Complement-dependent Cytotoxicity Assay Research Articles

Interchain disulfide engineering enables the efficient production of functional HLA-DQ-Fc fusion proteins

HLA-DQ molecules drive unwanted alloimmune responses after solid-organ transplants and several autoimmune diseases, including Type1 Diabetes and celiac disease. Biologics with HLA molecules as part of the design are emerging therapeutic options for these allo- and autoimmune conditions. However, the soluble α and β chains of class II HLA molecules do not dimerize efficiently without their transmembrane domains, which hinders their production. In this study, we examined the feasibility of inter-chain disulfide engineering by introducing paired cysteines to juxtaposed positions in the α and β chains of HLA-DQ7, encoded by HLA-DQA1*05:01 and HLA-DQB1*03:01 respectively. We identified three variant peptide-HLA-DQ7-Fc fusion proteins (DQ7Fc) with increased expression and production yield, namely Y19C-D6C (YCDC), A83C-E5C (ACEC), and A84C-N33C (ACNC). The mutated residues were conserved across all HLA-DQ proteins and had limited solvent exposure. Further characterizations of the YCDC variant showed that the expression of the fusion protein is peptide-dependent; inclusion of a higher-affinity peptide correlated with increased protein expression. However, high-affinity peptide alone was insufficient for stabilizing the DQ7 complex without the engineered disulfide bond. Multiple DQ7Fc variants demonstrated expected binding characteristics with commercial anti-DQ antibodies in two immunoassays and by a cell-based assay. Lastly, DQ7Fc variants demonstrated dose-dependent killing of DQ7-specific B cell hybridomas in a flow cytometric, complement-dependent cytotoxicity assay. These data support inter-chain disulfide engineering as a novel approach to efficiently producing functional HLA-DQ molecules and potentially other class II HLA molecules as candidate therapeutic agents.

Abstract 2737: LIS22, a first in class polyclonal antibody immunotherapy in T cell blood cancers

Abstract Background: Several approaches such as antibody drug conjugate (ADC), chimeric antigen receptor T cells (CAR-T) and recently bispecific antibodies are being introduced as innovative weapon in B-Cell lymphoma treatment. In the opposite, very limited therapeutic options are available for the treatment of rare refractory T cell malignancies such as PTCL. The 5-year survival rate of PTCL is lower than 30% according to the recent clinical data. Developing new effective treatment strategies for PTCL is an urgent problem. LIS22, is a first in class glyco-humanized polyclonal antibody (GH-pAb), targeting multiple tumor-associated antigens simultaneously. In this study, we characterized the safety and efficacy of LIS22 in preclinical models of T cell malignancies. Material and Methods: LIS22 ability to induce Antibody-Dependent Cell Cytotoxicity (ADCC), Antibody-Dependent Cellular Phagocytosis (ADCP), Complement Dependent Cytotoxicity (CDC), and apoptosis was tested in a panel of hematologic malignancy cell lines and peripheral blood mononuclear cell (PBMC). To assess the targeting and recognition of LIS22 in PTCL patients, we evaluated the immunolabelling of LIS22 on patients’ biopsies (n=119) using tissue microarray. LIS22 efficacy in vivo were evaluated in NMRI nude mice and SRG rats using T1301 and Jurkat cancer cell lines. Pharmacokinetics and safety of this drug were assessed in cynomolgus monkeys after single and repeated IV dosing up to 50mg/kg. Results: LIS22 acts via several mechanisms, at 30µg/ml, it induced cytotoxicity via CDC (in 70%), ADCP (in 49%), ADCC (in 41%) and apoptosis (in 30%) of HPB-ALL human T blood cancer cell line but not in PBMC. LIS22 showed a potent in vitro antitumor activity in a panel of hematologic malignancy cell lines, it induced specific tumor cell CDC (EC50=41.4±28.9µg/mL). In both CDC and apoptosis cytotoxicity assays, LIS22 displayed a significantly higher potency on T cell blood cancers and no toxicity on healthy blood cells compared to PTCL clinically active drug Alemtuzumab (Anti-CD52). It was able to kill up to 100% of the cancer cells without affecting PBMC. In immunolabeling assay, LIS22 demonstrated cross-reaction to PTCL patient biopsy (staining up to 93%). In vivo efficacy studies in different mice and rat xenograft models, LIS22 induced a significant reduction of tumor growth up to 90% across several tested tumors. LIS22 was also characterized by high tolerance profile and satisfactory exposure in monkeys after repeated dosing (up to 250mg/kg of cumulative dose). Conclusion: LIS22 appeared as a novel and promising cancer immunotherapy against T Cell hematologic malignancies. It has already been administered to human in another indication and showed a satisfactory PK and safety profile (no signs of immunotoxicity or systemic cytokine release). LIS22 is now ready to enter Phase I/II in the coming months for the treatment of peripheral T cell lymphoma (PTCL). Citation Format: Carine Ciron, Francoise Shneiker, Ophélie Dauphouy, Juliette Rousse, Pierre-Joseph Royer, Gwenaelle Evanno, Odile Duvaux, Firas Bassissi. LIS22, a first in class polyclonal antibody immunotherapy in T cell blood cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2737.

