T-cell Dependent Antibody Research Articles

Ascorbic acid alleviates rheumatoid arthritis by inhibiting the production of autoantibodies

BackgroundAscorbic acid can regulate the function of the immune system. This study aimed to investigate the underlying mechanisms of ascorbic acid in plasma cell differentiation and rheumatoid arthritis (RA).MethodsMice were intraperitoneally injected with either ascorbic acid or an equivalent volume of phosphate-buffered saline (PBS). To elucidate the effects of ascorbic acid on arthritis, we utilized a collagen induced arthritis mouse model (CIA). To investigate the effects of ascorbic acid on antibody response, mice were immunized with (4-Hydroxy-3-nitrophenylacetyl)-Ficoll (NP-Ficoll) or (4-hydroxy-3-nitrophenyl) acetyl-keyhole limpet hemocyanin (NP-KLH) to elicit a T-cell independent (TI) or T-cell dependent (TD) antibody response. To clarify the ability of ascorbic acid on plasma cell production, we tracked the B cell differentiation fate on the NP-specific B1-8hi BCR transgenic background.ResultsAscorbic acid-injected mice demonstrated significantly delayed disease incidence and decreased disease severity compared to PBS-injected mice. Ascorbic acid can reduce the titers of autoantibodies in both arthritis and lupus mice models. Ascorbic acid can significantly reduce the number of plasma cells and the production of antigen-specific antibodies in TI and TD antibody response. In addition, ascorbic acid can disrupt the antibody affinity maturation. Through B1-8hi adoptive transfer experiments, it has been demonstrated that ascorbic acid restrains B cell differentiation into plasma cells in a cell-intrinsic manner. After in-depth exploration, we found that ascorbic acid can block the cell cycle of B cells and promote cell apoptosis. Mechanistically, ascorbic acid inhibited the production of autoreactive plasma cells by inhibiting the Stat3 signaling pathway.ConclusionOur study demonstrates that ascorbic acid has the ability to suppress the generation of autoreactive plasma cells, diminish the production of autoantibodies, and consequently delay the onset of rheumatoid arthritis.

IL-15/IL-15Rα-Fc-Fusion Protein XmAb24306 Potentiates Activity of CD3 Bispecific Antibodies through Enhancing T-Cell Expansion.

An insufficient quantity of functional T cells is a likely factor limiting the clinical activity of T-cell bispecific antibodies, especially in solid tumor indications. We hypothesized that XmAb24306 (efbalropendekin alfa), a lymphoproliferative interleukin (IL)-15/IL-15 receptor α (IL-15Rα) Fc-fusion protein, may potentiate the activity of T-cell dependent (TDB) antibodies. The activation of human peripheral T cells by cevostamab, an anti-FcRH5/CD3 TDB, or anti-HER2/CD3 TDB resulted in the upregulation of the IL-2/15Rβ (CD122) receptor subunit in nearly all CD8+ and majority of CD4+ T cells, suggesting that TDB treatment may sensitize T cells to IL-15. XmAb24306 enhanced T-cell bispecific antibody-induced CD8+ and CD4+ T-cell proliferation and expansion. In vitro combination of XmAb24306 with cevostamab or anti-HER2/CD3 TDB resulted in significant enhancement of tumor cell killing, which was reversed when T-cell numbers were normalized, suggesting that T-cell expansion is the main mechanism of the observed benefit. Pretreatment of immunocompetent mice with a mouse-reactive surrogate of XmAb24306 (mIL-15-Fc) resulted in a significant increase of T cells in the blood, spleen, and tumors and converted transient anti-HER2/CD3 TDB responses to complete durable responses. In summary, our results support the hypothesis that the number of tumor-infiltrating T cells is rate limiting for the activity of solid tumor-targeting TDBs. Upregulation of CD122 by TDB treatment and the observed synergy with XmAb24306 and T-cell bispecific antibodies support clinical evaluation of this novel immunotherapy combination.

Preclinical immunogenicity risk assessment of biotherapeutics using CD4 T cell assays.

