Enhancing IgA-mediated neutrophil cytotoxicity against neuroblastoma by CD47 blockade.

BACKGROUND: Approximately half of the neuroblastoma patients develop high-risk neuroblastoma. Current treatment involves a multimodal strategy, including immunotherapy with dinutuximab (IgG ch14.18) targeting GD2. Despite achieving promising results, the recurrence rate remains high and poor survival persists. The therapeutic efficacy of dinutuximab is compromised by suboptimal activation of neutrophils and severe neuropathic pain, partially induced by complement activation. METHODS: To enhance neutrophil cytotoxicity, IgG ch14.18 was converted to the IgA isotype, resulting in potent neutrophil-mediated antibody-dependent cell-mediated cytotoxicity (ADCC), without complement activation. However, myeloid checkpoint molecules hamper neutrophil cytotoxicity, for example through CD47 that is overexpressed on neuroblastomas and orchestrates an immunosuppressive environment upon ligation to signal regulatory protein alpha (SIRPα) expressed on neutrophils. In this study, we combined IgA therapy with CD47 blockade. RESULTS: In vitro killing assays showed enhanced IgA-mediated ADCC by neutrophils targeting neuroblastoma cell lines and organoids in comparison to IgG. Notably, when combined with CD47 blockade, both IgG and IgA therapy were enhanced, though the combination with IgA resulted in the greatest improvement of ADCC. Furthermore, in a neuroblastoma xenograft model, we systemically blocked CD47 with a SIRPα fusion protein containing an ablated IgG1 Fc, and compared IgA therapy to IgG therapy. Only IgA therapy combined with CD47 blockade increased neutrophil influx to the tumor microenvironment. Moreover, the IgA combination strategy hampered tumor outgrowth most effectively and prolonged tumor-specific survival. CONCLUSION: These promising results highlight the potential to enhance immunotherapy efficacy against high-risk neuroblastoma through improved neutrophil cytotoxicity by combining IgA therapy with CD47 blockade.

Combining Cellular Immunization and Phage Display Screening Results in Novel, FcγRI-Specific Antibodies.

Antibodies that specifically bind to individual human fragment crystallizable γ receptors (FcγRs) are of interest as research tools in studying immune cell functions, as well as components in bispecific antibodies for immune cell engagement in cancer therapy. Monoclonal antibodies for human low-affinity FcγRs have been successfully generated by hybridoma technology and are widely used in pre-clinical research. However, the generation of monoclonal antibodies by hybridoma technology that specifically bind to the high-affinity receptor FcγRI is challenging. Monomeric mouse IgG2a, IgG2b, and IgG3 bind human FcγRI with high affinity via the Fc part, leading to an Fc-mediated rather than a fragment for antigen binding (Fab)-mediated selection of monoclonal antibodies. Blocking the Fc-binding site of FcγRI with an excess of human IgG or Fc during screening decreases the risk of Fc-mediated interactions but can also block the potential epitopes of new antibody candidates. Therefore, we replaced hybridoma technology with phage display of a single-chain fragment variable (scFv) antibody library that was generated from mice immunized with FcγRI-positive cells and screened it with a cellular panning approach assisted by next-generation sequencing (NGS). Seven new FcγRI-specific antibody sequences were selected with this methodology, which were produced as Fc-silent antibodies showing FcγRI-restricted specificity.

IgA antibody immunotherapy targeting GD2 is effective in preclinical neuroblastoma models.