Abstract 2739: XON27, a novel and potent immunotherapy against hematologic malignancies

Abstract Background: Over the past decades, intensive research has led to the development of innovative therapies (monoclonal and bispecific antibodies, CAR T cells) that have increased the survival rate of patients with multiple myeloma (MM) and B cell lymphoma. However, despite this progress, the majority of patients relapse and become refractory due to the development of resistance. Polyclonal antibodies, with their ability to target multiple antigens, can counteract this resistance. Here we evaluate the safety and efficacy of XON27, a first-in-class glyco-humanized polyclonal antibody (GH-pAb), in MM and other blood cancers. Material and Methods: The ability of XON27 to induce antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) and apoptosis was tested in a panel of hematological malignancy cells and peripheral blood mononuclear cells (PBMC). Specific binding to human blood cancers was assessed using tissue microarrays. To study resistance mechanisms, MM cell lines were cocultured with macrophages in the presence or absence of XON27 and macrophage. To study relapse mechanisms and tumorigenicity, repeated in vitro administration of XON27 was evaluated in KMS-12-BM. The in vivo efficacy of XON27 was evaluated in KMS-12-BM NMRI nude mice model. Pharmacokinetics and safety of this drug were assessed in non-human primate after repeated IV dosing up to 60mg/kg.Results: XON27 induces potent and specific cytotoxicity in several blood malignancy cell lines including human MM cells via multiple mechanisms: CDC (EC50=35.84 ug/ml), apoptosis (IC50=100 ug/ml) and ADCP (up to 40% of cell death at 50 ug/ml). The cytotoxicity induced by XON27 in the KMS-12-BM was significantly more potent compared to daratumumab (anti-CD38) in both CDC and apoptosis assays, XON27 was able to kill up to 100% of cancer cells without affecting PBMC. XON27 showed cross-reactivity with blood cancer patient biopsies without staining healthy tissue. In addition, repeated administrations of XON27 demonstrate its ability to effectively kill tumor cells after each administration. No resistance is induced after repeated treatments. Interestingly, XON27 blocked the proliferation of residual tumor cells for up to 45 days after the second treatment at 50 ug/ml compared to the control condition, demonstrating sustainable effect of XON27 on tumorigenicity. The in vitro efficacy of XON27 was translated into in vivo efficacy in a murine MM xenograft model. It induced a significant reduction in tumor growth of up to 75%. XON27 was also characterized by a high tolerability profile and satisfactory exposure in primates. Conclusion: Although many therapeutic advances have been made in the field of blood cancers, myeloma remains an incurable disease with a real need for new innovative therapies. XON27 represents a promising and selective immunotherapy which may provide an effective and safe treatment against refractory myeloma. Citation Format: Carine Ciron, Pierre Morice, Françoise Shneiker, Odile Duvaux, Firas Bassissi. XON27, a novel and potent immunotherapy against hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2739.