T-cell dependent antibody responses to biotherapeutics remain a challenge to the optimal clinical application of biotherapeutics because of their capacity to impair drug efficacy and their potential to cause safety issues. To minimize this clinical immunogenicity risk, preclinical assays measuring the capacity of biotherapeutics to elicit CD4 T cell response in vitro are commonly used. However, there is considerable variability in assay formats and a general poor understanding of their respective predictive value. In this study, we evaluated the performance of three different CD4 T cell proliferation assays in their capacity to predict clinical immunogenicity: a CD8 T cell depleted peripheral blood mononuclear cells (PBMC) assay and two co-culture-based assays between dendritic cells (DCs) and autologous CD4 T cells with or without restimulation with monocytes. A panel of 10 antibodies with a wide range of clinical immunogenicity was selected. The CD8 T cell depleted PBMC assay predicted the clinical immunogenicity in four of the eight highly immunogenic antibodies included in the panel. Similarly, five antibodies with high clinical immunogenicity triggered a response in the DC: CD4 T cell assay but the responses were of lower magnitude than the ones observed in the PBMC assay. Remarkably, three antibodies with high clinical immunogenicity did not trigger any response in either platform. The addition of a monocyte restimulation step to the DC: CD4 T cell assay did not further improve its predictive value. Overall, these results indicate that there are no CD4 T cell assay formats that can predict the clinical immunogenicity of all biotherapeutics and reinforce the need to combine results from various preclinical assays assessing antigen uptake and presentation to fully mitigate the immunogenicity risk of biotherapeutics.

Mechanisms of antibody mediated immunity – Distinct in early life

Immune responses in early life are characterized by a failure to robustly generate long-lasting protective responses against many common pathogens or upon vaccination. This is associated with a reduced ability to generate T-cell dependent high affinity antibodies. This review highlights the differences in T-cell dependent antibody responses observed between infants and adults, in particular focussing on the alterations in immune cell function that lead to reduced T follicular helper cell-B cell crosstalk within germinal centres in early life. Understanding the distinct functional characteristics of early life humoral immunity, and how these are regulated, will be critical in guiding age-appropriate immunological interventions in the very young.

Determination of in vitro immunotoxic potencies of a series of perfluoralkylsubstances (PFASs) in human Namalwa B lymphocyte and human Jurkat T lymphocyte cells.

Exposure to PFASs is associated to several adverse health effects, such as immunotoxicity. Immunotoxic effects of PFOA and PFOS, including a reduced antibody response in both experimental animals and humans, have been reported. However, there is limited understanding of the underlying mechanisms involved. Moreover, there is only a restricted amount of immunotoxicity data available for a limited number of PFASs. In the current study the effects of 15 PFASs, including short- and long-chain perfluorinated carboxylic and sulfonic acids, fluorotelomer alcohols, and perfluoralkyl ether carboxylic acids were studied on the expression of recombinant activating gene 1 (RAG1) and RAG2 in the Namalwa human B lymphoma cell line, and on the human IL-2 promotor activity in Jurkat T-cells. Concentration-response data were subsequently used to derive in vitro relative potencies through benchmark dose analysis. In vitro relative potency factors (RPFs) were obtained for 6 and 9 PFASs based on their effect on RAG1 and RAG2 gene expression in Namalwa B-cells, respectively, and for 10 PFASs based on their inhibitory effect on IL-2 promotor activity in Jurkat T-cells. The most potent substances were HFPO-TA for the reduction of RAG1 and RAG2 gene expression in Namalwa cells (RPFs of 2.1 and 2.3 respectively), and PFDA on IL-2 promoter activity (RPF of 9.1). RAG1 and RAG2 play a crucial role in V (D)J gene recombination, a process for acquiring a varied array of antibodies crucial for antigen recognition. Hence, the effects observed in Namalwa cells might indicate a PFAS-induced impairment of generating a diverse range of B-cells essential for antigen recognition. The observed outcomes in the Jurkat T-cells suggest a possible PFAS-induced reduction of T-cell activation, which may contribute to a decline in the T-cell dependent antibody response. Altogether, the present study provides potential mechanistic insights into the reported PFAS-induced decreased antibody response. Additionally, the presented in vitro models may represent useful tools for assessing the immunotoxic potential of PFASs and prioritization for further risk assessment.

B-cell-specific MhcII regulates microbiota composition in a primarily IgA-independent manner.

