Search

The 'Antibodies to Watch' article series provides an annual summary of commercially sponsored monoclonal antibody therapeutics currently in late-stage clinical development, regulatory review, and those recently granted a first approval in any country. In this installment, we discuss key details for 16 antibody therapeutics granted a first approval in 2023, as of November 17 (lecanemab (Leqembi), rozanolixizumab (RYSTIGGO), pozelimab (VEOPOZ), mirikizumab (Omvoh), talquetamab (Talvey), elranatamab (Elrexfio), epcoritamab (EPKINLY), glofitamab (COLUMVI), retifanlimab (Zynyz), concizumab (Alhemo), lebrikizumab (EBGLYSS), tafolecimab (SINTBILO), narlumosbart (Jinlitai), zuberitamab (Enrexib), adebrelimab (Arelili), and divozilimab (Ivlizi)). We briefly review 26 product candidates for which marketing applications are under consideration in at least one country or region, and 23 investigational antibody therapeutics that are forecast to enter regulatory review by the end of 2024 based on company disclosures. These nearly 50 product candidates include numerous innovative bispecific antibodies, such as odronextamab, ivonescimab, linvoseltamab, zenocutuzumab, and erfonrilimab, and antibody-drug conjugates, such as trastuzumab botidotin, patritumab deruxtecan, datopotamab deruxtecan, and MRG002, as well as a mixture of two immunocytokines (bifikafusp alfa and onfekafusp alfa). We also discuss clinical phase transition and overall approval success rates for antibody therapeutics, which are crucial to the biopharmaceutical industry because these rates inform decisions about resource allocation. Our analyses indicate that these molecules have approval success rates in the range of 14-32%, with higher rates associated with antibodies developed for non-cancer indications. Overall, our data suggest that antibody therapeutic development efforts by the biopharmaceutical industry are robust and increasingly successful.

B cells are known to contribute to the anti-tumor immune response, especially in immunogenic tumors such as melanoma, yet humoral immunity has not been characterized in these cancers to detail. Here we show comprehensive phenotyping in samples of circulating and tumor-resident B cells as well as serum antibodies in melanoma patients. Memory B cells are enriched in tumors compared to blood in paired samples and feature distinct antibody repertoires, linked to specific isotypes. Tumor-associated B cells undergo clonal expansion, class switch recombination, somatic hypermutation and receptor revision. Compared with blood, tumor-associated B cells produce antibodies with proportionally higher levels of unproductive sequences and distinct complementarity determining region 3 properties. The observed features are signs of affinity maturation and polyreactivity and suggest an active and aberrant autoimmune-like reaction in the tumor microenvironment. Consistent with this, tumor-derived antibodies are polyreactive and characterized by autoantigen recognition. Serum antibodies show reactivity to antigens attributed to autoimmune diseases and cancer, and their levels are higher in patients with active disease compared to post-resection state. Our findings thus reveal B cell lineage dysregulation with distinct antibody repertoire and specificity, alongside clonally-expanded tumor-infiltrating B cells with autoimmune-like features, shaping the humoral immune response in melanoma., (© 2023. The Author(s).)

Outcomes for half of patients with melanoma remain poor despite standard-of-care checkpoint inhibitor therapies. The prevalence of the melanoma-associated antigen chondroitin sulfate proteoglycan 4 (CSPG4) expression is ~70%, therefore effective immunotherapies directed at CSPG4 could benefit many patients. Since IgE exerts potent immune-activating functions in tissues, we engineer a monoclonal IgE antibody with human constant domains recognizing CSPG4 to target melanoma. CSPG4 IgE binds to human melanomas including metastases, mediates tumoricidal antibody-dependent cellular cytotoxicity and stimulates human IgE Fc-receptor-expressing monocytes towards pro-inflammatory phenotypes. IgE demonstrates anti-tumor activity in human melanoma xenograft models engrafted with human effector cells and is associated with enhanced macrophage infiltration, enriched monocyte and macrophage gene signatures and pro-inflammatory signaling pathways in the tumor microenvironment. IgE prolongs the survival of patient-derived xenograft-bearing mice reconstituted with autologous immune cells. No ex vivo activation of basophils in patient blood is measured in the presence of CSPG4 IgE. Our findings support a promising IgE-based immunotherapy for melanoma., (© 2023. The Author(s).)

Background: Survival rates for ovarian cancer remain poor, and monitoring and prediction of therapeutic response may benefit from additional markers. Ovarian cancers frequently overexpress Folate Receptor alpha (FRα) and the soluble receptor (sFRα) is measurable in blood. Here we investigated sFRα as a potential biomarker., Methods: We evaluated sFRα longitudinally, before and during neo-adjuvant, adjuvant and palliative therapies, and tumour FRα expression status by immunohistrochemistry. The impact of free FRα on the efficacy of anti-FRα treatments was evaluated by an antibody-dependent cellular cytotoxicity assay., Results: Membrane and/or cytoplasmic FRα staining were observed in 52.7% tumours from 316 ovarian cancer patients with diverse histotypes. Circulating sFRα levels were significantly higher in patients, compared to healthy volunteers, specifically in patients sampled prior to neoadjuvant and palliative treatments. sFRα was associated with FRα cell membrane expression in the tumour. sFRα levels decreased alongside concurrent tumour burden in patients receiving standard therapies. High concentrations of sFRα partly reduced anti-FRα antibody tumour cell killing, an effect overcome by increased antibody doses., Conclusions: sFRα may present a non-invasive marker for tumour FRα expression, with the potential for monitoring patient response to treatment. Larger, prospective studies should evaluate FRα for assessing disease burden and response to systemic treatments., (© 2022. The Author(s).)

In this 14th installment of the annual Antibodies to Watch article series, we discuss key events in commercial monoclonal antibody therapeutics development that occurred in 2022 and forecast events that might occur in 2023. As of mid-November, 12 antibody therapeutics had been granted first approvals in either the United States or European Union (tebentafusp (Kimmtrak), faricimab (Vabysmo), sutimlimab (Enjaymo), relatlimab (Opdualag), tixagevimab/cilgavimab (Evusheld), mosunetuzumab (Lunsumio), teclistamab (TECVAYLI), spesolimab (SPEVIGO), tremelimumab (Imjudo; combo with durvalumab), nirsevimab (Beyfortus), mirvetuximab soravtansine (ELAHERE™), and teplizumab (TZIELD)), including 4 bispecific antibodies and 1 ADC. Based on FDA action dates, several additional product candidates could be approved by the end of 2022. An additional seven were first approved in China or Japan in 2022, including two bispecific antibodies (cadonilimab and ozoralizumab). Globally, at least 24 investigational antibody therapeutics are undergoing review by regulatory agencies as of mid-November 2022. Our data show that, with antibodies for COVID-19 excluded, the late-stage commercial clinical pipeline grew by ~20% in the past year to include nearly 140 investigational antibody therapeutics that were designed using a wide variety of formats and engineering techniques. Of those in late-stage development, marketing application submissions for at least 23 may occur by the end of 2023, of which 5 are bispecific (odronextamab, erfonrilimab, linvoseltamab, zanidatamab, and talquetamab) and 2 are ADCs (datopotamab deruxtecan, and tusamitamab ravtansine).

