (1G9), PD-1 (8H3), PD-L1 (6A2), TIGIT + PD-1, TIGIT + PD-L1 mAbs, or IgG control. (B) A) Kaplan-Meier estimated overall survival (measured from day of intracranial tumor implantation) of CT2A-luc mice treated with TIGIT (1G9), PD-1 (8H3), TIGIT + PD-1 mAbs, or IgG control. For both experiments, all treatments were started on day 6 following implantation, with the following dosing: anti-TIGIT was given as 200μg every 3days for 4 doses. Both anti-PD-1 and anti-PD-L1 were given as an initial 500μg dose followed by 250μg every 3days for 7 doses.4 The n per group and number at risk table is included underneath each graph, along with the corresponding Cox regression analysis. Treatment groups with significantly different OS from the combination TIGIT + PD-1 combination-treated reference group were highlighted in yellow. HR = hazard ratio, CI = confidence interval.Abstract 256 Figure 4How depletion of CD8+ T cells, CD4+ T cells, or NK cells affects survival associated with TIGIT-targeting mAb therapy with/without clinically-analogous PD-1 blockade. Kaplan-Meier estimated overall survival (measured from day of intracranial tumor implantation) of GL261-luc2 mice treated with TIGIT (1G9), PD-1 (8H3), TIGIT + PD-1 mAbs, or IgG control; and compared to groups that additional had CD8+, CD4+, or NK1.1+ antibody-based depletion. The treatment and dosing characteristics were the same as Figure 3. The n per group and number at risk table is included underneath the graph, along with the corresponding Cox regression analysis. Treatment groups with significantly different OS from the combination TIGIT + PD-1 combination-treated reference group were highlighted in yellow. HR = hazard ratio, CI = confidence interval.ConclusionsOur results recapitulate published findings regarding the synergistic benefits of combining TIGIT 1G9 mAb with anti-PD-1 using the clinically-relevant 8H3 mAb in syngeneic mouse glioblastoma, and extend those findings to anti-TIGIT+anti-PDL1 combinations. TIGIT/CD226 was highly co-expressed by immuno-responsive GL261-luc2’s tumor-infiltrating lymphocytes (TILs); wheres CD155/PD-L1 expression predominated in tumor-infiltrating myeloid cells. Depletion of CD8+ or CD4+ TILs modestly reduced anti-TIGIT+anti-PD1’s efficacy—suggesting a mechanism that is at least partially independent of T (and NK) cells. Our preliminary results suggest a complex interplay between TIGIT/CD226/CD155 and PD-1/PD-L1/PD-L2 axes in tumors and their microenvironmental constituents that warrants further investigation; plus, careful consideration of antibody clones’ functionality is necessary for designing immunotherapy combinations.AcknowledgementsWe gratefully acknowledge the support of the The Jennifer Oppenheimer Cancer Research Initiative; The Ben and Catherine Ivy Foundation; Hope It’s A Beach Thing; and the Pan Mass Challenge (Erica’s Entourage and CRUS11TOUR), and the NCI (P01CA236749; K12CA090354).ReferencesDixon KO, Schorer M, Nevin J, Etminan Y, Amoozgar Z, Kondo T, Kurtulus S, Kassam N, Sobel RA, Fukumura D, Jain RK, Anderson AC, Kuchroo VK, Joller N. Functional anti-TIGIT antibodies regulate development of autoimmunity and antitumor immunity. J Immunol 2018 April 15;200(8):3000–3007.Hung AL, Maxwell R, Theodros D, Belcaid Z, Mathios D, Luksik AS, Kim E, Wu A, Xia Y, Garzon-Muvdi T, Jackson C, Ye X, Tyler B, Selby M, Korman A, Barnhart B, Park SM, Youn JI, Chowdhury T, Park CK, Brem H, Pardoll DM, Lim M. TIGIT and PD-1 dual checkpoint blockade enhances antitumor immunity and survival in GBM. Oncoimmunology 2018 May 24;7(8):e1466769.Raphael I, Kumar R, McCarl LH, Shoger K, Wang L, Sandlesh P, Sneiderman CT, Allen J, Zhai S, Campagna ML, Foster A, Bruno TC, Agnihotri S, Hu B, Castro BA, Lieberman FS, Broniscer A, Diaz AA, Amankulor NM, Rajasundaram D, Pollack IF, Kohanbash G. TIGIT and PD-1 immune checkpoint pathways are associated with patient outcome and anti-tumor immunity in glioblastoma. Front Immunol 2021 May 7;12:637146.Reardon DA, Gokhale PC, Klein SR, Ligon KL, Rodig SJ, Ramkissoon SH, Jones KL, Conway AS, Liao X, Zhou J, Wen PY, Van Den Abbeele AD, Hodi FS, Qin L, Kohl NE, Sharpe AH, Dranoff G, Freeman GJ. Glioblastoma eradication following immune checkpoint blockade in an orthotopic, immunocompetent model. Cancer Immunol Res 2016 February;4(2):124–35. Iorgulescu JB, Gokhale PC, Speranza MC, Eschle BK, Poitras MJ, Wilkens MK, Soroko KM, Chhoeu C, Knott A, Gao Y, Lim-Fat MJ, Baker GJ, Bonal DM, Nguyen QD, Grant GRL, Ligon KL, Sorger PK, Chiocca EA, Anderson AC, Kirschmeier PT, Sharpe AH, Freeman GJ, Reardon DA. Concurrent dexamethasone limits the clinical benefit of immune checkpoint blockade in glioblastoma. Clin Cancer Res 2021 January 1;27(1):276–287.