Pretransplant Screening for Prevention of Hyperacute Graft Loss in Pig-to-primate Kidney Xenotransplantation.

Xenotransplantation using pig organs is now a clinical reality. However, the process for xenograft recipient screening lacks clarity and scientific rigor: no established thresholds exist to determine which levels of preformed antipig natural antibodies (Nabs) will be safe for clinical xenograft transplantation, and hyperacute rejection (HAR) or acute humoral xenograft rejection (AHXR), which still impacts pig-to-primate kidney xenograft survivals, may impede broader application of pig-to-human clinical xenograft transplantation. We retrospectively examined 28 cases of pig-to-baboon kidney xenotransplantation using GalTKO±human complement regulatory protein (hCRP)-transgenic (Tg) pig donors, as well as 6 cases of triple-KO multi-Tg (10GE) pig donors, and developed screening algorithms to predict risk of HAR/AHXR based on recipient antipig Nab levels. Preformed Nabs were evaluated using both complement-dependent cytotoxicity and antibody (IgM and IgG) binding flow-cytometry assays. High complement-dependent cytotoxicity was associated with HAR/AHXR as expected. However, we also found that high levels of IgG were independently associated with HAR/AHXR, and we developed 2 indices to interpret and predict the risk of IgG-mediated HAR/AHXR. Based on the data in this study, we have established a new 2-step screening, which will be used for future clinical kidney xenotransplantation trials.

Dynamics of HLA and angiotensin II type 1 receptor antibodies during pregnancy

BackgroundAlloantibodies, especially anti-human leukocyte antigen antibodies (HLA antibodies), and autoantibodies, as angiotensin II type 1 receptor antibodies (AT1R antibodies), may complicate the access and the course of transplantation. Pregnancy is a known source of HLA antibodies, with most studies evaluating pregnancy-induced sensitization by complement-dependent cytotoxicity assays, mainly after childbirth. AT1R antibodies have been evaluated in the context of preeclampsia. We aimed to evaluate pregnancy as a natural source of HLA antibodies and AT1R antibodies, their dynamics along gestation and the potential factors involved in antibody appearance. MethodsSerum samples from pregnant women were collected during the three trimesters of pregnancy (1T, 2T, 3T). Presence of HLA antibodies was assessed by screening beads on Luminex and AT1R antibodies by ELISA. ResultsA cohort of 138 pregnant women were included. Samples from all were tested in 1T, 127 in 2T and 102 in 3T. HLA antibodies increased from 29.7 % (1T) to 38.2 % (3T). AT1R antibodies were stable around 30 % along pregnancy. Up to 43.2 % multiparous women had HLA antibodies, with a similar proportion of class I and class II antibodies. In primiparous women HLA antibodies increased along pregnancy (from 17.6 % to 34.1 %), with predominance of class II HLA antibodies. AT1R antibodies were not different in primiparous and multiparous women. ConclusionsPregnancy is a relevant source of HLA antibodies sensitization, but not of AT1R antibodies. HLA antibodies increased clearly in primiparous women with predominance of class II. The use of newer solid-phase techniques on Luminex evidence a higher degree of HLA sensitization during pregnancy.

Development of therapeutic monoclonal antibodies against DKK1 peptide-HLA-A2 complex to treat human cancers

BackgroundTargeted immunotherapy with monoclonal antibodies (mAbs) is an effective and safe method for the treatment of malignancies. Development of mAbs with improved cytotoxicity, targeting new and known tumor-associated antigens, therefore...