Efficient characterization of IgE antibodies and their glycan structures is required for understanding their function in allergy and in the emerging AllergoOncology field for antibody immunotherapy. We report the generation, glyco-profiling and functional analysis of native and sialic acid-deficient glyco-engineered human IgE. The antibodies produced from human embryonic kidney cells were purified via a human IgE class-specific affinity matrix and structural integrity was confirmed by SDS-PAGE and size-exclusion chromatography (SEC). Purified IgEs specific for the tumor-associated antigens Chondroitin Sulfate Proteoglycan 4 (CSPG4-IgE) and Human Epidermal Growth Factor Receptor 2 (HER2-IgE) were devoid of by-products such as free light chains. Using neuraminidase-A, we generated sialic acid-deficient CSPG4-IgE as example glyco-engineered antibody. Comparative glycan analyses of native and glyco-engineered IgEs by Hydrophilic interaction liquid chromatography (HILIC)-high performance liquid chromatography (HPLC) indicated loss of sialic acid terminal residues and differential glycan profiles. Native and glyco-engineered CSPG4-IgEs recognized Fc receptors on the surface of human FcεRI-expressing rat basophilic leukemia RBL-SX38 cells, and of CD23/FcεRII-expressing human RPMI-8866 B-lymphocytes and bound to CSPG4-expressing A2058 human melanoma cells, confirming Fab-mediated recognition. When cross-linked on the cell surface, both IgEs triggered RBL-SX38 degranulation. We demonstrate efficient generation and functional competence of recombinant native and sialic acid-deficient IgEs.

B cells are emerging as key players of anti-tumor adaptive immune responses. We investigated regulatory and pro-inflammatory cytokine-expressing B cells in patients with melanoma by flow cytometric intracellular cytokine, CyTOF, transcriptomic, immunofluorescence, single-cell RNA-seq, and B:T cell co-culture analyses. We found enhanced circulating regulatory (TGF-β + and PD-L1 + ) and reduced pro-inflammatory TNF-α + B cell populations in patients compared with healthy volunteers (HVs), including lower IFN-γ + :IL-4 +  and higher TGF-β + :TNF-α + B cell ratios in patients. TGF-β-expressing B cells in the melanoma tumor microenvironment assembled in clusters and interacted with T cells via lymphoid recruitment (SELL, CXCL13, CCL4, CD74) signals and with Tregs via CD47:SIRP-γ, and FOXP3-promoting Galectin-9:CD44. While reduced in tumors compared to blood, TNF-α-expressing B cells engaged in crosstalk with Tregs via TNF-α signaling and the ICOS/ICOSL axis. Patient-derived B cells promoted FOXP3 + Treg differentiation in a TGF-β-dependent manner, while sustaining expression of IFN-γ and TNF-α by autologous T-helper cells and promoting T-helper cell proliferation ex vivo , an effect further enhanced with anti-PD-1 checkpoint blockade. Our findings reveal cytokine-expressing B cell compartments skewed toward regulatory phenotypes in patient circulation and melanoma lesions, intratumor spatial localization, and bidirectional crosstalk between B and T cell subsets with immunosuppressive attributes., Competing Interests: S. N. Karagiannis and J. Spicer are founders and shareholders of Epsilogen Ltd. All other authors have declared that no conflict of interest exists., (© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Increasing evidence has linked the humoral immune response with the development of various cancers. Therefore, there is growing interest in investigating the predictive value of antibodies to assess overall and tissue site-specific cancer risk. Given the large amount of antibody types and the broad scope of the search (i.e. cancer risk), the primary aim of this systematic review was to present an overview of the most researched antibodies (i.e. immunoglobulin (Ig) isotypes (IgG, IgM, IgA, and IgE), tumour and self-antigen-reactive antibodies, infection-related antibodies) in relation to overall and site-specific cancer risk. We identified various antibody types that have been associated with the risk of cancer. While no significant associations were found for IgM serum levels, studies found an inconsistent association among IgE, IgA, and IgG serum levels in relation to cancer risk. When evaluating antibodies against infectious agents, most studies reported a positive link with specific cancers known to be associated with the specific agent recognized by serum antibodies (i.e. helicobacter pylori and gastric cancer, hepatitis B virus and hepatocellular carcinoma, and human papillomavirus and cervical cancer). Several reports identified autoantibodies, as single biomarkers (e.g. anti-p53, anti-MUC1, and anti-CA125) but especially in panels of multiple autoantibodies, to have potential as diagnostic biomarkers for specific cancer types. Overall, there is emerging evidence associating certain antibodies to cancer risk, especially immunoglobulin isotypes, tumour-associated antigen-specific, and self-reactive antibodies. Further experimental studies are necessary to assess the efficacy of specific antibodies as markers for the early diagnosis of cancer., (© The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Immunology.)

Human B cells and their expressed antibodies are crucial in conferring immune protection. Identifying pathogen-specific antibodies following infection is possible due to enhanced humoral immunity against well-described molecules on the pathogen surface. However, screening for cancer-reactive antibodies remains challenging since target antigens are often not identified a priori and the frequency of circulating B cells recognizing cancer cells is likely very low. We investigated whether combined ex vivo culture of human B cells with three innate stimuli, interleukin-17 (IL-17), B-cell activation factor (BAFF), and the toll-like receptor 9 (TLR-9) agonist DNA motif CpG ODN 2006 (CpG), each known to activate B cells through different signalling pathways, promote cell activation, proliferation, and antibody production. Combined IL-17+BAFF+CpG prolonged B-cell survival and increased proliferation compared with single stimuli. IL-17+BAFF+CpG triggered higher IgG secretion, likely by activating differentiated, memory and class-switched CD19+CD20+CD27+IgD- B cells. Regardless of anti-FOLR antibody seropositive status, IL-17+BAFF+CpG combined with a monovalent tumour-associated antigen (folate receptor alpha [FOLR]) led to secreted antibodies recognizing the antigen and the antigen-expressing IGROV1 cancer cells. In a seropositive individual, FOLR stimulation favoured class-switched memory B-cell precursors (CD27-CD38-IgD-), class-switched memory B cells and anti-FOLR antibody production, while IL-17+BAFF+CpG combined with FOLR, promoted class-switched memory B-cell precursors and antibody-secreting (CD138+IgD-) plasma cells. Furthermore, IL-17+BAFF+CpG stimulation of peripheral blood B cells from patients with melanoma revealed tumour cell-reactive antibodies in culture supernatants. These findings suggest that innate signals stimulate B-cell survival and antibody production and may help identify low-frequency antigen-reactive humoral responses., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Immunology.)