The expression of major histocompatibility complex class II (MhcII) molecules on B cells is required for the development of germinal centers (GCs) in lymphoid follicles; the primary sites for the generation of T-cell-dependent (TD) antibody responses. Peyer's patches (PPs) are secondary lymphoid tissues (SLOs) in the small intestine (SI) that give rise to high-affinity, TD antibodies (mainly immunoglobulin A (IgA)) generated against the microbiota. While several studies have demonstrated that MhcII antigen presentation by other immune cells coordinate TD IgA responses and regulate microbiota composition, whether or not B-cell-specific MhcII influences gut microbial ecology is unknown. Here, we developed a novel Rag1 -/- adoptive co-transfer model to answer this question. In this model, Rag1 -/- mice were reconstituted with naïve CD4+ T cells and either MhcII-sufficient or MhcII-deficient naïve B cells. Subsequent to this, resulting shifts in microbiota composition was characterized via 16S rRNA gene sequencing of SI-resident and fecal bacterial communities. Results from our experiments indicate that SLO development and reconstitution of an anti-commensal TD IgA response can be induced in Rag1 -/- mice receiving T cells and MhcII-sufficient B cells, but not in mice receiving T cells and MhcII-deficient B cells. Results from our 16S experiments confirmed that adaptive immunity is a relevant host factor shaping microbial ecology in the gut, and that its impact was most pronounced on SI-resident bacterial communities. Our data also clearly establishes that MhcII-mediated cognate interactions between B cells and T cells regulates this effect by maintaining species richness in the gut, which is a phenotype commonly associated with good health. Finally, contrary to expectations, our experimental results indicate that IgA was not responsible for driving any of the effects on the microbiota ascribed to the loss of B cell-specific MhcII. Collectively, results from our experiments support that MhcII-mediated antigen presentation by B cells regulates microbiota composition and promotes species richness through an IgA-independent mechanism.

T cell redirecting and TLR7/8 agonist encapsulated liposomes targeting HER2+ tumors: Preclinical efficacy, pharmacokinetics and safety evaluations.

e14590 Background: T-cell dependent bispecific antibodies (TDBs) have shown limited success in solid tumor treatments. We hypothesized that it may be due to TDBs limited distribution in solid tumor tissues. We therefore propose to take advantage of the EPR (enhanced permeability and retention) effect of the liposome system to improve the PKPD properties of TDBs. The liposomes are named TRAFsomes (T cell Redirecting Antibody Fragment-anchored Liposomes). HF5050 is a TRAFsome containing on average 6 anti-CD3 and 10 anti-Her2 on each liposome surface and the toll-like TLR-7/8 agonist Resiquimod encapsulated inside at specific lipid to drug ratios. It preclinical efficacy, pharmacokinetics and safety evaluation studies are conducted to support the IND application. Methods: The T cell redirecting and target specific cytotoxic effect of HF5050 were evaluated in five PBMC co-cultured cancer cell models with different Her2 expression. Tumor cells derived from adenoid cystic carcinoma tumor tissues was also included. T cell redirection and activation are examined by T cell surface markers CD25/CD69 and secretion of IFN gamma, Perforin, and granzymes B in the absence and presence of cancer cells. For the incorporation of TLR7/8 agonist Resiquimod, we evaluated the effect of drug dose by varying the drug loading ratios in order to achieve a synergistic effect. For the in vivo efficacy studies, the N87 gastric cancer CDX model and an adenoid cystic carcinoma tumor PDX model were set on PBMC reconstructed humanized NCG-B2M mice. Pharmacokinetic and tissue distribution studies are carried out in tumor bearing mouse models as well. Results: HF5050 has shown to be a highly effective anti-cancer agent in both in vitro and in vivo studies. It mediates T cells-tumor cells redirection and cytotoxicity in a dose-dependent manner, especially in Her-2 highly expressing cell lines. HF5050 also effectively stimulates T cell activation and displays high efficacy in PBMC reconstruct humanized NCG-B2M tumor-bearing mice. Pharmacokinetic research confirms liposome successfully maintains drug levels in the bloodstream, and improves drug accumulation within tumor tissues. Conclusions: Overall, these data demonstrate that HF5050 processing a novel cell bridging function between T cells and tumor cells, and with the capability of activating immune cells. Based on the encouraging preclinical results, HF5050 exhibits promising development potential.

Abstract 2269: Bexmarilimab induces B-cell activation and autoantibody production