BACKGROUND: Immunotherapy targeting GD2 is very effective against high-risk neuroblastoma, though administration of anti-GD2 antibodies induces severe and dose-limiting neuropathic pain by binding GD2-expressing sensory neurons. Previously, the IgG1 ch14.18 (dinutuximab) antibody was reformatted into the IgA1 isotype, which abolishes neuropathic pain and induces efficient neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) via activation of the Fc alpha receptor (FcαRI/CD89). METHODS: To generate an antibody suitable for clinical application, we engineered an IgA molecule (named IgA3.0 ch14.18) with increased stability, mutated glycosylation sites and substituted free (reactive) cysteines. The following mutations were introduced: N45.2G and P124R (CH1 domain), C92S, N120T, I121L and T122S (CH2 domain) and a deletion of the tail piece P131-Y148 (CH3 domain). IgA3.0 ch14.18 was evaluated in binding assays and in ADCC and antibody-dependent cellular phagocytosis (ADCP) assays with human, neuroblastoma patient and non-human primate effector cells. We performed mass spectrometry analysis of N-glycans and evaluated the impact of altered glycosylation in IgA3.0 ch14.18 on antibody half-life by performing pharmacokinetic (PK) studies in mice injected intravenously with 5 mg/kg antibody solution. A dose escalation study was performed to determine in vivo efficacy of IgA3.0 ch14.18 in an intraperitoneal mouse model using 9464D-GD2 neuroblastoma cells as well as in a subcutaneous human xenograft model using IMR32 neuroblastoma cells. Binding assays and PK studies were compared with one-way analysis of variance (ANOVA), ADCC and ADCP assays and in vivo tumor outgrowth with two-way ANOVA followed by Tukey's post-hoc test. RESULTS: ADCC and ADCP assays showed that particularly neutrophils and macrophages from healthy donors, non-human primates and patients with neuroblastoma are able to kill neuroblastoma tumor cells efficiently with IgA3.0 ch14.18. IgA3.0 ch14.18 contains a more favorable glycosylation pattern, corresponding to an increased antibody half-life in mice compared with IgA1 and IgA2. Furthermore, IgA3.0 ch14.18 penetrates neuroblastoma tumors in vivo and halts tumor outgrowth in both 9464D-GD2 and IMR32 long-term tumor models. CONCLUSIONS: IgA3.0 ch14.18 is a promising new therapy for neuroblastoma, showing (1) increased half-life compared to natural IgA antibodies, (2) increased protein stability enabling effortless production and purification, (3) potent CD89-mediated tumor killing in vitro by healthy subjects and patients with neuroblastoma and (4) antitumor efficacy in long-term mouse neuroblastoma models.

Characterization of human Fc alpha receptor transgenic mice: comparison of CD89 expression and antibody-dependent tumor killing between mouse strains.

Since mice do not express a homologue of the human Fc alpha receptor (FcαRI or CD89), a transgenic mouse model was generated in four different backgrounds (C57BL/6, BALB/c, SCID and NXG) expressing the FcαRI under the endogenous human promoter. In this study, we describe previously unknown characteristics of this model, such as the integration site of the FCAR gene, the CD89 expression pattern in healthy male and female mice and in tumor-bearing mice, expression of myeloid activation markers and FcγRs and IgA/CD89-mediated tumor killing capacity. In all mouse strains, CD89 expression is highest in neutrophils, intermediate on other myeloid cells such as eosinophils and DC subsets and inducible on, among others, monocytes, macrophages and Kupffer cells. CD89 expression levels are highest in BALB/c and SCID, lower in C57BL/6 and lowest in NXG mice. Additionally, CD89 expression on myeloid cells is increased in tumor-bearing mice across all mouse strains. Using Targeted Locus Amplification, we determined that the hCD89 transgene has integrated in chromosome 4. Furthermore, we established that wildtype and hCD89 transgenic mice have a similar composition and phenotype of immune cells. Finally, IgA-mediated killing of tumor cells is most potent with neutrophils from BALB/c and C57BL/6 and less with neutrophils from SCID and NXG mice. However, when effector cells from whole blood are used, SCID and BALB/c are most efficient, since these strains have a much higher number of neutrophils. Overall, hCD89 transgenic mice provide a very powerful model to test the efficacy of IgA immunotherapy against infectious diseases and cancer.

Effective, Long-Term, Neutrophil Depletion Using a Murinized Anti-Ly-6G 1A8 Antibody.

Neutrophils are crucial innate immune cells but also play key roles in various diseases, such as cancer, where they can perform both pro- and anti-tumorigenic functions. To study the function of neutrophils in vivo, these cells are often depleted using Ly-6G or Gr-1 depleting antibodies or genetic "knockout" models. However, these methods have several limitations, being only partially effective, effective for a short term, and lacking specificity or the ability to conditionally deplete neutrophils. Here, we describe the use of a novel murinized Ly-6G (1A8) antibody. The murinized Ly-6G antibody is of the mouse IgG2a isotype, which is the only isotype that can bind all murine Fcγ receptors and C1q and is, therefore, able to activate antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) pathways. We show that this mouse-Ly-6G antibody shows efficient, long-term, and near-complete (>90%) neutrophil depletion in the peripheral blood of C57Bl6/J, Balb/c, NXG and SCID mice for up to at least four weeks, using a standardized neutrophil depletion strategy. In addition, we show that neutrophils are efficiently depleted in the blood and tumor tissue of IMR32 tumor-bearing SCID mice, analyzed six weeks after the start of the treatment.