In this 13th annual installment of the annual 'Antibodies to Watch' article series, we discuss key events in commercial antibody therapeutics development that occurred in 2021 and forecast events that might occur in 2022. Regulatory review of antibody therapeutics that target the SARS-CoV-2 coronavirus proceeded at an unprecedented pace in 2021, resulting in both emergency use authorizations and full approvals for sotrovimab, regdanvimab, REGEN-COV2, as well as others, in numerous countries. As of November 1, a total of 11 antibody therapeutics had been granted first approvals in either the United States or European Union in 2021 (evinacumab, dostarlimab loncastuximab tesirine, amivantamab, aducanumab, tralokinumab, anifrolumab, bimekizumab, tisotumab vedotin, regdanvimab, REGEN-COV2). The first global approvals of seven products, however, were granted elsewhere, including Japan (pabinafusp alfa), China (disitamab vedotin, penpulimab, zimberelimab), Australia (sotrovimab, REGEN-COV2), or the Republic of Korea (regdanvimab). Globally, at least 27 novel antibody therapeutics are undergoing review by regulatory agencies. First actions by the Food and Drug Administration on the biologics license applications for faricimab, sutimlimab, tebentafusp, relatlimab, sintilimab, ublituximab and tezepelumab are expected in the first quarter of 2022. Finally, our data show that, with antibodies for COVID-19 excluded, the late-stage commercial clinical pipeline of antibody therapeutics grew by over 30% in the past year. Of those in late-stage development, marketing applications for at least 22 may occur by the end of 2022.

IgE antibodies elicit powerful immune responses, recruiting effector cells to tumors more efficiently and with greater cytotoxicity than IgG antibodies. Consequently, IgE antibodies are a promising alternative to conventional IgG-based therapies in oncology (AllergoOncology). As the pharmacokinetics of IgE antibodies are less well understood, we used molecular imaging in mice to compare the distribution and elimination of IgE and IgG antibodies targeting the human tumor-associated antigen chondroitin sulfate proteoglycan 4 (CSPG4). Anti-CSPG4 IgE and IgG1 antibodies with human Fc domains were radiolabeled with 111 In. CSPG4-expressing A375 human melanoma xenografts implanted in NOD- scid IL2rg -/- mice were also engrafted with human immune cells by intravenous administration. 111 In-anti-CSPG4 antibodies were administered intravenously. Their distribution was determined by single-photon emission computed tomography (SPECT) and ex vivo gamma-counting over 120 h. SPECT imaging was conducted from 0 to 60 min after antibody administration to precisely measure the early phase of IgE distribution. 111 In-labeled anti-CSPG4 IgG and IgE showed serum stability in vitro of >92% after 5 days. In A375 xenograft-bearing mice, anti-CSPG4 IgE showed much faster blood clearance and higher accumulation in the liver compared to anti-CSPG4 IgG. However, tumor-to-blood and tumor-to-muscle ratios were similar between the antibody isotypes and higher compared with a non-tumor-targeting isotype control IgE. IgE excretion was much faster than IgG. In non-tumor-bearing animals, early SPECT imaging revealed a blood clearance half-life of 10 min for IgE. Using image-based quantification, we demonstrated that the blood clearance of IgE is much faster than that of IgG while the two isotypes showed comparable tumor-to-blood ratios., Competing Interests: SNK and JS are founders and shareholders of Epsilogen Ltd. HJB is currently employed through a fund provided by Epsilogen Ltd. The other author(s) report no potential competing interest., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

IgE, the predominant antibody class of the allergic response, is known for its roles in protecting against parasites; however, a growing body of evidence indicates a significant role for IgE and its associated effector cells in tumour immunosurveillance, highlighted by the field of AllergoOncology and the successes of the first-in-class IgE cancer therapeutic MOv18. Supporting this concept, substantial epidemiological data ascribe potential roles for IgE, allergy, and atopy in protecting against specific tumour types, with a corresponding increased cancer risk associated with IgE immunodeficiency. Here, we consider how epidemiological data in combination with functional data reveals a complex interplay of IgE and allergy with cancer, which cannot be explained solely by one of the existing conventional hypotheses. We furthermore discuss how, in turn, such data may be used to inform future therapeutic approaches, including the clinical management of different patient groups. With epidemiological findings highlighting several high-risk cancer types protected against by high IgE levels, it is possible that use of IgE-based therapeutics for a range of malignant indications may offer efficacy to complement that of established IgG-class antibodies.

In breast cancer, humoral immune responses may contribute to clinical outcomes, especially in more immunogenic subtypes. Here, we investigated B lymphocyte subsets, immunoglobulin expression, and clonal features in breast tumors, focusing on aggressive triple-negative breast cancers (TNBC). In samples from patients with TNBC and healthy volunteers, circulating and tumor-infiltrating B lymphocytes (TIL-B) were evaluated. CD20 + CD27 + IgD - isotype-switched B lymphocytes were increased in tumors, compared with matched blood. TIL-B frequently formed stromal clusters with T lymphocytes and engaged in bidirectional functional cross-talk, consistent with gene signatures associated with lymphoid assembly, costimulation, cytokine-cytokine receptor interactions, cytotoxic T-cell activation, and T-cell-dependent B-cell activation. TIL-B-upregulated B-cell receptor (BCR) pathway molecules FOS and JUN, germinal center chemokine regulator RGS1, activation marker CD69, and TNFα signal transduction via NFκB, suggesting BCR-immune complex formation. Expression of genes associated with B lymphocyte recruitment and lymphoid assembly, including CXCL13, CXCR4, and DC-LAMP, was elevated in TNBC compared with other subtypes and normal breast. TIL-B-rich tumors showed expansion of IgG but not IgA isotypes, and IgG isotype switching positively associated with survival outcomes in TNBC. Clonal expansion was biased toward IgG, showing expansive clonal families with specific variable region gene combinations and narrow repertoires. Stronger positive selection pressure was present in the complementarity determining regions of IgG compared with their clonally related IgA in tumor samples. Overall, class-switched B lymphocyte lineage traits were conspicuous in TNBC, associated with improved clinical outcomes, and conferred IgG-biased, clonally expanded, and likely antigen-driven humoral responses. SIGNIFICANCE: Tumor-infiltrating B lymphocytes assemble in clusters, undergoing B-cell receptor-driven activation, proliferation, and isotype switching. Clonally expanded, IgG isotype-biased humoral immunity associates with favorable prognosis primarily in triple-negative breast cancers., (©2021 The Authors; Published by the American Association for Cancer Research.)