Background Cancer is the leading cause of death in the geriatric dog population. Currently, the use of immune checkpoint inhibitors (ICIs) such as anti-CTLA4 antibodies has markedly improved the prognosis of several cancers in their advanced stages. However, ICIs targeting CTLA4 blockade to treat canine cancer patients are yet to define. In this study, we sought to develop, characterize and assess whether chimeric heavy chain only antibodies (cHcAbs) against CTLA4 are viable therapeutic candidates for the treatment of canine cancers.Methods Anti-CTLA4 nanobodies (Nbs) were identified from a yeast nanobody (Nb) library using magnetic-assisted cell sorting (MACS) and flow cytometry. cHcAbs were engineered by genetically fusing the DNA sequences coding for anti-CTLA4 Nbs with the Fc domain of the subclass B of canine IgG. Recombinant cHcAbs were purified from ExpiCHO-S cells. Stable cell lines expressing canine CTLA4 and FcγRI were used to elucidate the binding ability and specificity of cHcAbs. PBMCs isolated from healthy dogs were used to evaluate the ability of cHcAbs to activate canine PBMCs.Results Novel Nbs were identified using the extracellular domain of canine CTLA4 protein to screen a fully synthetic yeast nanobody library. Purified Nbs bind specifically to natïve canine CTLA4. We report that chimeric HcAbs, which were engineered by fusing the anti-CTLA4 Nbs and Fc region of subclass B of canine IgG, were half the size of a conventional mAb and formed dimers. The chimeric HcAbs specifically binds both with canine CTLA4 and FcƳ receptors. As the binding of Nbs overlapped with the MYPPPY motif of canine CTLA4, these Nbs were expected to sterically disrupt the interaction of canine CTLA4 to B-7s. Like their human counterpart, canine CTLA4 was expressed on helper T cells and a small subset of cytotoxic T cells. Canine Tregs also constitutively expressed CTLA4, and stimulation with PMA/Ionomycin dramatically increased expression of CTLA4 on the cell surface. Stimulation of canine PBMCs in the presence of agonistic anti-CD3 Ab and cHcAb6 significantly increased the expression of IFN-γ as compared to the isotype control.Conclusions This study identifies a novel nanobody-based CTLA4 inhibitor for the treatment of canine cancer patients. Furthermore, this approach provides a critical proof-of-concept for developing nanobody-based humanized anti-CTLA4 therapy for advanced stages of cancers.

Monoclonal antibodies (mAbs) are promising alternatives to treat infectious diseases, especially given their potential for applications in combination therapies with antimicrobial drugs to enhance the antifungal efficacy. Protection mediated by mAbs used to treat experimental paracoccidioidomycosis (PCM) has been demonstrated previously. Our aim in the present work was to characterize a monoclonal antibody (mAbF1.4) raised against a cell wall glycoconjugate fraction of Paracoccidioides spp. and to analyze its efficacy combined with trimethoprim-sulfamethoxazole (TMP/SMX) as treatment for experimental PCM. We demonstrated that the epitope recognized by mAbF1.4 is consistent with branched glucose residues present on a cell wall β-glucan polymer. In vitro, mAbF1.4 increased the phagocytic capacity and nitric oxide concentration induced by the macrophage cell line J774.1A, and this resulted in a significant reduction in the viability of the opsonophagocytized yeasts. In vivo, we detected a significant reduction in pulmonary fungal burdens of mice treated with mAbF1.4 in association with TMP/SMX, which correlated with increased pulmonary concentrations (determined by ELISA) of IFN- γ, TNF-α, IL-10 and IL-17. In parallel, we observed a decrease in IL-4, suggesting that the treatment was associated with a mixed Th1-Th17 type immune response. Histopathology of lung segments from mice receiving the combination therapy showed a significant reduction in granulomas, which were well-defined, and improved maintenance of lung architecture. These findings demonstrate that mAbF1.4 + TMP/SMX therapy is a promising approach to combat PCM as well as decrease disease sequelae and highlights the potential benefits of immune mediators in PCM combined immunotherapy.