Abstract Introduction: Bexmarilimab, a Clever-1 targeting humanized antibody, is a macrophage checkpoint inhibitor promoting antigen presentation and pro-inflammatory cytokine secretion. Data from the first-in-human clinical Phase I/II study (MATINS; NCT03733990) demonstrate that single agent bexmarilimab is able to ignite an interferon response with survival benefit in 30% of patients with advanced gastric cancer, cutaneous melanoma and cholangiocarcinoma (ASCO 2022). Since Clever-1 knockout mice have been reported to have enhanced T-cell antibody production and bexmarilimab increases peripheral B-cell populations, we investigated B-cell phenotype, clonality and autoantibody formation in bexmarilimab treatmented MATINS patients. Methods: We first performed comprehensive phenotyping of peripheral B-cells from a colorectal carcinoma patient showing partial response (per RECIST v1.1). Autoantibody reactivities were measured in 80 serum samples of bexmarilimab treated patients (37 pre-treatment and 43 post-treatment samples from cycles 3 and 4) and compared to serums from 53 healthy individuals using Oncimmune's SeroTag multiplex technology with an immune-oncology (IO) specific protein array comprised of 1162 antigens. Results: Single-cell sequencing together with BCR sequencing of the responder’s B-cells during cycle 4 revealed an induced and activated B-cell population consisting of four transcriptionally similar clusters expressing IGHM, IGHD, CD23 but not CD43 and one cluster expressing CD79B, PLD4 and MZB1, which was distant from the naïve CCR7 expressing B-cells and IGHA1 and IGHG1 expressing plasma blasts. The expansion of B-cells was not due to clonal expansion as no overlapping BCR clones were identified between B-cells at pre-dose and cycle 4. Autoantibody profiling revealed great inter-individual heterogeneity in the number and targets of induced antibodies among patients treated with bexmarilimab reflecting individual patterns in self- and tumor antigens. However, shared autoantibody changes against targets such as cancer testis antigens (GAGE2), typical autoimmune disease antigens (SNRPC, TPO, TOP1) and antigens related to the induction of innate immunity and interferon responses, e.g. TRIM21 were observed. Importantly, we identified a set of pre-treatment autoantibodies that were associated with longer time to disease progression and were predictive of clinical response (disease control rate [DCR], consisting of CR, PR, SD) and progression-free survival (PFS) on bexmarilimab therapy. Conclusions: Our data implies that bexmarilimab can induce activation and secondary Ig rearrangements in mature B-cells, which has been reported to occur in germinal centres during T-cell dependent antibody responses to increase B-cell diversity and affinity of antigen receptors. B cell diversity and activation was reflected in patient autoantibody production and may enhance cancer immune recognition. Citation Format: Elisa M. Vuorinen, Mari L. Björkman, Reetta Virtakoivu, Juho Jalkanen, Sofia Aakko, Akira Takeda, Petra Budde, Hans-Dieter Zucht, Manuel Brautigam, Behnaz Ahangarianabhari, Petri Bono, Maija Hollmén. Bexmarilimab induces B-cell activation and autoantibody production [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2269.

Laboratory safety evaluation of lokivetmab, a canine anti-interleukin-31 monoclonal antibody, in dogs

Lokivetmab (Cytopoint®, Zoetis) is a canine monoclonal antibody that specifically binds and neutralizes interleukin (IL)-31. Lokivetmab is approved for use in dogs for the treatment of atopic dermatitis (AD) and allergic dermatitis. The laboratory safety of lokivetmab was evaluated in 2 studies by adapting the science-based, case-by-case approach used for preclinical and early clinical safety evaluation of human biopharmaceuticals. The main objectives were to demonstrate the safety of lokivetmab in healthy laboratory Beagle dogs by using integrated clinical, morphologic, and functional evaluations. In Study 1, dogs were treated s.c. with saline or lokivetmab at 3.3 mg/kg (1X, label dose) or 10 mg/kg (3X intended dose) for 7 consecutive monthly doses, with terminal pathology and histology assessments. In Study 2, the functional immune response was demonstrated in naïve dogs using the T-cell dependent antibody response (TDAR) test with 2 different dose levels of unadjuvanted keyhole limpet hemocyanin (KLH) as the model immunogen. The primary endpoint was anti-KLH IgG antibody titer, and secondary endpoints were ex vivo IL-2 enzyme-linked immunospot (ELISpot) and peripheral blood mononuclear cell lymphoproliferation assays. Both studies included monitoring general health, periodic veterinary clinical evaluations, serial clinical pathology and toxicokinetics, and monitoring for anti-drug antibodies.In both studies, the health of dogs receiving lokivetmab was similar to controls, with no treatment-related changes uncovered. Extensive pathology evaluations of immune tissues (Study 1) revealed no lokivetmab-related morphologic changes, and in dogs treated at 10 mg/kg lokivetmab, immunization with the model antigen KLH did not impair the functional antibody or T-cell recall responses. There were no immunogenicity-related or hypersensitivity-related responses observed in either study. These studies in healthy laboratory dogs showed that lokivetmab was well-tolerated, did not produce any treatment-related effects, and had no effect on immune system morphology or its functional response. These studies also demonstrated the utility of a science-based case-by-case approach to the safety evaluation of a veterinary biopharmaceutical product.

Historical Review and Future of Cardiac Xenotransplantation.