mAbs, either mono- or bispecific (bsAb), represent one of the most successful approaches to treat many types of malignancies. However, there are certain limitations to the use of full length mAbs for clinical applications, which can be overcome by engineered antibody fragments. The aim of this study was to develop a small bsAb, in the format of a single-chain diabody (scDb), to efficiently target two proteins, the hERG1 potassium channel and the β1 subunit of integrin receptors, which specifically form a macromolecular complex in cancer cells. We provide evidence that the scDb we produced binds to the hERG1/β1 complex in cancer cells and tissues, but does not bind to the hERG1 channel in nonpathologic tissues, in particular the heart. The scDb-hERG1-β1 (i) downregulates the formation of the hERG1/β1 complex, (ii) inhibits Akt phosphorylation and HIF-1α expression, and (iii) decreases cell survival, proliferation, and migration in vitro These effects only occur in cancer cells (either colon, pancreatic, or breast), but not in normal cells. In vivo , the scDb-hERG1-β1 shows a good pharmacokinetic profile, with a half-life of 13.5 hours and no general, cardiac, or renal toxicity when injected intravenously up to the dose of 8 mg/kg. The scDb-hERG1-β1 accumulates into subcutaneous xenografted tumors, arising from either colon or pancreatic human cancer cells, and induces a reduction of tumor growth and vascularization. Overall, the scDb-hERG1-β1 represents an innovative single-chain bispecific antibody for therapeutic applications in solid cancers that overexpress the hERG1/β1 integrin signaling complex., (©2021 American Association for Cancer Research.)

Background: Cancer immunotherapy with monoclonal antibodies and chimeric antigen receptor (CAR) T cell therapies can benefit from selection of new targets with high levels of tumor specificity and from early assessments of efficacy and safety to derisk potential therapies., Methods: Employing mass spectrometry, bioinformatics, immuno-mass spectrometry and CRISPR/Cas9 we identified the target of the tumor-specific SF-25 antibody. We engineered IgE and CAR T cell immunotherapies derived from the SF-25 clone and evaluated potential for cancer therapy., Results: We identified the target of the SF-25 clone as the tumor-associated antigen SLC3A2, a cell surface protein with key roles in cancer metabolism. We generated IgE monoclonal antibody, and CAR T cell immunotherapies each recognizing SLC3A2. In concordance with preclinical and, more recently, clinical findings with the first-in-class IgE antibody MOv18 (recognizing the tumor-associated antigen Folate Receptor alpha), SF-25 IgE potentiated Fc-mediated effector functions against cancer cells in vitro and restricted human tumor xenograft growth in mice engrafted with human effector cells. The antibody did not trigger basophil activation in cancer patient blood ex vivo, suggesting failure to induce type I hypersensitivity, and supporting safe therapeutic administration. SLC3A2-specific CAR T cells demonstrated cytotoxicity against tumor cells, stimulated interferon-γ and interleukin-2 production in vitro. In vivo SLC3A2-specific CAR T cells significantly increased overall survival and reduced growth of subcutaneous PC3-LN3-luciferase xenografts. No weight loss, manifestations of cytokine release syndrome or graft-versus-host disease, were detected., Conclusions: These findings identify efficacious and potentially safe tumor-targeting of SLC3A2 with novel immune-activating antibody and genetically modified cell therapies., Competing Interests: Competing interests: SNK and JS are founders and shareholders of Epsilogen Ltd., and HJB is now employed through a fund provided by Epsilogen Ltd., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

Cytokine therapy for cancer has indicated efficacy in certain diseases but is generally accompanied by severe toxicity. The field of antibody-cytokine fusion proteins (immunocytokines) arose to target these effector molecules to the tumor environment in order to expand the therapeutic window of cytokine therapy. Pre-clinical evidence has shown the increased efficacy and decreased toxicity of various immunocytokines when compared to their cognate unconjugated cytokine. These anti-tumor properties are markedly enhanced when combined with other treatments such as chemotherapy, radiotherapy, and checkpoint inhibitor antibodies. Clinical trials that have continued to explore the potential of these biologics for cancer therapy have been conducted. This review covers the in vitro, in vivo, and clinical evidence for the application of immunocytokines in immuno-oncology.

The contributions of the humoral immune response to melanoma are now widely recognized, with reports of positive prognostic value ascribed to tumor-infiltrating B cells (TIL-B) and increasing evidence of B cells as key predictors of patient response to treatment. There are disparate views as to the pro- and anti-tumor roles of B cells. B cells appear to play an integral role in forming tumor-associated tertiary lymphoid structures (TLSs) which can further modulate T cell activation. Expressed antibodies may distinctly influence tumor regulation in the tumor microenvironment, with some isotypes associated with strong anti-tumor immune response and others with progressive disease. Recently, B cells have been evaluated in the context of cancer immunotherapy. Checkpoint inhibitors (CPIs), targeting T cell effector functions, have revolutionized the management of melanoma for many patients; however, there remains a need to accurately predict treatment responders. Increasing evidence suggests that B cells may not be simple bystanders to CPI immunotherapy. Mature and differentiated B cell phenotypes are key positive correlates of CPI response. Recent evidence also points to an enrichment in activatory B cell phenotypes, and the contribution of B cells to TLS formation may facilitate induction of T cell phenotypes required for response to CPI. Contrastingly, specific B cell subsets often correlate with immune-related adverse events (irAEs) in CPI. With increased appreciation of the multifaceted role of B cell immunity, novel therapeutic strategies and biomarkers can be explored and translated into the clinic to optimize CPI immunotherapy in melanoma., Competing Interests: SNK and JFS are founders and shareholders of Epsilogen Ltd. HJB is now employed through a fund provided by Epsilogen Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Willsmore, Harris, Crescioli, Hussein, Kakkassery, Thapa, Cheung, Chauhan, Bax, Chenoweth, Laddach, Osborn, McCraw, Hoffmann, Nakamura, Geh, MacKenzie-Ross, Healy, Tsoka, Spicer, Papa, Barber, Lacy and Karagiannis.)