Anti-TNFα and anti-IL-23 antibodies are highly effective therapies for Crohn’s disease or ulcerative colitis in a proportion of patients. V56B2 is a novel bispecific domain antibody in which a llama-derived IL-23p19-specific domain antibody, humanised and engineered for intestinal protease resistance, V900, was combined with a previously-described TNFα-specific domain antibody, V565. V56B2 contains a central protease-labile linker to create a single molecule for oral administration. Incubation of V56B2 with trypsin or human faecal supernatant resulted in a complete separation of the V565 and V900 monomers without loss of neutralising potency. Following oral administration of V900 and V565 in mice, high levels of each domain antibody were detected in the faeces, demonstrating stability in the intestinal milieu. In ex vivo cultures of colonic biopsies from IBD patients, treatment with V565 or V900 inhibited tissue phosphoprotein levels and with a combination of the two, inhibition was even greater. These results support further development of V56B2 as an oral therapy for IBD with improved safety and efficacy in a greater proportion of patients as well as greater convenience for patients compared with traditional monoclonal antibody therapies.

Multiple myeloma is the second most common hematological malignancy in adults, accounting for 2% of all cancer-related deaths in the UK. Current chemotherapy-based regimes are insufficient, as most patients relapse and develop therapy resistance. This review focuses on current novel antibody- and aptamer-based therapies aiming to overcome current therapy limitations, as well as their respective limitations and areas of improvement. The use of computer modeling methods, as a tool to study and improve ligand-receptor alignments for the use of novel therapy development will also be discussed, as it has become a rapid, reliable and comparatively inexpensive method of investigation.

Targeting the immunological checkpoint PD-1/PD-L1 with antibodies has shown opportunities to improve cancer treatment in recent years. However, antibody therapy is a double-edged sword with high cost, low patient tolerance, lack of oral bioavailability, and a reaction to most solid tumors that prevents the adoption of antibodies. Advancement of small-molecule PD-1/PD-L1 inhibitors that could overwhelm these drawbacks is sluggish because of the poor pharmacodynamic properties and shallow pocket of the PD-1/PD-L1 binding interface. Recently, a number of compounds have been discovered to bind the PD-L1/PD-L1 dimer interface, providing an excellent alternative to inhibit the interaction between PD-1/PD-L1 and small molecules. Quantitative characterization of PD-L1 interactions with these inhibitors will advance the design of novel and efficient inhibitors in the future. Here, the binding free energies of 35 PD-L1 dimer inhibitors have been calculated using the alanine-scanning-interaction-entropy (AS-IE) method. Hotspot residues on PD-L1 and potential modification groups on the inhibitors were identified. The experimental results for the AS-IE method were better correlated than the classical MM/GBSA method. These results may set the stage for the design the more powerful PD-L1 inhibitors.Communicated by Ramaswamy H. Sarma.

The successive emergences and accelerating spread of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages and evolved resistance to some ongoing clinical therapeutics increase the risks associated with the coronavirus disease 2019 (COVID-19) pandemic. An urgent intervention for broadly effective therapies to limit the morbidity and mortality of COVID-19 and future transmission events from SARS-related coronaviruses (SARSr-CoVs) is needed. Here, we isolate and humanize an angiotensin-converting enzyme-2 (ACE2)-blocking monoclonal antibody (MAb), named h11B11, which exhibits potent inhibitory activity against SARS-CoV and circulating global SARS-CoV-2 lineages. When administered therapeutically or prophylactically in the hACE2 mouse model, h11B11 alleviates and prevents SARS-CoV-2 replication and virus-induced pathological syndromes. No significant changes in blood pressure and hematology chemistry toxicology were observed after injections of multiple high dosages of h11B11 in cynomolgus monkeys. Analysis of the structures of the h11B11/ACE2 and receptor-binding domain (RBD)/ACE2 complexes shows hindrance and epitope competition of the MAb and RBD for the receptor. Together, these results suggest h11B11 as a potential therapeutic countermeasure against SARS-CoV, SARS-CoV-2, and escape variants., Here the authors report the isolation and structural and biological characterization of a humanized angiotensin-converting enzyme 2 (ACE2)-blocking antibody, which exterts potent inhibitory activity against SARS-CoV and circulating global SARS-CoV-2 lineages both in vitro and in hACE2 mouse model.