Analysing the protection from respiratory tract infections and allergic diseases early in life by human milk components: the PRIMA birth cohort.

BACKGROUND: Many studies support the protective effect of breastfeeding on respiratory tract infections. Although infant formulas have been developed to provide adequate nutritional solutions, many components in human milk contributing to the protection of newborns and aiding immune development still need to be identified. In this paper we present the methodology of the "Protecting against Respiratory tract lnfections through human Milk Analysis" (PRIMA) cohort, which is an observational, prospective and multi-centre birth cohort aiming to identify novel functions of components in human milk that are protective against respiratory tract infections and allergic diseases early in life. METHODS: For the PRIMA human milk cohort we aim to recruit 1000 mother-child pairs in the first month postpartum. At one week, one, three, and six months after birth, fresh human milk samples will be collected and processed. In order to identify protective components, the level of pathogen specific antibodies, T cell composition, Human milk oligosaccharides, as well as extracellular vesicles (EVs) will be analysed, in the milk samples in relation to clinical data which are collected using two-weekly parental questionnaires. The primary outcome of this study is the number of parent-reported medically attended respiratory infections. Secondary outcomes that will be measured are physician diagnosed (respiratory) infections and allergies during the first year of life. DISCUSSION: The PRIMA human milk cohort will be a large prospective healthy birth cohort in which we will use an integrated, multidisciplinary approach to identify the longitudinal effect human milk components that play a role in preventing (respiratory) infections and allergies during the first year of life. Ultimately, we believe that this study will provide novel insights into immunomodulatory components in human milk. This may allow for optimizing formula feeding for all non-breastfed infants.

Anti-GD2 IgA kills tumors by neutrophils without antibody-associated pain in the preclinical treatment of high-risk neuroblastoma.

BACKGROUND: The addition of monoclonal antibody therapy against GD2 to the treatment of high-risk neuroblastoma led to improved responses in patients. Nevertheless, administration of GD2 antibodies against neuroblastoma is associated with therapy-limiting neuropathic pain. This severe pain is evoked at least partially through complement activation on GD2-expressing sensory neurons. METHODS: To reduce pain while maintaining antitumor activity, we have reformatted the approved GD2 antibody ch14.18 into the IgA1 isotype. This novel reformatted IgA is unable to activate the complement system but efficiently activates leukocytes through the FcαRI (CD89). RESULTS: IgA GD2 did not activate the complement system in vitro nor induced pain in mice. Importantly, neutrophil-mediated killing of neuroblastoma cells is enhanced with IgA in comparison to IgG, resulting in efficient tumoricidal capacity of the antibody in vitro and in vivo. CONCLUSIONS: Our results indicate that employing IgA GD2 as a novel isotype has two major benefits: it halts antibody-induced excruciating pain and improves neutrophil-mediated lysis of neuroblastoma. Thus, we postulate that patients with high-risk neuroblastoma would strongly benefit from IgA GD2 therapy.

The selection of variable regions affects effector mechanisms of IgA antibodies against CD20.

Blockade of the CD47-SIRPα axis improves lymphoma cell killing by myeloid effector cells, which is an important effector mechanism for CD20 antibodies in vivo. The approved CD20 antibodies rituximab, ofatumumab, and obinutuzumab are of human immunoglobulin G1 (IgG1) isotype. We investigated the impact of the variable regions of these 3 CD20 antibodies when expressed as human IgA2 isotype variants. All 3 IgA2 antibodies mediated antibody-dependent cellular phagocytosis (ADCP) by macrophages and antibody-dependent cellular cytotoxicity (ADCC) by polymorphonuclear cells. Both effector mechanisms were significantly enhanced in the presence of a CD47-blocking antibody or by glutaminyl cyclase inhibition to interfere with CD47-SIRPα interactions. Interestingly, an IgA2 variant of obinutuzumab (OBI-IgA2) was consistently more potent than an IgA2 variant of rituximab (RTX-IgA2) or an IgA2 variant of ofatumumab (OFA-IgA2) in triggering ADCC. Furthermore, we observed more effective direct tumor cell killing by OBI-IgA2 compared with RTX-IgA2 and OFA-IgA2, which was caspase independent and required a functional cytoskeleton. IgA2 variants of all 3 antibodies triggered complement-dependent cytotoxicity, with OBI-IgA2 being less effective than RTX-IgA2 and OFA-IgA2. When we investigated the therapeutic efficacy of the CD20 IgA2 antibodies in different in vivo models, OBI-IgA2 was therapeutically more effective than RTX-IgA2 or OFA-IgA2. In vivo efficacy required the presence of a functional IgA receptor on effector cells and was independent of complement activation or direct lymphoma cell killing. These data characterize the functional activities of human IgA2 antibodies against CD20, which were affected by the selection of the respective variable regions. OBI-IgA2 proved particularly effective in vitro and in vivo, which may be relevant in the context of CD47-SIRPα blockade.