Birch pollen allergy is among the most prevalent pollen allergies in Northern and Central Europe. This IgE-mediated disease can be treated with allergen immunotherapy (AIT), which typically gives rise to IgG antibodies inducing tolerance. Although the main mechanisms of allergen immunotherapy (AIT) are known, questions regarding possible Fc-mediated effects of IgG antibodies remain unanswered. This can mainly be attributed to the unavailability of appropriate tools, i.e., well-characterised recombinant antibodies (rAbs). We hereby aimed at providing human rAbs of several classes for mechanistic studies and as possible candidates for passive immunotherapy. We engineered IgE, IgG 1 , and IgG 4 sharing the same variable region against the major birch pollen allergen Bet v 1 using Polymerase Incomplete Primer Extension (PIPE) cloning. We tested IgE functionality and IgG blocking capabilities using appropriate model cell lines. In vitro studies showed IgE engagement with FcεRI and CD23 and Bet v 1-dependent degranulation. Overall, we hereby present fully functional, human IgE, IgG 1 , and IgG 4 sharing the same variable region against Bet v 1 and showcase possible applications in first mechanistic studies. Furthermore, our IgG antibodies might be useful candidates for passive immunotherapy of birch pollen allergy.

Antibody-Drug Conjugates (ADCs) developed as a targeted treatment approach to deliver toxins directly to cancer cells are one of the fastest growing classes of oncology therapeutics, with eight ADCs and two immunotoxins approved for clinical use. However, selection of an optimum target and payload combination, to achieve maximal therapeutic efficacy without excessive toxicity, presents a significant challenge. We have developed a platform to facilitate rapid and cost-effective screening of antibody and toxin combinations for activity and safety, based on streptavidin-biotin conjugation. For antibody selection, we evaluated internalization by target cells using streptavidin-linked antibodies conjugated to biotinylated saporin, a toxin unable to cross cell membranes. For payload selection, we biotinylated toxins and conjugated them to antibodies linked to streptavidin to evaluate antitumour activity and pre-clinical safety. As proof of principle, we compared trastuzumab conjugated to emtansine via streptavidin-biotin (Trastuzumab-SB-DM1) to the clinically approved trastuzumab emtansine (T-DM1). We showed comparable potency in reduction of breast cancer cell survival in vitro and in growth restriction of orthotopic breast cancer xenografts in vivo. Our findings indicate efficient generation of functionally active ADCs. This approach can facilitate the study of antibody and payload combinations for selection of promising candidates for future ADC development.

Despite emerging targeted and immunotherapy treatments, no monoclonal antibodies or antibody-drug conjugates (ADCs) directly targeting tumor cells are currently approved for melanoma therapy. The tumor-associated antigen chondroitin sulphate proteoglycan 4 (CSPG4), a neural crest glycoprotein over-expressed on 70% of melanomas, contributes to proliferative signaling pathways, but despite highly tumor-selective expression it has not yet been targeted using ADCs. We developed a novel ADC comprising an anti-CSPG4 antibody linked to a DNA minor groove-binding agent belonging to the novel pyrridinobenzodiazepine (PDD) class. Unlike conventional DNA-interactive pyrrolobenzodiazepine (PBD) dimer payloads that cross-link DNA, PDD-based payloads are mono-alkylating agents but have similar efficacy and substantially enhanced tolerability profiles compared to PBD-based cross-linkers. We investigated the anti-tumor activity and safety of the anti-CSPG4-(PDD) ADC in vitro and in human melanoma xenografts. Anti-CSPG4-(PDD) inhibited CSPG4-expressing melanoma cell growth and colony formation and triggered apoptosis in vitro at low nanomolar to picomolar concentrations without off-target Fab-mediated or Fc-mediated toxicity. Anti-CSPG4-(PDD) restricted xenograft growth in vivo at 2 mg/kg doses. One 5 mg/kg injection triggered tumor regression in the absence of overt toxic effects or of acquired residual tumor cell resistance. This anti-CSPG4-(PDD) can deliver a highly cytotoxic DNA mono-alkylating payload to CSPG4-expressing tumors at doses tolerated in vivo.

IgE monoclonal antibodies hold great potential for cancer therapy. Preclinical in vivo systems, particularly those in which the antibody recognizes the host species target antigen and binds to cognate Fc receptors, are often the closest approximation to human exposure and represent a key challenge for evaluating the safety of antibody-based therapies. We sought to develop an immunocompetent rat system to assess the safety of a rodent anti-tumor IgE, as a surrogate for the human therapeutic candidate. We generated a rat IgE against the human tumor-associated antigen chondroitin sulfate proteoglycan 4 (CSPG4) and cross-reactive for the rat antigen. We analyzed CSPG4 distribution in normal rat and human tissues and investigated the in vivo safety of the antibody by monitoring clinical signs and molecular biomarkers after systemic administration to immunocompetent rats. Human and rat CSPG4 expression in normal tissues were comparable. Animals receiving antibody exhibited transient mild to moderate adverse events accompanied by mild elevation of serum tryptase, but not of angiotensin II or cytokines implicated in allergic reactions or cytokine storm. In the long term, repeated antibody administration was well tolerated, with no changes in animal body weight, liver and kidney functions or blood cell counts. This model provides preclinical support for the safety profiling of IgE therapeutic antibodies. Due to the comparable antigen tissue distribution in human and rat, this model may also comprise an appropriate tool for proof-of-concept safety evaluations of different treatment approaches targeting CSPG4.