Iberdomide Enhances Dara Mediated Cytotoxicity through Upregulation of CDC Activity and Elevated NK Cell Mediated ADCC

Background: Multiple Myeloma (MM) is an incurable hematological malignancy with current treatments comprising combination regimens, typically featuring immunomodulatory agents (IMiDs) due to their potent anti-tumor activity and immunomodulatory effects. Clinical studies utilizing the combination of IMiD agents and daratumumab (DARA), an anti-CD38 monoclonal antibody, have shown improved patient outcomes for MM. However, patients ultimately relapse demonstrating the need for novel therapies. Iberdomide (IBER) is a novel cereblon E3 ligase modulator. It promotes faster and deeper degradation of the substrate targets compared to IMiDs, resulting in enhanced anti-proliferative, pro-apoptotic and immune stimulatory activity. In early clinical studies, IBER has been shown to overcome resistance to IMiDs and in the ongoing Phase 3 EXCALIBER study (NCT04975997) is being explored for treatment of newly diagnosed (ND) and relapsed/refractory (RR) MM in combination with DARA and dexamethasone. Here, we investigate the potential mechanisms of synergy between IBER and DARA in preclinical models. Methods: Complement dependent cytotoxicity (CDC) of DARA was assessed with 15% human serum for human complement components and assays were performed by titrating either single agent or combinations in a tritiated thymidine assay. Antibody dependent cellular cytotoxicity (ADCC) was performed by co-culturing MM cells with varying expression levels of CD38 and effector cells and assessing target cell viability with annexin-v/topro-3 flow. A humanized PBMC adoptive transfer mouse model, utilizing human PBMC engraftment 14 days prior to implantation, was used to assess in vivo activity of IBER, DARA and IBER + DARA. Results: In CDC assays, we observed variable sensitivity of MM cells to complement-specific CDC in the presence of DARA (0.1-1 µg/ml), regardless of cell surface CD38 expression. Notably, IBER treatment resulted in upregulation of CD38 surface expression in 60% of MM cell lines tested. LP1 and MOLP8 displayed susceptibility to DARA-mediated CDC. DARA showed a dose-dependent increase in CDC activity in both cell lines. Notably, IBER+DARA also showed ~35% more synergistic CDC activity in both cell lines, than the single agents. In ADCC assays, we observed pretreatment of PBMC with IBER (vs. DMSO) significantly increased DARA mediated ADCC in H929 and LP-1 cells. To confirm that this effect was directed by NK cells, we cultured NK cells for 24 hr in supernatants from stimulated PBMC in presence of IBER prior to co-culture with MM cells. We noted that IBER-treated PBMC supernatant significantly enhanced the NK ADCC activity of DARA suggesting that IBER enhancement of cytokine secretion from PBMC increases NK cell mediated ADCC. Orthogonally, we pre-treated MM cells with IBER or DMSO prior to co-culture in ADCC assay. Notably, IBER treatment of MM cells, without treating immune cells, also increased DARA-mediated ADCC, indicating that IBER directly increases sensitivity to DARA-directed ADCC. To investigate the MOA of IBER-DARA synergy in ADCC, we analyzed gene expression and cell surface changes of a panel of NK-specific receptor ligands on MM cells following treatment with IBER. We observed IBER-induced upregulation of MICA mRNA levels (~2x) and surface expression (~40%) in MM target cells. We used RNAi to knock down Ikaros, an IBER substrate, which resulted in increased MICA expression (~3x) in MM cells. Furthermore, we observed that RNAi knock down of MICA in MM cells resulted in a significant decrease (60%, p<0.0001) in MM cell killing by NK cells, suggesting IBER-enhanced sensitivity of MM cells to NK-cell killing was in part, due to the upregulation of MICA through degradation of Ikaros. Lastly, we examined the anti-MM effects of IBER in a humanized xenograft model with a lenalidomide resistant cell line. Longitudinal analysis identified persistence of human PBMC engraftment and activation of immune subsets (NK cells and T-cells) that were increased upon IBER treatments, consistent with clinical observations. The combination of IBER/DARA resulted in ~22% tumor growth inhibition (TGI) in comparison to single agents, DARA and IBER and ~37% TGI in comparison to vehicle. Conclusion: Taken together, these data demonstrate that IBER and DARA work synergistically in preclinical models primarily through enhancement of both ADCC and CDC. This combination has the potential to improve outcomes in MM.