Bovine IgG Prevents Experimental Infection With RSV and Facilitates Human T Cell Responses to RSV.

Respiratory syncytial virus (RSV) infections represent a major burden of disease in infants and are the second most prevalent cause of death worldwide. Human milk immunoglobulins provide protection against RSV. However, many infants depend on processed bovine milk-based nutrition, which lacks intact immunoglobulins. We investigated the potential of bovine antibodies to neutralize human RSV and facilitate-cell immune activation. We show cow's milk IgG (bIgG) and Intravenous Immunoglobulin (IVIG) have a similar RSV neutralization capacity, even though bIgG has a lower pre-F to post-F binding ratio compared to human IVIG, with the majority of bIgG binding to pre-F. RSV is better neutralized with human IVIG. Consequently, we enriched RSV specific T cells by culturing human PBMC with a mixture of RSV peptides, and used these T cells to study the effect of bIgG and IVIG on the activation of pre-F-pecific T cells. bIgG facilitated in vitro T cell activation in a similar manner as IVIG. Moreover, bIgG was able to mediate T cell activation and internalization of pathogens, which are prerequisites for inducing an adaptive viral response. Using in vivo mouse experiments, we showed that bIgG is able to bind the murine activating IgG Fc Receptors (FcγR), but not the inhibiting FcγRII. Intranasal administration of the monoclonal antibody palivizumab, but also of bIgG and IVIG prevented RSV infection in mice. The concentration of bIgG needed to prevent infection was ~5-fold higher compared to IVIG. In conclusion, the data presented here indicate that functionally active bIgG facilitates adaptive antiviral T cell responses and prevents RSV infection in vitro and in vivo.

Novel chimerized IgA CD20 antibodies: Improving neutrophil activation against CD20-positive malignancies.

Current combination therapies elicit high response rates in B cell malignancies, often using CD20 antibodies as the backbone of therapy. However, many patients eventually relapse or develop progressive disease. Therefore, novel CD20 antibodies combining multiple effector mechanisms were generated. To study whether neutrophil-mediated destruction of B cell malignancies can be added to the arsenal of effector mechanisms, we chimerized a panel of five previously described murine CD20 antibodies to the human IgG1, IgA1 and IgA2 isotype. Of this panel, we assessed in vitro antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and direct cell death induction capacity and studied the efficacy in two different in vivo mouse models. IgA antibodies outperformed IgG1 antibodies in neutrophil-mediated killing in vitro, both against CD20-expressing cell lines and primary patient material. In these assays, we observed loss of CD19 with both IgA and IgG antibodies. Therefore, we established a novel method to improve the assessment of B-cell depletion by CD20 antibodies by including CD24 as a stable cell marker. Subsequently, we demonstrated that only IgA antibodies were able to reduce B cell numbers in this context. Additionally, IgA antibodies showed efficacy in both an intraperitoneal tumor model with EL4 cells expressing huCD20 and in an adoptive transfer model with huCD20-expressing B cells. Taken together, we show that IgA, like IgG, can induce ADCC and CDC, but additionally triggers neutrophils to kill (malignant) B cells. We conclude that antibodies of the IgA isotype offer an attractive repertoire of effector mechanisms for the treatment of CD20-expressing malignancies.

Functional diversification of hybridoma-produced antibodies by CRISPR/HDR genomic engineering.