Introduction: It is acknowledged that any claim of efficacy of allergen immunotherapy must be done for each specific product, and this remains true also for venom immunotherapy (VIT). Thus, we evaluated the efficacy and safety of a specific tyrosine-adsorbed VIT for vespula spp. and honeybee in real-life., Methods: Consecutive patients diagnosed with hymenoptera allergy, and receiving VIT for either vespula or honeybee with a tyrosine-adsorbed preparation were observed to evaluate the grade of reaction (according to Muller) at the first field re-sting. A modified ultra-rush protocol was used., Results: A total of 247 patients (73 female) were observed (102 honeybee, group H, 145 vespula, group V). Seventy-five patients in group H had a re-sting, and 74/75 had a lower grade reaction at re-sting as compared to the pre-VIT reaction. Considering systemic reactions, protection was achieved in 89% of patients. In group V 118 patients were re-stung, and 76/118 patients with previous grade III-IV reaction had no more systemic reaction under VIT. Overall, considering systemic reactions, protection was achieved in 92% of subjects. Of note, in both groups there was a clear inverse correlation between the severity of pre-VIT and during VIT reactions. The duration of VIT at the time of re-sting did not affect the efficacy. The safety was overall good, with 18% ad 15.4% local reactions in groups H and V, respectively., Discussion: Modified extracts, including tyrosine-absorbed, have the aim of improving the safety of VIT still yet maintaining the efficacy. Field re-sting is the best way to assess the efficacy in real life. In this observational study we could confirm the protective efficacy of the tyrosine-adsorbed extract, with a good safety expecially in the build-up using a modified-rush protocol., Conclusion: The tyrosine-adsorbed VIT used herein is a viable and advantageous form of treatment for hymenoptera allergy., Competing Interests: None to declare for the present work., (© 2019 The Author(s).)

Human immunoglobulin E (IgE) is the most extensively glycosylated antibody isotype so glycans attached to the seven N -glycosites (NGS) in its Fab and Fc domains may modulate its functions. However, targeted modification of glycans in multiply glycosylated proteins remains a challenge. Here, we applied an in vivo approach that allows the manipulation of IgE N -glycans, using a trastuzumab equivalent IgE (HER2-IgE) as a model. Taking advantage of plant inherent features, i.e., synthesis of largely homogeneous complex N -glycans and susceptibility to glycan engineering, we generated targeted glycoforms of HER2-IgE largely resembling those found in serum IgE. Plant-derived HER2-IgE exhibited N -glycans terminating with GlcNAc, galactose or sialic acid, lacking, or carrying core fucose and xylose. We were able to not only modulate the five NGSs naturally decorated with complex N -glycans, but to also induce targeted glycosylation at the usually unoccupied NGS6, thus increasing the overall glycosylation content of HER2-IgE. Recombinant human cell-derived HER2-IgE exhibited large N -glycan heterogeneity. All HER2-IgE variants demonstrated glycosylation-independent binding to the target antigen and the high affinity receptor FcεRI, and subsequent similar capacity to trigger mast cell degranulation. In contrast, binding to the low affinity receptor CD23 (FcεRII) was modulated by the glycan profile, with increased binding to IgE variants with glycans terminating with GlcNAc residues. Here we offer an efficient in planta approach to generate defined glycoforms on multiply glycosylated IgE, allowing the precise exploration of glycosylation-dependent activities., (Copyright © 2019 Montero-Morales, Maresch, Crescioli, Castilho, Ilieva, Mele, Karagiannis, Altmann and Steinkellner.)

Background: Antibody Fc-driven engagement of macrophages is critical for evoking cellular activation and effector functions and influencing tumour-associated macrophage (TAM) recruitment. We previously reported that IgE class antibodies promote restriction of cancer growth in rodent models associated with significant TAM infiltration. However, the human macrophage-associated IgE-Fc Receptor (FcεR) axis remains unexplored. We investigated the effects of anti-tumour IgE stimulation on human macrophage activation., Methods: Human blood monocyte-differentiated quiescent (M0), classically-(M1) and alternatively-(M2) activated macrophages were crosslinked with IgE and polyclonal antibodies to mimic immune complex formation. We examined surface marker expression, cytokine secretion, protein kinase phosphorylation and gene expression in IgE-stimulated macrophages and IgE antibody-dependent macrophage-mediated cytotoxicity (ADCC) against tumour cells., Findings: A proportion (40%) of M2 and (<20%) M0 and M1 macrophages expressed the high-affinity IgE receptor FcεRI. IgE crosslinking triggered upregulation of co-stimulatory CD80, increased TNFα, IFNγ, IL-1β, IL-12, IL-10, IL-13, CXCL9, CXCL11 and RANTES secretion by M0 and M2 and additionally enhanced MCP-1 by M2 macrophages. IgE-stimulated M1 macrophages retained secretion of pro-inflammatory cytokines. IgE crosslinking enhanced the FcεRI-dependent signalling pathway, including phosphorylation of the Lyn kinase, ERK1/2 and p38 in M2 macrophages and upregulated Lyn gene expression by M1 and M2 macrophages. Anti-tumour IgE engendered ADCC of cancer cells by all macrophage subsets., Interpretation: IgE can engage and re-educate alternatively-activated macrophages towards pro-inflammatory phenotypes and prime all subsets to mediate anti-tumour functions. This points to IgE-mediated cascades with potential to activate immune stroma and may be significant in the clinical development of strategies targeting tumour-resident macrophages., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

The immune system employs several checkpoint pathways to regulate responses, maintain homeostasis and prevent self-reactivity and autoimmunity. Tumor cells can hijack these protective mechanisms to enable immune escape, cancer survival and proliferation. Blocking antibodies, designed to interfere with checkpoint molecules CTLA-4 and PD-1/PD-L1 and counteract these immune suppressive mechanisms, have shown significant success in promoting immune responses against cancer and can result in tumor regression in many patients. While inhibitors to CTLA-4 and the PD-1/PD-L1 axis are well-established for the clinical management of melanoma, many patients do not respond or develop resistance to these interventions. Concerted efforts have focused on combinations of approved therapies aiming to further augment positive outcomes and survival. While CTLA-4 and PD-1 are the most-extensively researched targets, results from pre-clinical studies and clinical trials indicate that novel agents, specific for checkpoints such as A2AR, LAG-3, IDO and others, may further contribute to the improvement of patient outcomes, most likely in combinations with anti-CTLA-4 or anti-PD-1 blockade. This review discusses the rationale for, and results to date of, the development of inhibitory immune checkpoint blockade combination therapies in melanoma. The clinical potential of new pipeline therapeutics, and possible future therapy design and directions that hold promise to significantly improve clinical prognosis compared with monotherapy, are discussed.