Along with the development of immunosuppressive drugs, major advances on xenotransplantation were achieved by understanding the immunobiology of xenograft rejection. Most importantly, three predominant carbohydrate antigens on porcine endothelial cells were key elements provoking hyperacute rejection: α1,3-galactose, SDa blood group antigen, and N-glycolylneuraminic acid. Preformed antibodies binding to the porcine major xenoantigen causes complement activation and endothelial cell activation, leading to xenograft injury and intravascular thrombosis. Recent advances in genetic engineering enabled knock-outs of these major xenoantigens, thus producing xenografts with less hyperacute rejection rates. Another milestone in the history of xenotransplantation was the development of co-stimulation blockaded strategy. Unlike allotransplantation, xenotransplantation requires blockade of CD40-CD40L pathway to prevent T-cell dependent B-cell activation and antibody production. In 2010s, advanced genetic engineering of xenograft by inducing the expression of multiple human transgenes became available. So-called 'multi-gene' xenografts expressing human transgenes such as thrombomodulin and endothelial protein C receptor were introduced, which resulted in the reduction of thrombotic events and improvement of xenograft survival. Still, there are many limitations to clinical translation of cardiac xenotransplantation. Along with technical challenges, zoonotic infection and physiological discordances are major obstacles. Social barriers including healthcare costs also need to be addressed. Although there are several remaining obstacles to overcome, xenotransplantation would surely become the novel option for millions of patients with end-stage heart failure who have limited options to traditional therapeutics.

Twelve-month in utero safety assessment of gardenia blue, a natural food colorant.

Toxicity assessment of the food colorant Gardenia jasminoides Ellis at dietary exposures of 0.0%, 0.1%, 0.5%, 1.5%, 3.0% and 5.0% included measures of T-cell- dependent antibody response, neurotoxicity, and clinical and anatomic pathology in Sprague Dawley rats during mating, gestation, lactation, postnatal development, and following weaning for up to 12 months including 3- and 6-month interim evaluations. Blue coloration of the gastrointestinal tract, mesenteric lymph nodes and kidneys was present in treated rats only at necropsy with minimal blue coloration at the lowest dose and without histopathological correlates in any of the tissues. There was good survival with no consistent treatment-related changes in hematology, clinical chemistry, enhanced evaluation of lymphoid tissues, or tissue histopathology at interim and final time points. T-cell dependent antibody response and neurotoxicity screening were negative in treated rats. The no-observed-adverse-effect level (NOAEL) was determined to be 5.0% gardenia blue (2,854.5 and 3,465.4 mg/kg/day in parental males and females, respectively, prior to mating; 3,113.5 and 4,049.6 mg/kg/day in male and female offspring, respectively, following up to 12 months of exposure.

S1PR4 deficiency results in reduced germinal center formation but only marginally affects antibody production.

Splenic B cells exhibit a high expression of the G protein-coupled sphingosine-1-phosphate (S1P) receptor type 4 (S1PR4). Little is known about the functional relevance of S1PR4 expression on those cells. In this study, S1PR4-deficient mice were used to study the role of S1PR4-mediated S1P signaling in B cell motility in vitro and for the maintenance of the splenic architecture under steady state conditions as well as in polymicrobial abdominal sepsis in vivo. Finally, the impact of S1PR4 deficiency on antibody production after immunization with T cell dependent antigens was assessed. Loss of S1PR4 resulted in minor alterations of the splenic architecture concerning the presence of B cell follicles. After sepsis induction, the germinal center response was severely impaired in S1PR4-deficient animals. Splenic B cells showed reduced motility in the absence of S1PR4. However, titres of specific antibodies showed only minor reductions in S1PR4-deficient animals. These observations suggest that S1P signaling mediated by S1PR4 modifies chemokine-induced splenic B cell chemotaxis, thus modulating splenic microarchitecture, GC formation and T-cell dependent antibody production.

Pretreatment with Tocilizumab Prior to the CD3 Bispecific Cevostamab in Patients with Relapsed/Refractory Multiple Myeloma (RRMM) Showed a Marked Reduction in Cytokine Release Syndrome Incidence and Severity