Preclinical and Translational Biomarker Analyses to Inform Clinical Development of Mezigdomide (CC-92480) in Combination with Dexamethasone and Daratumumab in Multiple Myeloma

Introduction: Mezigdomide (MEZI) is a novel, oral cereblon (CRBN) E3 ligase modulator (CELMoD) that induces rapid and potent degradation of lkaros and Aiolos. Reduction of these transcription factors results in direct tumoricidal and immunomodulatory effects in multiple myeloma (MM). MEZI showed promising efficacy and safety in combination with dexamethasone in the phase 1/2 CC-92480-MM-001 trial (NCT03374085) in Relapsed/Refractory MM. MEZI is currently being investigated in combination with daratumumab and dexamethasone (MEZI + DARA + DEX or MEZI-Dd) in the phase 1/2 CC-92480-MM-002 trial (NCT03989414). In MEZI-Dd cohort, MEZI is administered at three different schedules (B1 for 21/28 days; B2 for 14/21 days; B3 for 7/14 days), and at two different doses (0.3mg; 0.6mg). Here we report preclinical data and pharmacodynamic (PD) biomarker analyses from blood and bone marrow samples collected in CC-92480-MM-002 to support dose and schedule optimization of MEZI-Dd. Methods:Preclinical studies on the pro-apoptotic effects of MEZI on daratumumab-mediated complement-dependent cytotoxicity (CDC) and peripheral blood mononuclear cell-based antibody-dependent cell-mediated cytotoxicity (ADCC) were performed in MM cell lines treated with MEZI-D in comparison to single-agent MEZI or DARA. Clinical PD biomarker analyses included peripheral blood samples collected on treatment Cycle (C)1 Day (D)1 and mid C1-C3 for the expression of Aiolos in T-cells and immunomodulation by flow cytometry. Bone marrow samples were collected for immunohistochemistry at screening and mid C2. Serum free light chain (sFLC) and soluble B-cell maturation antigen (BCMA) were analyzed as biomarkers for tumor burden from C1-C6. Results: Preclinically, combinations of MEZI-D induced synergistic anti-tumor activity in MOLP-8 cells. The MEZI-D combination showed greater anti-MM activities across the dosing gradient than either single-agent DARA (~2-4.5 fold) or MEZI (~1.7-2.4 fold) in a CDC assay. MEZI-D also induced significantly more cell apoptosis than POM-D (p<0.05) in an ADCC assay. In the clinic, MEZI-Dd was sufficient to induce rapid Aiolos degradation in the peripheral blood T-cells for doses as low as 0.3mg MEZI, but 0.6mg MEZI was necessary to result in >50% substrate degradation, suggesting 0.6mg MEZI induced deeper substrate degradation. While 0.3mg MEZI-Dd decreased total CD56+/CD16+ NK-cell counts (Median -83.8%) by mid C1 via immunophenotyping analyses, the combination increased %Ki67+ proliferative NK-cell (Median 251.9%). 0.3mg MEZI-Dd also showed consistent trends of schedule-dependent PD immune effects by mid C3, including increased %Ki67+ proliferative CD3+ T-cell (Median B1: 113.1%, B2: 365.1%, B3: 37.4%), decreased CD45RA+CCR7+ naïve CD4+ T-cell (Median B1: -82.0%, B2: -62.3%, B3: -43.9%), increased CD45RO+CCR7- effector memory CD4+ T-cell (Median B1: 142.3%, B2: 82.9%, B3: 13.9%), and increased HLA-DR expressing CD4+ T-cell activation (Median B1: 149.3%, B2: 107.9%, B3: 17.1%). Thus, greater PD immune effects were observed with schedules of 21/28 and 14/21 days, compared to the 7/14 schedule. At 0.6mg MEZI-Dd, greater median changes of PD immune effects were induced with the 7/14 schedule, compared to 0.3mg MEZI-Dd. Conversely, at continuous dosing schedules longer than 7 days, MEZI-Dd PD immune effects of 0.6mg MEZI were similar to 0.3mg MEZI. In addition, greater reduction of tumor burden was observed at 0.6mg MEZI-Dd, compared to 0.3mg MEZI-Dd across different schedules, as assessed by sFLC and soluble BCMA analyses. Conclusions: Our data show dose- and schedule-dependent PD effects of MEZI-Dd. PD analyses at 0.3mg MEZI suggest at least two weeks of continuous dosing may be needed for greater PD activities in combination with daratumumab, compared to one week of dosing. For continuous dosing of one week, a higher dose at 0.6mg MEZI may be needed to achieve greater PD activities. Further investigation of doses at 0.6mg and 1mg MEZI is underway to determine the optimal dose. Moreover, MEZI induction of NK-cell proliferation and/or activation may provide mechanistic rationale for possible MEZI-D synergy in the clinic.