Purpose: Highly aggressive triple-negative breast cancers (TNBCs) lack validated therapeutic targets and have high risk of metastatic disease. Folate receptor alpha (FRα) is a central mediator of cell growth regulation that could serve as an important target for cancer therapy. Experimental Design: We evaluated FRα expression in breast cancers by genomic ( n = 3,414) and IHC ( n = 323) analyses and its association with clinical parameters and outcomes. We measured the functional contributions of FRα in TNBC biology by RNA interference and the antitumor functions of an antibody recognizing FRα (MOv18-IgG1), in vitro , and in human TNBC xenograft models. Results: FRα is overexpressed in significant proportions of aggressive basal like/TNBC tumors, and in postneoadjuvant chemotherapy-residual disease associated with a high risk of relapse. Expression is associated with worse overall survival. TNBCs show dysregulated expression of thymidylate synthase, folate hydrolase 1, and methylenetetrahydrofolate reductase, involved in folate metabolism. RNA interference to deplete FRα decreased Src and ERK signaling and resulted in reduction of cell growth. An anti-FRα antibody (MOv18-IgG1) conjugated with a Src inhibitor significantly restricted TNBC xenograft growth. Moreover, MOv18-IgG1 triggered immune-dependent cancer cell death in vitro by human volunteer and breast cancer patient immune cells, and significantly restricted orthotopic and patient-derived xenograft growth. Conclusions: FRα is overexpressed in high-grade TNBC and postchemotherapy residual tumors. It participates in cancer cell signaling and presents a promising target for therapeutic strategies such as ADCs, or passive immunotherapy priming Fc-mediated antitumor immune cell responses. Clin Cancer Res; 24(20); 5098-111. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Modern molecular imaging techniques have greatly improved tumor detection and post-treatment follow-up of cancer patients. In this context, antibody-based imaging is rapidly becoming the gold standard, since it combines the unique specificity of antibodies with the sensitivity of the different imaging technologies. The aim of this study was to generate and characterize antibodies in single chain Fragment variable (scFv) format directed to an emerging cancer biomarker, the human ether-à-go-go-related gene-1 (hERG1) potassium channel, and to obtain a proof of concept for their potential use for in vivo molecular imaging. The anti-hERG1scFv was generated from a full length monoclonal antibody and then mutagenized, substituting a Phenylalanine residue in the third framework of the V H domain with a Cysteine residue. The resulting scFv-hERG1-Cys showed much higher stability and protein yield, increased affinity and more advantageous binding kinetics, compared to the "native" anti-hERG1scFv. The scFv-hERG1-Cys was hence chosen and characterized: it showed a good binding to the native hERG1 antigen expressed on cells, was stable in serum and displayed a fast pharmacokinetic profile once injected intravenously in nude mice. The calculated half-life was 3.1 hours and no general toxicity or cardiac toxic effects were detected. Finally, the in vivo distribution of an Alexa Fluor 750 conjugated scFv-hERG1-Cys was evaluated both in healthy and tumor-bearing nude mice, showing a good tumor-to-organ ratio, ideal for visualizing hERG1-expressing tumor masses in vivo . In conclusion, the scFv-hERG1-Cys possesses features which make it a suitable tool for application in cancer molecular imaging., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

Overexpression of the chondroitin sulfate proteoglycan 4 (CSPG4) has been associated with the pathology of multiple types of such as melanoma, breast cancer, squamous cell carcinoma, mesothelioma, neuroblastoma, adult and pediatric sarcomas, and some hematological cancers. CSPG4 has been reported to exhibit a role in the growth and survival as well as in the spreading and metastasis of tumor cells. CSPG4 is overexpressed in several malignant diseases, while it is thought to have restricted and low expression in normal tissues. Thus, CSPG4 has become the target of numerous anticancer treatment approaches, including monoclonal antibody-based therapies. This study reviews key potential anti-CSPG4 antibody and immune-based therapies and examines their direct antiproliferative/metastatic and immune activating mechanisms of action.

Antibody-drug conjugates (ADCs) are emerging as effective tools in cancer therapy, combining the antibody's exquisite specificity for the target antigen-expressing cancer cell together with the cytotoxic potency of the payload. Much success stems from the rational design of "toxic warheads", chemically linked to antibodies, and from fine-tuning the intricate properties of chemical linkers. Here, we focus on the antibody moiety of ADCs, dissecting the impact of Fab, linkers, isotype and Fc structure on the anti-tumoral and immune-activating functions of ADCs. Novel design approaches informed by antibody structural attributes present opportunities that may contribute to the success of next generation ADCs.

Monoclonal antibodies find broad application as therapy for various types of cancer by employing multiple mechanisms of action against tumors. Manipulating the Fc-mediated functions of antibodies that engage immune effector cells, such as NK cells, represents a strategy to influence effector cell activation and to enhance antibody potency and potentially efficacy. We developed a novel approach to generate and ascertain the functional attributes of Fc mutant monoclonal antibodies. This entailed coupling single expression vector (pVitro1) antibody cloning, using polymerase incomplete primer extension (PIPE) polymerase chain reaction, together with simultaneous Fc region point mutagenesis and high yield transient expression in human mammalian cells. Employing this, we engineered wild type, low (N297Q, NQ), and high (S239D/I332E, DE) FcR-binding Fc mutant monoclonal antibody panels recognizing two cancer antigens, HER2/ neu and chondroitin sulfate proteoglycan 4. Antibodies were generated with universal mutagenic primers applicable to any IgG1 pVitro1 constructs, with high mutagenesis and transfection efficiency, in small culture volumes, at high yields and within 12 days from design to purified material. Antibody variants conserved their Fab-mediated recognition of target antigens and their direct anti-proliferative effects against cancer cells. Fc mutations had a significant impact on antibody interactions with Fc receptors (FcRs) on human NK cells, and consequently on the potency of NK cell activation, quantified by immune complex-mediated calcium mobilization and by antibody-dependent cellular cytotoxicity (ADCC) of tumor cells. This strategy for manipulation and testing of Fc region engagement with cognate FcRs can facilitate the design of antibodies with defined effector functions and potentially enhanced efficacy against tumor cells.

Identification of mutations in the gene encoding the serine/threonine-protein kinase, BRAF, and constitutive activation of the mitogen-activated protein kinase (MAPK) pathway in around 50% of malignant melanomas have led to the development and regulatory approval of targeted pathway inhibitor drugs. A proportion of patients are intrinsically resistant to BRAF inhibitors, and most patients who initially respond, acquire resistance within months. In this review, we discuss pathway inhibitors and their mechanisms of resistance, and we focus on numerous efforts to improve clinical benefits through combining agents with disparate modes of action, including combinations with checkpoint inhibitor antibodies. We discuss the merits of combination strategies based on enhancing immune responses or overcoming tumor-associated immune escape mechanisms. Emerging insights into mechanisms of action, resistance pathways and their impact on host-tumor relationships will inform the design of optimal combinations therapies to improve outcomes for patients who currently do not benefit from recent treatment breakthroughs., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Evidence of tumor-resident mature B cell and antibody compartments and reports of associations with favorable prognosis in malignant melanoma suggest that humoral immunity could participate in antitumor defense. Likely striving to confer immunological protection while being subjected to tumor-promoting immune tolerance, B cells may engender multiple functions, including antigen processing and presentation, cytokine-mediated signaling, antibody class switching, expression and secretion. We review key evidence in support of multifaceted immunological mechanisms by which B cells may counter or contribute to malignant melanoma, and we discuss their potential translational implications. Dissecting the contributions of tumor-associated humoral responses can inform future treatment avenues.