Hybridoma technology is instrumental for the development of novel antibody therapeutics and diagnostics. Recent preclinical and clinical studies highlight the importance of antibody isotype for therapeutic efficacy. However, since the sequence encoding the constant domains is fixed, tuning antibody function in hybridomas has been restricted. Here, we demonstrate a versatile CRISPR/HDR platform to rapidly engineer the constant immunoglobulin domains to obtain recombinant hybridomas, which secrete antibodies in the preferred format, species, and isotype. Using this platform, we obtained recombinant hybridomas secreting Fab' fragments, isotype-switched chimeric antibodies, and Fc-silent mutants. These antibody products are stable, retain their antigen specificity, and display their intrinsic Fc-effector functions in vitro and in vivo. Furthermore, we can site-specifically attach cargo to these antibody products via chemoenzymatic modification. We believe that this versatile platform facilitates antibody engineering for the entire scientific community, empowering preclinical antibody research.

Potent Fc Receptor Signaling by IgA Leads to Superior Killing of Cancer Cells by Neutrophils Compared to IgG.

Antibody therapy of cancer is increasingly used in the clinic and has improved patient's life expectancy. Except for immune checkpoint inhibition, the mode of action of many antibodies is to recognize overexpressed or specific tumor antigens and initiate either direct F(ab')2-mediated tumor cell killing, or Fc-mediated effects such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity/phagocytosis (ADCC/P) after binding to activating Fc receptors. All antibodies used in the clinic are of the IgG isotype. The IgA isotype can, however, also elicit powerful anti-tumor responses through engagement of the activating Fc receptor for monomeric IgA (FcαRI). In addition to monocytes, macrophages and eosinophils as FcαRI expressing immune cells, neutrophils are especially vigorous in eliminating IgA opsonized tumor cells. However, with IgG as single agent it appears almost impossible to activate neutrophils efficiently, as we have visualized by live cell imaging of tumor cell killing. In this study, we investigated Fc receptor expression, binding and signaling to clarify why triggering of neutrophils by IgA is more efficient than by IgG. FcαRI expression on neutrophils is ~2 times and ~20 times lower than that of Fcγ receptors FcγRIIa and FcγRIIIb, but still, binding of neutrophils to IgA- or IgG-coated surfaces was similar. In addition, our data suggest that IgA-mediated binding of neutrophils is more stable compared to IgG. IgA engagement of neutrophils elicited stronger Fc receptor signaling than IgG as indicated by measuring the p-ERK signaling molecule. We propose that the higher stoichiometry of IgA to the FcαR/FcRγ-chain complex, activating four ITAMs (Immunoreceptor Tyrosine-based Activating Motifs) compared to a single ITAM for FcγRIIa, combined with a possible decoy role of the highly expressed FcγRIIIb, explains why IgA is much better than IgG at triggering tumor cell killing by neutrophils. We anticipate that harnessing the vast population of neutrophils by the use of IgA monoclonal antibodies can be a valuable addition to the growing arsenal of antibody-based therapeutics for cancer treatment.

Breast Milk Prefusion F Immunoglobulin G as a Correlate of Protection Against Respiratory Syncytial Virus Acute Respiratory Illness.

Background: Transplacental respiratory syncytial virus (RSV) antibody transfer has been characterized, but little is known about the protective effect of breast milk RSV-specific antibodies. Serum antibodies against the prefusion RSV fusion protein (pre-F) exhibit high neutralizing activity. We investigate protection of breast milk pre-F antibodies against RSV acute respiratory infection (ARI). Methods: Breast milk at 1, 3, and 6 months postpartum and midnasal swabs during infant illness episodes were collected in mother-infant pairs in Nepal. One hundred seventy-four infants with and without RSV ARI were matched 1:1 by risk factors for RSV ARI. Pre-F immunoglobulin A (IgA) and immunoglobulin G (IgG) antibody levels were measured in breast milk. Results: The median breast milk pre-F IgG antibody concentration before illness was lower in mothers of infants with RSV ARI (1.4 [interquartile range {IQR}, 1.1-1.6] log10 ng/mL) than without RSV ARI (1.5 [IQR, 1.3-1.8] log10 ng/mL) (P = .001). There was no difference in median maternal pre-F IgA antibody concentrations in cases vs controls (1.7 [IQR, 0.0-2.2] log10 ng/mL vs 1.7 [IQR, 1.2-2.2] log10 ng/mL, respectively; P = .58). Conclusions: Low breast milk pre-F IgG antibodies before RSV ARI support a potential role for pre-F IgG as a correlate of protection against RSV ARI. Induction of breast milk pre-F IgG may be a mechanism of protection for maternal RSV vaccines.