Adaptation of HLA testing to characterize the cynomolgus macaque MHC polymorphisms and alloantibody signatures.

Nonhuman primates are the closest animal models to humans with respect to genetics and physiology. Consequently, a critical component of immunogenetics research relies on drawing inferences from the cynomolgus macaque to inform human trials. Despite the conserved organization of the Major Histocompatibility Complex (MHC) between cynomolgus macaques and humans, MHC genotyping of cynomolgus macaques is challenging due to high rates of copy number variants, duplications, and rearrangements, particularly at the MHC class I loci. Furthermore, the limited availability of commercial reagents specific to cynomolgus macaques that can be used to characterize anti-MHC class I and class II antibody (Ab) specificities in cynomolgus macaques presents a major bottleneck in translational research. Here we successfully characterized cynomolgus macaque Mafa class I and class II serologic specificities in 86 animals originating from various geographical regions using the complement dependent cytotoxicity (CDC) assay with human HLA class I and class II monoclonal antibody (mAb) typing trays. Further, we successfully induced and characterized anti-Mafa class I and class II alloantibody specificity using HLA single antigen bead assays. We also subsequently tracked the alloAb burden in the animals during treatment with anti-B lymphocyte stimulator (BLyS) treatment. Altogether, these methods can be easily used in translational research to serotype MHC class I and class II specificity in macaques, characterize their alloAb specificity, and evaluate the efficacy of novel therapeutic modalities in depleting circulating alloAbs in these animals.

New Complement Dependent Cytotoxicity Assay and its application to COVID-19 patient sera harboring anti-lymphocyte antibodies.

Abstract Complement dependent cytotoxicity (CDC) is one of the ways IgM and IgG antibodies can mediate the lysis of target cells, being IgM the antibody (Ab) isotype with the highest CDC activity. In a cohort of hospitalized COVID-19 patients we identified that 27% carried IgM anti-lymphocyte Ab in their sera. To quantify the sera potential lytic activity against healthy donor (HD) PBMC we developed a flow-based CDC assay with an internal negative control. The assay allows for normalization of the results and therefore inter-experiment data comparison without the need of using counting beads. In short, after CFSE labelling, 2 million PBMC are incubated with either complete media (CM) or with the different experimental conditions (Ab, sera, plasma), then they are washed thoroughly before adding them to equal numbers of unlabeled PBMC (internal negative control). This mixture of hot CFSE+ and cold CFSE-targets is then incubated with rabbit complement for an hour, stained and flow. A “ratio tube” containing the mixture with the CM incubated CFSE cells, is left without rabbit complement at 4 degrees and used to calculate the initial CFSE+/CFSE− ratio. We calculate the percentage of CDC lysis based on the CFSE+/CFSE− ratio in each tube after normalizing it to the ratio tube. Deploying this assay, we found CDC activity against B and T cells in five and two COVID-19 patients’ sera, respectively, out of 23 patients tested. This improved CDC assay shows high reproducibility and allows for quick, reliable, and easy test of multiple conditions. This work was supported by the Divisions of Intramural Research of NIAID, NIH