Outcomes in chronic lymphocytic leukemia (CLL) have been dramatically improved with the addition of anti-CD20 antibodies to chemotherapy, defining a new standard of care for many years. More recently, therapies targeting fundamental signaling and anti-apoptotic pathways within the CLL cell have demonstrated dramatic clinical responses, including in patients with high-risk prognostic markers, thus emerging as preferred therapy for many patients. While the addition of anti-CD20 antibodies to traditional chemotherapy resulted in significant improvements in outcomes, the role of monoclonal antibodies in the era of targeted agents remains an active area of investigation. Furthermore, since the advent of next-generation anti-CD20 antibodies, the role of specific anti-CD20 antibodies remains an open question.In this review, we highlight the important role that anti-CD20 antibody therapy has had in the field of CLL, both when used with chemotherapy and in combination with targeted therapy, as well as the current studies that are further exploring this treatment paradigm in the modern era.While anti-CD20 antibodies have played a pivotal role in the treatment of CLL, additional studies will be required to determine the optimal application of these therapies in combination with targeted therapy.

SARS-CoV-2 causes severe acute respiratory syndrome, which has led to significant morbidity and mortality around the world. Since its emergence, extensive prophylactic and therapeutic countermeasures have been employed to successfully prevent the spread of COVID-19. Extensive work has been undertaken on using monoclonal antibody therapies, mass vaccination programs, and antiviral drugs to prevent and treat COVID-19. However, since antiviral drugs could take years to become widely available, immunotherapy and vaccines currently appear to be the most feasible option. In December 2020, the first vaccine against SARS-CoV-2 was approved by the World Health Organization (WHO) and, subsequently, many other vaccines were approved for use by different international regulators in different countries. Most monoclonal antibodies (mAbs) and vaccines target the SARS-CoV-2 surface spike (S) protein. Recently, mutant (or variant) SARS-CoV-2 strains with increased infectivity and virulence that evade protective host antibodies present either due to infection, antibody therapy, or vaccine administration have emerged. In this manuscript, we discuss the different monoclonal antibody and vaccine therapies available against COVID-19 and how the efficacy of these therapies is affected by the emergence of variants of SARS-CoV-2. We also discuss strategies that might help society cope with variants that could neutralize the effects of immunotherapy and escape the protective immunity conferred by vaccines.

Monoclonal antibodies (mAbs) are emerging as safe and effective therapeutics against SARS-CoV-2. However, variant strains of SARS-CoV-2 have evolved, with early studies showing that some mAbs may not sustain their efficacy in the face of escape mutants. Also, from the onset of the COVID-19 pandemic, concern has been raised about the potential for Fcγ receptor-mediated antibody-dependent enhancement (ADE) of infection. In this study, plaque reduction neutralization assays demonstrated that mAb 1741-LALA neutralizes SARS-CoV-2 strains B.1.351, D614 and D614G. MAbs S1D2-hIgG1 and S1D2-LALA mutant (STI-1499-LALA) did not neutralize B.1.351, but did neutralize SARS-CoV-2 strains D614 and D614G. LALA mutations did not result in substantial differences in neutralizing abilities between clones S1D2-hIgG1 vs STI-1499-LALA. S1D2-hIgG1, STI-1499-LALA, and convalescent plasma showed minimal ability to induce ADE in human blood monocyte-derived macrophages. Further, no differences in pharmacokinetic clearance of S1D2-hIgG1 vs STI-1499-LALA were observed in mice expressing human FcRn. These findings confirm that SARS-CoV-2 has already escaped some mAbs, and identify a mAb candidate that may neutralize multiple SARS-CoV-2 variants. They also suggest that risk of ADE in macrophages may be low with SARS-CoV-2 D614, and LALA Fc change impacts neither viral neutralization nor Ab clearance.

Activation of the tyrosine kinase with Ig and epidermal growth factor homology domain 2 (Tie2) receptor by angiopoietin-1 (Ang1) is critical for vascular stabilization: it promotes survival signal transduction via auto-phosphorylation and reduces vascular permeability by strengthening tight junctions between endothelial cells. Thus, Tie2/Ang1 signaling is a promising therapeutic target for vascular diseases. However, in vivo use of existing Tie2 signaling modulators, such as recombinant Ang1, is restricted by limitations in manufacturability and stability. Here, we present a novel engineered tetra-valent agonistic antibody, ASP4021, which can specifically and fully activate the Tie2 receptor in an equivalent manner to Ang1. ASP4021 induced Tie2 self-phosphorylation and inhibited apoptosis in a human primary endothelial cell line. Additionally, single administration of ASP4021 significantly suppressed mustard-oil-induced vascular permeability in rats. ASP4021 may thus be a potential therapeutic candidate for diseases associated with vascular weakness such as diabetic retinopathy, diabetic macular edema and critical limb ischemia.

Donanemab, an antibody directed toward an N-terminal pyroglutamate Aβ epitope, was recently tested in a phase 2 trial of early symptomatic Alzheimer's disease patients (n=257) treated for 76 weeks [1]. The antibody therapy reduced plaque load by 80%. This accompanied a significant (P=0.04) delay in decline (~6 month) in the integrated Alzheimer's Disease Rating Scale (iADRS) scale, but not 4 other cognitive/functional scales. However, donanemab treated patients had accelerated brain volume loss (~5ml) as assessed by MRI.