Reformatting palivizumab and motavizumab from IgG to human IgA impairs their efficacy against RSV infection in vitro and in vivo.

Respiratory syncytial virus (RSV) infection is a leading cause of hospitalization and mortality in young children. Protective therapy options are limited. Currently, palivizumab, a monoclonal IgG1 antibody, is the only licensed drug for RSV prophylaxis, although other IgG antibody candidates are being evaluated. However, at the respiratory mucosa, IgA antibodies are most abundant and act as the first line of defense against invading pathogens. Therefore, it would be logical to explore the potential of recombinant human IgA antibodies to protect against viral respiratory infection, but very little research on the topic has been published. Moreover, it is unknown whether human antibodies of the IgA isotype are better suited than those of the IgG isotype as antiviral drugs to combat respiratory infections. To address this, we generated various human IgA antibody formats of palivizumab and motavizumab, two well-characterized human IgG1 anti-RSV antibodies. We evaluated their efficacy to prevent RSV infection in vitro and in vivo and found similar, but somewhat decreased efficacy for different IgA subclasses and formats. Thus, reformatting palivizumab or motavizumab into IgA reduces the antiviral potency of either antibody. Moreover, our results indicate that the efficacy of intranasal IgA prophylaxis against RSV infection in human FcαRI transgenic mice is independent of Fc receptor expression.

The Therapeutic CD38 Monoclonal Antibody Daratumumab Induces Programmed Cell Death via Fcγ Receptor-Mediated Cross-Linking.

Emerging evidence suggests that FcγR-mediated cross-linking of tumor-bound mAbs may induce signaling in tumor cells that contributes to their therapeutic activity. In this study, we show that daratumumab (DARA), a therapeutic human CD38 mAb with a broad-spectrum killing activity, is able to induce programmed cell death (PCD) of CD38(+) multiple myeloma tumor cell lines when cross-linked in vitro by secondary Abs or via an FcγR. By comparing DARA efficacy in a syngeneic in vivo tumor model using FcRγ-chain knockout or NOTAM mice carrying a signaling-inactive FcRγ-chain, we found that the inhibitory FcγRIIb as well as activating FcγRs induce DARA cross-linking-mediated PCD. In conclusion, our in vitro and in vivo data show that FcγR-mediated cross-linking of DARA induces PCD of CD38-expressing multiple myeloma tumor cells, which potentially contributes to the depth of response observed in DARA-treated patients and the drug's multifaceted mechanisms of action.

An Anti-EGFR IgA That Displays Improved Pharmacokinetics and Myeloid Effector Cell Engagement In Vivo.

Antibodies of IgA isotype effectively engage myeloid effector cells for cancer immunotherapy. Here, we describe preclinical studies with an Fc engineered IgA2m(1) antibody containing the variable regions of the EGFR antibody cetuximab. Compared with wild-type IgA2m(1), the engineered molecule lacked two N-glycosylation sites (N166 and N337), two free cysteines (C311 and C472), and contained a stabilized heavy and light chain linkage (P221R mutation). This novel molecule displayed improved production rates and biochemical properties compared with wild-type IgA. In vitro, Fab- and Fc-mediated effector functions, such as inhibition of ligand binding, receptor modulation, and engagement of myeloid effector cells for antibody-dependent cell-mediated cytotoxicity, were similar between wild-type and engineered IgA2. The engineered antibody displayed lower levels of terminal galactosylation leading to reduced asialoglycoprotein-receptor binding and to improved pharmacokinetic properties. In a long-term in vivo model against EGFR-positive cancer cells, improved serum half-life translated into higher efficacy of the engineered molecule, which required myeloid cells expressing human FcαRI for its full efficacy. However, Fab-mediated effector functions contributed to the in vivo efficacy because the novel IgA antibody demonstrated therapeutic activity also in non-FcαRI transgenic mice. Together, these results demonstrate that engineering of an IgA antibody can significantly improve its pharmacokinetics and its therapeutic efficacy to inhibit tumor growth in vivo.

Improved in vivo anti-tumor effects of IgA-Her2 antibodies through half-life extension and serum exposure enhancement by FcRn targeting.