Background: Blockade of the cytokine IL-6 receptor by monoclonal antibodies, such as tocilizumab (TCZ), is a key strategy for the management of both CAR-T and T-cell dependent bispecific monoclonal antibody (TDB) induced cytokine release syndrome (CRS). Preclinical data indicate that inhibition of IL-6, IL-1, or TNF-α signaling prior to TDB administration can prevent development of CRS without any impact on anti-tumor activity (Li et al. Sci Transl Med 2019). To test this hypothesis in the clinic, a dedicated TCZ pretreatment arm was added to the ongoing GO39775 Phase I study (NCT03275103) of cevostamab, an FcRH5xCD3 TDB, to determine whether a single dose of TCZ administered prior to the first dose of cevostamab can reduce the incidence of CRS. Methods: Patients (pts) who have RRMM for which no established therapy is available, appropriate or tolerable were enrolled in a dedicated TCZ pretreatment arm. Cevostamab was administered intravenously (IV) in a 21-day Cycle (C). A single 3.6mg priming dose was given on Day (D) 1 of C1 followed by a target dose of 90mg on C1D8 and then C2D1 and beyond. A single IV 8mg/kg dose of TCZ was administered as a pretreatment on C1D1 two hours prior to the administration of cevostamab. Patient data from the previously enrolled non-TCZ 3.6/90mg dosing arm served as a retrospective comparator. Pts in both arms received corticosteroid, antihistamine and acetaminophen premedication prior to cevostamab. CRS is reported using ASTCT criteria (Lee et al. Biol Blood Marrow Transplant 2019). Results: At data cut-off (March 8, 2022), 28 pts had enrolled in the TCZ pretreatment arm (median age: 64.5 years) and 44 pts in the non-TCZ comparator (median age: 63.0 years). Median prior lines of therapy were 4 (2.0-7.0) and 6 (2.0-11.0) in the TCZ pretreatment and non-TCZ arms, respectively. Twenty-two pts (78.6%) were triple-class refractory and 12 pts (42.9%) were penta-refractory in the TCZ pretreatment arm, vs. 38 (86.4%) triple-class refractory and 32 (72.7%) penta-refractory pts in the non-TCZ arm. Pts in the TCZ arm had a median time on study of 4.7 months (range: 0.3-11.3) vs. 13.1 months (range: 0.2-30.8) in the non-TCZ arm. All patients in the TCZ arm experienced at least one adverse event (AE). Overall, 35.7% of pts in the TCZ arm experienced CRS as compared to 90.9% in the non-TCZ 3.6/90mg group (see figure). One patient in each group experienced G3 CRS and no G4/G5 events were observed. Fewer pts required TCZ to treat CRS (5/28) in the TCZ arm than the non-TCZ arm (16/44). Only two pts on the TCZ arm had more than one CRS event (vs. 12/44 non-TCZ) and no CRS events were observed at C2 and beyond (vs. 5/44 non-TCZ). Rates of non-CRS AEs (notably thrombocytopenia, infections, and liver enzyme elevations) in the TCZ arm were similar to the non-TCZ arm with the exception of neutropenia (Trudel et al. ASH 2021). In the TCZ arm 64.3% of pts experienced G3/G4 neutropenia as compared to 38.6% in the comparator arm. In both arms, neutropenia events were reversible, manageable with growth factor as indicated, and none led to the discontinuation of cevostamab. Consistent with preclinical data, no apparent negative impact on anti-tumor activity was observed. The overall response rate in the TCZ arm with 90mg target dose was 50% (13/26) [95% CI: 28.9-71.2] compared to 37.2% (16/43) [95% CI: 21.6-52.8] in pts receiving 90mg without TCZ pretreatment. Very good partial response or better (VGPR+) rates were 26.9% vs. 25.6%, respectively. Available pharmacodynamic data did not demonstrate any apparent change in measures of T-cell activation and proliferation in pts receiving TCZ pretreatment. Consistent with the known mechanism of action of TCZ, levels of C-reactive protein, a downstream product of hepatic IL-6 activation, were heavily suppressed in the vast majority of pts in TCZ arm suggestive of near-complete inhibition of the IL-6 signaling pathway. Conclusions: Clinical data from the GO39775 study shows for the first time that TCZ pretreatment can significantly reduce the risk of developing TDB-induced CRS without an apparent impact on anti-myeloma activity. The data support additional investigation of the use of anti-cytokine pretreatment with the goal of substantially reducing the frequency and potentially the severity of CRS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 1338: Humanized mouse model for in vivo evaluation of bispecific antibody efficacy