There is an urgent need for effective therapeutic interventions against SARS-CoV-2, including new variants that continue to arise. Neutralizing monoclonal antibodies have shown promise in clinical studies. We investigated the therapeutic efficacy of a combination of two potent monoclonal antibodies, C135-LS and C144-LS that carry half-life extension mutations, in the rhesus macaque model of COVID-19. Twelve young adult macaques (three groups of four animals) were inoculated intranasally and intra-tracheally with a high dose of SARS-CoV-2 and 24 hours later, treated intravenously with a high (40 mg/kg) or low (12 mg/kg) dose of the C135-LS and C144-LS antibody combination, or a control monoclonal antibody. Animals were monitored for 7 days. Compared to the control animals, animals treated with either dose of the anti-SARS-CoV-2 antibodies showed similarly improved clinical scores, lower levels of virus replication in upper and lower respiratory tract, and significantly reduced interstitial pneumonia, as measured by comprehensive lung histology. In conclusion, this study provides proof-of-concept in support of further clinical development of these monoclonal antibodies against COVID-19 during early infection., Author summary Monoclonal antibodies that neutralize SARS-CoV-2 have shown promise in treating recently infected individuals who are at high risk of progression to severe COVID-19 disease. Although several monoclonal antibodies are currently being used in the clinic, there is an ongoing need to develop additional antibodies. The ideal monoclonal antibodies, or combinations, should be potent and durable, and maintain activity against emerging viral variants. In this study, we tested a combination of two potent monoclonal antibodies, C135-LS and C-144-LS, engineered to have long half-lives, in the macaque model of SARS-CoV-2 infection. Animals treated early after infection fared better than placebo-treated controls, manifesting fewer clinical signs, less virus replication in the respiratory tract, and reduced lung inflammation. These promising data support clinical testing of these monoclonal antibodies in humans and further development of similar antibody-based prophylactic and therapeutic strategies.

The use of antibodies in the treatment of lung diseases is of increasing interest especially as the search for COVID-19 therapies has unfolded. Historically, the use of antibody therapy was based on multiple targets including receptors involved in local hyper-reactivity in asthma, viruses and micro-organisms involved in a variety of pulmonary infectious disease. Generally, protein therapeutics pose challenges with respect to formulation and delivery to retain activity and assure therapy. The specificity of antibodies amplifies the need for attention to molecular integrity not only in formulation but also during aerosol delivery for pulmonary administration. Drug product development can be viewed from considerations of route of administration, dosage form, quality, and performance measures. Nebulizers and dry powder inhalers have been used to deliver protein therapeutics and each has its advantages that should be matched to the needs of the drug and the disease. This review offers insight into quality and performance barriers and the opportunities that arise from meeting them effectively.

Epidermal growth factor receptor (EGFR)-targeted cancer therapy such as anti-EGFR monoclonal antibodies and tyrosine kinase inhibitors have demonstrated clinical efficacy. However, there remains a medical need addressing limitations of these therapies, which include a narrow therapeutic window mainly due to skin and organ toxicity, and primary and secondary resistance mechanisms of the EGFR-signaling cascade (e.g., RAS-mutated colorectal cancer). Using the redirected optimized cell killing (ROCK®) antibody platform, we have developed AFM24, a novel bispecific, IgG1-scFv fusion antibody targeting CD16A on innate immune cells, and EGFR on tumor cells. We herein demonstrate binding of AFM24 to CD16A on natural killer (NK) cells and macrophages with KD values in the low nanomolar range and to various EGFR-expressing tumor cells. AFM24 was highly potent and effective for antibody-dependent cell-mediated cytotoxicity via NK cells, and also mediated antibody-dependent cellular phagocytosis via macrophages in vitro. Importantly, AFM24 was effective toward a variety of EGFR-expressing tumor cells, regardless of EGFR expression level and KRAS/BRAF mutational status. In vivo, AFM24 was well tolerated up to the highest dose (75 mg/kg) when administered to cynomolgus monkeys once weekly for 28 days. Notably, skin and other toxicities were not observed. A transient elevation of interleukin-6 levels was detected at all dose levels, 2–4 hours post-dose, which returned to baseline levels after 24 hours. These results emphasize the promise of bispecific innate cell engagers as an alternative cancer therapy and demonstrate the potential for AFM24 to effectively target tumors expressing varying levels of EGFR, regardless of their mutational status. Abbreviations: ADA: antidrug antibody; ADCC: antibody-dependent cell-mediated cytotoxicity; ADCP: antibody-dependent cellular phagocytosis; AUC: area under the curve; CAR: chimeric-antigen receptor; CD: Cluster of differentiation; CRC :colorectal cancer; ECD: extracellular domain; EGF: epidermal growth factorEGFR epidermal growth factor receptor; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell sorting; Fc: fragment, crystallizableFv variable fragment; HNSCC: head and neck squamous carcinomaIL interleukinm; Ab monoclonal antibody; MOA: mechanism of action; NK :natural killer; NSCLC: non-small cell lung cancer; PBMC: peripheral blood mononuclear cell; PBS: phosphate-buffered saline; PD: pharmacodynamic; ROCK: redirected optimized cell killing; RSV: respiratory syncytial virus; SABC: specific antibody binding capacity; SD: standard deviation; TAM: tumor-associated macrophage; TKI: tyrosine kinase inhibitor; WT: wildtype