Antibody therapy is a validated treatment approach for several malignancies. All currently clinically applied therapeutic antibodies (Abs) are of the IgG isotype. However, not all patients respond to this therapy and relapses can occur. IgA represents an alternative isotype for antibody therapy that engages FcαRI expressing myeloid effector cells, such as neutrophils and monocytes. IgA Abs have been shown to effectively kill tumor cells both in vitro and in vivo. However, due to the short half-life of IgA Abs in mice, daily injections are required to reach an effect comparable to IgG Abs. The relatively long half-life of IgG Abs and serum albumin arises from their capability of interacting with the neonatal Fc receptor (FcRn). As IgA Abs lack a binding site for FcRn, we generated IgA Abs with the variable regions of the Her2-specific Ab trastuzumab and attached an albumin-binding domain (ABD) to the heavy or light chain (HCABD/LCABD) to extend their serum half-life. These modified Abs were able to bind albumin from different species in vitro. Furthermore, tumor cell lysis of IgA-Her2-LCABD Abs in vitro was similar to unmodified IgA-Her2 Abs. Pharmacokinetic studies in mice revealed that the serum exposure and half-life of the modified IgA-Her2 Abs was extended. In a xenograft mouse model, the modified IgA1 Abs exhibited a slightly, but significantly, improved anti-tumor response compared to the unmodified Ab. In conclusion, empowering IgA Abs with albumin-binding capacity results in in vitro and in vivo functional Abs with an enhanced exposure and prolonged half-life.

Simultaneous Targeting of FcγRs and FcαRI Enhances Tumor Cell Killing.

Efficacy of anticancer monoclonal antibodies (mAb) is limited by the exhaustion of effector mechanisms. IgG mAbs mediate cellular effector functions through FcγRs expressed on effector cells. IgA mAbs can also induce efficient tumor killing both in vitro and in vivo. IgA mAbs recruit FcαRI-expressing effector cells and therefore initiate different effector mechanisms in vivo compared with IgG. Here, we studied killing of tumor cells coexpressing EGFR and HER2 by the IgG mAbs cetuximab and trastuzumab and their IgA variants. In the presence of a heterogeneous population of effector cells (leukocytes), the combination of IgG and IgA mAbs to two different tumor targets (EGFR and HER2) led to enhanced cytotoxicity compared with each isotype alone. Combination of two IgGs or two IgAs or IgG and IgA against the same target did not enhance cytotoxicity. Increased cytotoxicity relied on the presence of both the peripheral blood mononuclear cell and the polymorphonuclear (PMN) fraction. Purified natural killer cells were only cytotoxic with IgG, whereas cytotoxicity induced by PMNs was strong with IgA and poor with IgG. Monocytes, which coexpress FcγRs and FcαRI, also displayed increased cytotoxicity by the combination of IgG and IgA in an overnight killing assay. Coinjection of cetuximab and IgA2-HER2 resulted in increased antitumor effects compared with either mAb alone in a xenograft model with A431-luc2-HER2 cells. Thus, the combination of IgG and IgA isotypes optimally mobilizes cellular effectors for cytotoxicity, representing a promising novel strategy to improve mAb therapy.

A novel FcγRIIa Q27W gene variant is associated with common variable immune deficiency through defective FcγRIIa downstream signaling.

We identified a novel Q27W FcγRIIa variant that was found more frequently in common variable immunodeficiency (CVID) or CVID-like children. We analyzed the possible functional consequence of the Q27W FcγRIIa mutation in human cells. We used peripheral blood mononuclear cells from Q27W FcγRIIa patients and healthy controls, and cultured cells that overexpress the Q27W and common FcγRIIa variants. The Q27W FcγRIIa mutation does not disrupt FcγRIIa surface expression in peripheral blood mononuclear cells. Mononuclear cells express multiple FcγR, precluding careful analysis of Q27W FcγRIIa functional deviation. For functional analysis of FcγRIIa function, we therefore overexpressed the Q27W FcγRIIa and common FcγRIIa variant in IIA1.6 cells that are normally deficient in FcγR. We show that FcγRIIa triggering-induced signaling is obstructed, as measured by both decrease in calcium flux and defective MAPK phosphorylation. In conclusion, we here describe a novel Q27W FcγRIIa variant that causes delayed downstream signaling. This variant may contribute to CVID.