Abstract Bispecific antibodies have shown several promising advantages over monoclonal antibodies and are emerging as one of growing cancer immunotherapies. Preclinical in vivo models to assess efficacy associated with immune cell engagement are needed. Here we evaluated an EGFR bispecific T cell engager (EGFRXCD3 BiTE) using xenograft models in humanized NSG™ (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice that have been engrafted with human CD34+ HSC and have human T cells in circulation. Three EGFR+ tumor models were used in this report including LG1202 lung PDX and two triple negative breast cancer cell lines MDA-MB-231 with a high EGFR expression level and MDA-MB-453 with a low EGFR expression. Interestingly we found that all three models were resistant to EGFR monoclonal antibody drug cetuximab despite the expression of EGFR but responded to EGFRXCD3 BiTE. BiTE-induced T cell activation was evident through the analysis of tumor infiltrated lymphocytes by flow cytometry at the end of the study. BiTE treatment for 3 weeks effectively reduced the LG1202 tumor growth whereas the effect of cetuximab on the tumor growth was minimal. In the BiTE-treated group, CD3+ T cell percentage increased as compared to the vehicle group, and both CD4+ and CD8+ T cell subsets showed an increased frequency of CD69+ cells. We also observed elevated levels of human cytokines IFN-γ, IL-6, IL-2, and IL-10 in circulation 4 h after animals received the first BiTE dose. We evaluated the dose response of BiTE treatments on MDA-MB-231 model with a high EGFR expression. Low dose BiTE reduced tumor growth whereas high dose BiTE effectively prevented or even regressed some tumor growth. Flow analysis showed that tumor CD3+ T cell frequency remained similar to the vehicle group but CD8+ T frequency increased after high dose BiTE. High CD69+CD8+ T cell frequency was found in the vehicle group and the percentage further increased after high dose BiTE. For the low EGFR-expressing MDA-MB-453 model, BiTE treatment for 4 weeks also effectively reduced the tumor growth. In comparison, while cetuximab treatment decreased EGFR expression on tumor cells, it did not reduce the tumor growth. Flow analysis showed that BiTE-treated group had increased T cell infiltration into the tumors when compared to the PBS and cetuximab groups. The frequency and cell number of CD25+ and CD69+ CD4+ T and CD8+ T cells in the tumor increased after BiTE treatment. Together, these data support our humanized mouse model to be used to validate T-cell dependent bispecific antibody efficacy. Citation Format: Li-Chin Yao, James G. Keck, Danying cai. Humanized mouse model for in vivo evaluation of bispecific antibody efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1338.

Preclinical Immunopharmacologic Assessment of KPL-404, a Novel, Humanized, Non-Depleting Antagonistic Anti-CD40 Monoclonal Antibody

The CD40/CD40L pathway plays a major role in multiple inflammatory processes involving different immune and stromal cells. Abnormal activation of this pathway has been implicated in pathogenesis of complex autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, Graves' disease, and Sjogren's Syndrome. We completed in vitro and in vivo preclinical characterization of KPL-404, a novel humanized anti-CD40 IgG4 monoclonal antibody, to demonstrate its potency, efficacy, and pharmacokinetic profile; safety was also assessed. In vitro, KPL-404 bound recombinant human and cynomolgus monkey CD40 with comparable affinity in the nanomolar range. KPL-404 binding to cell surface CD40 did not induce antibody- or complement-mediated cytotoxicity of CD40-expressing cells. Pharmacological antagonistic activity of KPL-404 was demonstrated in vitro by inhibition of CD40-mediated downstream NF-kB activation. In the in vivo study with cynomolgus monkeys, KPL-404, administered intravenously as a single dose (10 mg/kg) or two monthly doses of 1 or 5 mg/kg, did not elicit observable safety findings, including thrombocytopenia over 8 weeks. KPL-404 engaged CD40 expressed on peripheral B cells for 2 and 4 weeks after a single administration of 5 or 10 mg/kg IV, respectively, without depletion of peripheral B cells. At 5 mg/kg IV, KPL-404 blocked both primary and secondary responses to T-cell dependent antibody responses to test antigens, KLH, and tetanus toxoid. These data illustrated the relationship between KPL-404 serum concentration and pharmacodynamic effects of CD40-targeting in circulation and in lymphoid tissues. These data support clinical development of KPL-404 in autoimmune diseases. SIGNIFICANCE STATEMENT: We aimed to develop a potent and efficacious CD40 antagonist. In vitro and in vivo findings show that KPL-404 blocks the anti-CD40 antibody that potently inhibits primary and secondary antibody responses at pharmacologically relevant concentrations, has a favorable pharmacokinetic profile, and does not deplete B cells by antibody-dependent cellular cytotoxicity or apoptosis ("nondepleting"). These findings support clinical development of KPL-404 as a potential therapeutic in autoimmune diseases.