Immune checkpoint inhibitors demonstrate clinical activity in many tumor types, however, only a fraction of patients benefit. Combining CD137 agonists with these inhibitors increases anti-tumor activity preclinically, but attempts to translate these observations to the clinic have been hampered by systemic toxicity. Here we describe a human CD137xPD-L1 bispecific antibody, MCLA-145, identified through functional screening of agonist- and immune checkpoint inhibitor arm combinations. MCLA-145 potently activates T cells at sub-nanomolar concentrations, even under suppressive conditions, and enhances T cell priming, differentiation and memory recall responses. In vivo, MCLA-145 anti-tumor activity is superior to immune checkpoint inhibitor comparators and linked to recruitment and intra-tumor expansion of CD8 + T cells. No graft-versus-host-disease is observed in contrast to other antibodies inhibiting the PD-1 and PD-L1 pathway. Non-human primates treated with 100 mg/kg/week of MCLA-145 show no adverse effects. The conditional activation of CD137 signaling by MCLA-145, triggered by neighboring cells expressing >5000 copies of PD-L1, may provide both safety and potency advantages., The anti-tumour effect of immune checkpoint inhibitors is potentiated by CD137 agonists in preclinical models, but translation of these results to the clinical practice is hampered by toxicity. Authors describe here a human CD137xPD-L1 bispecific antibody with improved anti-cancer activity whilst maintaining low toxicity in non-human primates.

Neutralizing antibodies to the SARS CoV-2 spike proteins have been issued Emergency Use Authorizations and are a likely mechanism of vaccines to prevent COVID-19. However, benefit of treatment with monoclonal antibodies has only been observed in clinical trials in outpatients with mild to moderate COVID-19 but not in patients who are hospitalized and/or have advanced disease. To address this observation, we evaluated the timing of anti SARS-CoV-2 antibody production in hospitalized patients with the use of a highly sensitive multiplexed bead-based immunoassay allowing for early detection of antibodies to SARS-CoV-2. We found significantly lower levels of antibodies to the SARS-CoV-2 spike protein in the first week after symptom onset in patients who expired as compared to patients who were discharged. We also developed a model to characterize the relationship between each patient’s individual antibody level trajectory and eventual COVID 19 outcome which can be adapted into a prediction model with more data., Author summary We evaluated antibodies to SARS-CoV-2 over time in patients that were hospitalized with COVID 19. Early detection of Anti-SARS-CoV-2 antibodies was associated with survival in patients hospitalized with COVID 19. Early antibody levels predicted outcome in our study. This result is consistent with the benefit of therapeutic antibodies early in the course of COVID 19 disease. With additional study, early antibody levels may be helpful in deciding on appropriate therapies.

The efficacy of convalescent plasma for coronavirus disease 2019 (COVID-19) is unclear. Although most randomized controlled trials have shown negative results, uncontrolled studies have suggested that the antibody content could influence patient outcomes. We conducted an open-label, randomized controlled trial of convalescent plasma for adults with COVID-19 receiving oxygen within 12 d of respiratory symptom onset (NCT04348656). Patients were allocated 2:1 to 500 ml of convalescent plasma or standard of care. The composite primary outcome was intubation or death by 30 d. Exploratory analyses of the effect of convalescent plasma antibodies on the primary outcome was assessed by logistic regression. The trial was terminated at 78% of planned enrollment after meeting stopping criteria for futility. In total, 940 patients were randomized, and 921 patients were included in the intention-to-treat analysis. Intubation or death occurred in 199/614 (32.4%) patients in the convalescent plasma arm and 86/307 (28.0%) patients in the standard of care arm—relative risk (RR) = 1.16 (95% confidence interval (CI) 0.94–1.43, P = 0.18). Patients in the convalescent plasma arm had more serious adverse events (33.4% versus 26.4%; RR = 1.27, 95% CI 1.02–1.57, P = 0.034). The antibody content significantly modulated the therapeutic effect of convalescent plasma. In multivariate analysis, each standardized log increase in neutralization or antibody-dependent cellular cytotoxicity independently reduced the potential harmful effect of plasma (odds ratio (OR) = 0.74, 95% CI 0.57–0.95 and OR = 0.66, 95% CI 0.50–0.87, respectively), whereas IgG against the full transmembrane spike protein increased it (OR = 1.53, 95% CI 1.14–2.05). Convalescent plasma did not reduce the risk of intubation or death at 30 d in hospitalized patients with COVID-19. Transfusion of convalescent plasma with unfavorable antibody profiles could be associated with worse clinical outcomes compared to standard care., A randomized trial in patients hospitalized with COVID-19 showed no benefit and potentially increased harm associated with the use of convalescent plasma, with subgroup analyses suggesting that the antibody profile in donor plasma is critical in determining clinical outcomes.