IgE antibodies are key mediators of antiparasitic immune responses, but their potential for cancer treatment via antibody-dependent cell-mediated cytotoxicity (ADCC) has been little studied. Recently, tumor antigen-specific IgEs were reported to restrict cancer cell growth by engaging high-affinity Fc receptors on monocytes and macrophages; however, the underlying therapeutic mechanisms were undefined and in vivo proof of concept was limited. Here, an immunocompetent rat model was designed to recapitulate the human IgE-Fcε receptor system for cancer studies. We also generated rat IgE and IgG mAbs specific for the folate receptor (FRα), which is expressed widely on human ovarian tumors, along with a syngeneic rat tumor model expressing human FRα. Compared with IgG, anti-FRα IgE reduced lung metastases. This effect was associated with increased intratumoral infiltration by TNFα + and CD80 + macrophages plus elevated TNFα and the macrophage chemoattractant MCP-1 in lung bronchoalveolar lavage fluid. Increased levels of TNFα and MCP-1 correlated with IgE-mediated tumor cytotoxicity by human monocytes and with longer patient survival in clinical specimens of ovarian cancer. Monocytes responded to IgE but not IgG exposure by upregulating TNFα, which in turn induced MCP-1 production by monocytes and tumor cells to promote a monocyte chemotactic response. Conversely, blocking TNFα receptor signaling abrogated induction of MCP-1, implicating it in the antitumor effects of IgE. Overall, these findings show how antitumor IgE reprograms monocytes and macrophages in the tumor microenvironment, encouraging the clinical use of IgE antibody technology to attack cancer beyond the present exclusive reliance on IgG. Cancer Res; 77(5); 1127-41. ©2017 AACR ., (©2017 American Association for Cancer Research.)

B cells participate in immune surveillance in human circulation and tissues, including tumors such as melanoma. By contrast, the role of humoral responses in cutaneous immunity is underappreciated. We report circulating skin-homing CD22+CLA+B cells in healthy volunteers and melanoma patients (n = 73) and CD22+ cells in melanoma and normal skin samples (n = 189). Normal and malignant skin featured mature IgG and CD22 mRNA, alongside mRNA for the transiently-expressed enzyme Activation-induced cytidine Deaminase (AID). Gene expression analyses of publically-available data (n = 234 GEO, n = 384 TCGA) confirmed heightened humoral responses (CD20, CD22, AID) in melanoma. Analyses of 51 melanoma-associated and 29 normal skin-derived IgG sequence repertoires revealed lower IgG1/IgGtotal representation compared with antibodies from circulating B cells. Consistent with AID, comparable somatic hypermutation frequencies and class-switching indicated affinity-matured antibodies in normal and malignant skin. A melanoma-associated antibody subset featured shorter complementarity-determining (CDR3) regions relative to those from circulating B cells. Clonal amplification in melanoma-associated antibodies and homology modeling indicated differential potential antigen recognition profiles between normal skin and melanoma sequences, suggesting distinct antibody repertoires. Evidence for IgG-expressing B cells, class switching and antibody maturation in normal and malignant skin and clonally-expanded antibodies in melanoma, support the involvement of mature B cells in cutaneous immunity.

The field of antibody therapeutics is rapidly growing, with over 210 antibodies currently approved or in regulatory review and ~ 1,250 antibodies in clinical development. Antibodies are highly versatile molecules that, with strategic design of their antigen-binding domain (Fab) and the domain responsible for mediating effector functions (Fc), can be used in a wide range of therapeutic indications. Building on many years of progress, the biopharmaceutical industry is now advancing innovative research and development by exploring new targets and new formats and using antibody engineering to fine-tune functions tailored to specific disease requirements. In addition to considering the target and the disease context, however, the unique features of each therapeutic antibody trigger a diverse set of Fc-mediated effector functions. To avoid unexpected results on safety and efficacy outcomes during the later stages of the development process, it is crucial to measure the impact of antibody design on Fc-mediated effector function early in the antibody development process. Given the breadth of effector functions antibodies can deploy and the close interplay between the antibody Fab and Fc functional domains, it is important to conduct a comprehensive evaluation of Fc-mediated functions using an array of antigen-specific biophysical and cell-mediated functional assays. Here, we review antibody and Fc receptor properties that influence Fc effector functions and discuss their implications on development of safe and efficacious antibody therapeutics.

The commercial development of antibody therapeutics is a global enterprise involving thousands of biopharmaceutical firms and supporting service organizations. To date, their combined efforts have resulted in over 200 marketed antibody therapeutics and a pipeline of nearly 1,400 investigational product candidates that are undergoing evaluation in clinical studies as treatments for a wide variety of diseases. Here, we discuss key events in antibody therapeutics development that occurred during 2024 and forecast key events related to the late-stage clinical pipeline that may occur in 2025. In particular, we report on 21 antibody therapeutics granted a first approval in at least one country or region during 2024, including bispecific antibodies tarlatamab (IMDELLTRA®), zanidatamab (Ziihera®), zenocutuzumab (BIZENGRI®), odronextamab (Ordspono®), ivonescimab (®), and antibody-drug conjugate (ADC) sacituzumab tirumotecan (®). We also discuss 30 investigational antibody therapeutics for which marketing applications were undergoing review by at least one regulatory agency, as of our last update on December 9, 2024, including ADCs datopotamab deruxtecan, telisotuzumab vedotin, patritumab deruxtecan, trastuzumab botidotin, becotatug vedotin, and trastuzumab rezetecan. Of 178 antibody therapeutics we include in the late-stage pipeline, we summarize key data for 18 for which marketing applications may be submitted by the end of 2025, such as bi- or multispecific antibodies denecimig, sonelokimab, erfonrilimab, and anbenitamab. Key trends in the development and approval of antibody formats such as bispecifics and ADCs, as well as clinical-phase transition and global approval success rates for these antibody formats, are reported.