Follicular lymphoma: the diminishing role of chemotherapy

Follicular lymphoma (FL) is the most common indolent non-Hodgkin lymphoma, accounting for 70% of cases in Western countries. Despite this unique name, FL is an extremely heterogeneous disease, both clinically and biologically. The basis of FL heterogeneity lies in the different biological pathways which can be activated, because of the variety of gene mutations that can occur. Today, there is a growing interest in the knowledge of these activated pathways, which is also testified by the presence of a new model that incorporates FL mutations to define patient’s prognosis (m7-FLIPI). These evaluations are also appealing because of the recent possibility of using “targeted therapies”. Targeted therapies are new tools, currently applicable in the setting of relapse/refractory (R/R) disease, where we can find a great variety of “chemo-free” combinations. As in other hematologic malignancies, “cellular therapy” enriches FL drug scenario, including T-cell dependent bispecific antibodies and chimeric antigen receptor (CAR) T-cell. Since FL heterogeneity is the basis of the difference in therapeutic efficacy and disease course among patients, the hope for the future is to understand FL biology more deeply, to better comprehend how to obtain more representative samples and pre-treatment prognostic information in order to individualize the treatment strategy as early as frontline therapy

Evaluation of immunotoxicitiy in biomedical research and development

Immunotoxicology aims at studying toxic effects of any substance on the immune system and its functions. In its various fields of application, this science is dependent on regulatory texts and guidelines. Studies are based on in vitro, ex vivo and in vivo techniques and are observational or functional allowing the identification of a toxic effect and its underlying mechanisms, respectively. Here, we review the various tests to perform in biomedical research and development, with a particular interest for the T-cell Dependent Antibody Response (TDAR) assay. We also briefly discuss the upcoming evolutions in this domain within a more ethically sound framework such as limiting the use of laboratory animals. These evolutions are represented by the development of relevant cell models.

Seroconversion after coronavirus disease 2019 vaccination in patients with immune deficiency

Safety and efficacy are 2 major drivers for any vaccination strategy and have come to the forefront in the setting of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination. Patients with underlying primary or secondary immunodeficiencies have variable response to vaccination, but inactivated or nonviable vaccines are generally considered safe in patients with immune deficiency.1 The efficacy of inactivated vaccines is variable in patients with humoral immune deficiencies, in which many can mount a protective T-cell–dependent antibody response to protein-conjugated vaccines, except for patients with congenital agammaglobulinemia, such as X-linked agammaglobulinemia (XLA), who may lack the ability to mount any antibody response.

B-cell-intrinsic MHCII signaling promotes microbiota diversity

Abstract The expression of major histocompatibility complex class II (MHCII) molecules is essential for the formation of germinal centers (GC) in lymphoid follicles, which are the primary sites for the generation of T-cell-dependent (TD) high-affinity antibody responses. Gut peyer's patches (PPs) are the dominant peripheral lymphoid tissues that give rise to TD antibody (primarily immunoglobulin A (IgA)) responses generated against the gut microbiota. While it is anticipated, a requirement of B-cell-intrinsic MHCII expression for GC formation has never been formally described. Additionally, while anti-commensal TD IgA responses have been shown to regulate microbiota composition and function, to what degree B-cell-intrinsic MHCII influences this process is also undefined. Here, we use a RAG1−/− adoptive transfer model, where RAG1−/− mice are either reconstituted with naive CD4+ T cells and MHCII+ B cells or naive CD4+ T cells and MHCII−B cells to address these gaps in our knowledge. Results from these experiments demonstrate that B-cell-intrinsic MHCII signaling is a strict requirement for GC-TFH cell development. Consequently, B-cell-intrinsic MHCII signaling promotes the generation of high-affinity anti-commensal IgA responses in the gut, which lead to increased species richness within the fecal but not small intestinal microbial community. Collectively, our data suggest that B-cell-intrinsic MHCII signaling is crucial for the generation of high-affinity anti-commensal IgA responses generated against the gut microbiota, and that this response favors a more diverse bacterial community.

A Comprehensive Overview of the Clinical Relevance and Treatment Options for Antibody-mediated Rejection Associated With Non-HLA Antibodies.

Although solid organ transplant results have improved significantly in recent decades, a pivotal cause of impaired long-term outcome is the development of antibody-mediated rejection (AMR), a condition characterized by the presence of donor-specific antibodies to HLA or non-HLA antigens. Highly HLA-sensitized recipients are treated with desensitization protocols to rescue the transplantation. These and other therapies are also applied for the treatment of AMR. Therapeutic protocols include removal of antibodies, depletion of plasma and B cells, inhibition of the complement cascade, and suppression of the T-cell–dependent antibody response. As mounting evidence illustrates the importance of non-HLA antibodies in transplant outcome, there is a need to evaluate the efficacy of treatment protocols on non-HLA antibody levels and graft function. Many reviews have been recently published that provide an overview of the literature describing the association of non-HLA antibodies with rejection in transplantation, whereas an overview of the treatment options for non-HLA AMR is still lacking. In this review, we will therefore provide such an overview. Most reports showed positive effects of non-HLA antibody clearance on graft function. However, monitoring non-HLA antibody levels after treatment along with standardization of therapies is needed to optimally treat solid organ transplant recipients.