The Ig superfamily protein glycoprotein A33 (GPA33) has been implicated in immune dysregulation, but little is known about its expression in the immune compartment. Here, we comprehensively determined GPA33 expression patterns on human blood leukocyte subsets, using mass and flow cytometry. We found that GPA33 was expressed on fractions of B, dendritic, natural killer and innate lymphoid cells. Most prominent expression was found in the CD4+ T cell compartment. Naïve and CXCR5+ regulatory T cells were GPA33high, and naïve conventional CD4+ T cells expressed intermediate GPA33 levels. The expression pattern of GPA33 identified functional heterogeneity within the CD4+ central memory T cell (Tcm) population. GPA33+ CD4+ Tcm cells were fully undifferentiated, bona fide Tcm cells that lack immediate effector function, whereas GPA33– Tcm cells exhibited rapid effector functions and may represent an early stage of differentiation into effector/effector memory T cells before loss of CD62L. Expression of GPA33 in conventional CD4+ T cells suggests a role in localization and/or preservation of an undifferentiated state. These results form a basis to study the function of GPA33 and show it to be a useful marker to discriminate between different cellular subsets, especially in the CD4+ T cell lineage., Among human blood leukocytes GPA33 is predominantly expressed on CD4+ T cells that lack effector function. GPA33 marks naïve CD4+ T cells and separates the CCR7+CD62L+ central memory compartment into an undifferentiated population and one that exhibits effector properties, suggesting GPA33 has a role in localization and/or preservation of quiescence.

Transthyretin amyloid (ATTR) cardiomyopathy is a debilitating disease leading to heart failure and death. It is characterized by the deposition of extracellular ATTR fibrils in the myocardium. Reducing myocardial ATTR load is a therapeutic goal anticipated to translate into restored cardiac function and improved patient survival. For this purpose, we developed the selective anti-ATTR antibody NI301A, a recombinant human monoclonal immunoglobulin G1. NI301A was cloned following comprehensive analyses of memory B cell repertoires derived from healthy elderly subjects. NI301A binds selectively with high affinity to the disease-associated ATTR aggregates of either wild-type or variant ATTR related to sporadic or hereditary disease, respectively. It does not bind physiological transthyretin. NI301A removes ATTR deposits ex vivo from patient-derived myocardium by macrophages, as well as in vivo from mice grafted with patient-derived ATTR fibrils in a dose- and time-dependent fashion. The biological activity of ATTR removal involves antibody-mediated activation of phagocytic immune cells including macrophages. These data support the evaluation of safety and tolerability of NI301A in an ongoing phase 1 clinical trial in patients with ATTR cardiomyopathy., Analyzing memory B cell repertoires of the healthy elderly enabled Michalon et al. to develop a recombinant human antibody selective for transthyretin amyloid. This antibody removes cardiac amyloid through recruitment of phagocytic immune cells.

The emergence of a new acute respiratory syndrome Coronavirus 2 (SARS‐CoV‐2), the cause of the 2019‐nCOV disease (COVID‐19), has caused a pandemic and a global health crisis. Rapid human‐to‐human transmission, even from asymptomatic individuals, has led to the quick spread of the virus worldwide, causing a wide range of clinical manifestations from cold‐like symptoms to severe pneumonia, acute respiratory distress syndrome (ARDS), multiorgan injury, and even death. Therefore, using rapid and accurate diagnostic methods to identify the virus and subsequently select appropriate and effective treatments can help improvement of patients and control the pandemic. So far, various treatment regimens along with prophylactic vaccines have been developed to manage COVID‐19‐infected patients. Among these, antibody‐based therapies, including neutralizing antibodies (against different parts of the virus), polyclonal and monoclonal antibodies, plasma therapy, and high‐dose intravenous immunoglobulin (IVIG) have shown promising outcomes in accelerating and improving the treatment process of patients, avoiding the viral spreading widely, and managing the pandemic. In the current review paper, different types and applications of therapeutic antibodies in the COVID‐19 treatment are comprehensively discussed., Graphical abstract antibody‐based therapies, including neutralizing antibodies (against different parts of the virus), polyclonal and monoclonal antibodies, plasma therapy, and High‐dose intravenous immunoglobulin (IVIG) have shown promising outcomes in accelerating and improving the treatment process of patients, avoiding the viral widespread, and managing the pandemic. In the current review paper, different types and applications of therapeutic antibodies in the COVID‐19 treatment are comprehensively discussed

In the current global emergency due to SARS-CoV-2 outbreak, passive immunotherapy emerges as a promising treatment for COVID-19. Among animal-derived products, equine formulations are still the cornerstone therapy for treating envenomations due to animal bites and stings. Therefore, drawing upon decades of experience in manufacturing snake antivenom, we developed and preclinically evaluated two anti-SARS-CoV-2 polyclonal equine formulations as potential alternative therapy for COVID-19. We immunized two groups of horses with either S1 (anti-S1) or a mixture of S1, N, and SEM mosaic (anti-Mix) viral recombinant proteins. Horses reached a maximum anti-viral antibody level at 7 weeks following priming, and showed no major adverse acute or chronic clinical alterations. Two whole-IgG formulations were prepared via hyperimmune plasma precipitation with caprylic acid and then formulated for parenteral use. Both preparations had similar physicochemical and microbiological quality and showed ELISA immunoreactivity towards S1 protein and the receptor binding domain (RBD). The anti-Mix formulation also presented immunoreactivity against N protein. Due to high anti-S1 and anti-RBD antibody content, final products exhibited high in vitro neutralizing capacity of SARS-CoV-2 infection, 80 times higher than a pool of human convalescent plasma. Pre-clinical quality profiles were similar among both products, but clinical efficacy and safety must be tested in clinical trials. The technological strategy we describe here can be adapted by other producers, particularly in low- and middle-income countries.

Simple Summary Antibodies with their high specificity to antigens have been widely used in cancer immunotherapy. Natural killer (NK) cells are a group of innate immune cells which have strong cytotoxicity against cancerous cells, virus infected cells, or transformed cells. NK cells express abundant Fc receptors that can bind tumor-specific antibodies, thus allowing them to precisely redirect and eliminate cancer cells. In this study, we demonstrated that NK cells cytotoxicity toward MUC1-positive hematologic and solid tumor can be further enhanced by a humanized 5E5 anti-MUC1 antibody. Furthermore, Fc defucosylation of the antibodies further boosted the kill capacity of NK cells. We believe that our humanized anti-MUC1 antibody is a promising therapeutic candidate for clinical cancer treatment. Abstract Antibodies are commonly used in cancer immunotherapy because of their high specificity for tumor-associated antigens. The binding of antibodies can have direct effects on tumor cells but also engages natural killer (NK) cells via their Fc receptor. Mucin 1 (MUC1) is a highly glycosylated protein expressed in normal epithelial cells, while the under-glycosylated MUC1 epitope (MUC1-Tn/STn) is only expressed on malignant cells, making it an interesting diagnostic and therapeutic target. Several anti-MUC1 antibodies have been tested for therapeutic applications in solid tumors thus far without clinical success. Herein, we describe the generation of fully humanized antibodies based on the murine 5E5 antibody, targeting the tumor-specific MUC1-Tn/STn epitope. We confirmed that these antibodies specifically recognize tumor-associated MUC1 epitopes and can activate human NK cells in vitro. Defucosylation of these newly developed anti-MUC1 antibodies further enhanced antigen-dependent cellular cytotoxicity (ADCC) mediated by NK cells. We show that endocytosis inhibitors augment the availability of MUC1-Tn/STn epitopes on tumor cells but do not further enhance ADCC in NK cells. Collectively, this study describes novel fully humanized anti-MUC1 antibodies that, especially after defucosylation, are promising therapeutic candidates for cellular immunotherapy.

Background: Human milk from coronavirus disease 2019 (COVID-19)-recovered women may be useful as oral antibody therapy to prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infect...

In contrast to AAV, Simian Virus 40 (rSV40) not inducing neutralizing antibodies (NAbs) allowing re-treatment seems a promising vector for neonatal treatment of inherited liver disorders. Several studies have reported efficacy of rSV40 in animal models for inherited liver diseases. In all studies the ubiquitous endogenous early promoter controlled transgene expression establishing expression in all transduced tissues. Restricting this expression to the target tissues reduces the risk of immune response to the therapeutic gene. In this study a liver specific rSV40 vector was generated by inserting a hepatocyte specific promoter. This increased the specificity of the expression of hUGT1A1 in vitro. However, in vivo the efficacy of rSV40 appeared too low to demonstrate tissue specificity while increasing the vector dose was not possible because of toxicity. In contrast to earlier studies, neutralizing antibodies were induced. Overall, the lack of a platform to produce high titered and pure rSV40 particles and the induction of NAbs, renders it a poor candidate for in vivo gene therapy.