In Memoriam: Soldano Ferrone MD, PhD (1940–2023)

In Memoriam: Soldano Ferrone MD, PhD (1940–2023)

Abstract 4531: Clinical translation of biomarkers for ZL-1211, an anti-CLDN18.2 antibody, in patients with advanced solid tumors from a phase I dose-escalation study

Abstract ZL-1211, a humanized monoclonal IgG1 antibody targeting CLDN18.2, induces cancer cell death through antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). We developed ex vivo ADCC and CDC assays to study the mechanism of actions of ZL-1211. Our preclinical data has demonstrated greater antitumor activities than the clinical leading benchmark via enhanced ADCC by mutation in Fcγ domain and potent CDC activities. We used those ex vivo assays to further profile the clinical specimens from patients collected at baseline (pre-infusion) and the following cycles (post-infusion) in our phase 1 study (NCT05065710). Preliminary results in clinical samples indicated that baseline ADCC and CDC capabilities were correlated with anti-tumor activity of ZL-1211 in patients with CLDN18.2-positive locally advanced or metastatic solid tumors, suggesting probable predictive biomarkers. Results also showed that changes in circulating ZL-1211 mediated ADCC and CDC activities were dose-dependent and in accordance with the pharmacokinetic (PK) profile. In addition, we performed differential gene expression analysis and real-time PCR to compare gene expression in ZL-1211 responder and non-responder PDX models and CCLE cell lines. Our findings reveal that several immune-related genes may serve to predict the responses to ZL-1211 treatment. Other potential predictive biomarkers such as Fcγ receptor CD16A polymorphism and baseline stromal CD8 expression were also evaluated in clinical samples. Taken together, analytical approaches developed and validated from preclinical studies were successfully translated to characterize clinical samples in the current Phase 1 trial. The exploratory analysis indicated the clinical relevance of multiple biomarkers and their potential as predictive biomarkers of ZL-1211. Citation Format: Zhen Luo, Xiao Wang, Xuehuo Zeng, Jiaqing Yi, Tegan Burns, Herman Liu, Xinyu Zhang, Wenyu Liu, Karl Hsu, Hua Gong. Clinical translation of biomarkers for ZL-1211, an anti-CLDN18.2 antibody, in patients with advanced solid tumors from a phase I dose-escalation study. [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 4531.

LALAPG variant of the Human Contraception Antibody (HCA) reduces Fc-mediated effector functions while maintaining sperm agglutination activity.

High rates of unintended pregnancies worldwide indicate a need for more accessible and acceptable methods of contraception. We have developed a monoclonal antibody, the Human Contraception Antibody (HCA), for use by women in vaginal films and rings for contraception. The divalent F(ab')2 region of HCA binds to an abundant male reproductive tract-specific antigen, CD52g, and potently agglutinates sperm. Certain other antibody activities mediated by the Fc region such as mucus trapping, complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP) could have beneficial or negative effects. The purpose of this study was to document HCA Fc effector functions and determine whether an engineered variant of HCA with a modified Fc region, HCA-LALAPG, retains desirable contraceptive activity while minimizing Fc-mediated effects. Fab and Fc functions were compared between HCA and HCA-LALAPG. Fab activity was assessed using sperm agglutination and modified swim-up ("sperm escape") assays. Fc functions were assessed by CDC (sperm immobilization), ADCP, and cervical mucus penetration assays. HCA and HCA-LALAPG showed equivalent activity in assays of Fab function. In the assays of Fc function, HCA supported strong CDC, ADCP, and sperm trapping in cervical mucus whereas HCA-LALAPG demonstrated little to no activity. HCA and the HCA-LALAPG variant were both highly effective in the sperm agglutination assays but differed in Fc mediated functions. Use of the HCA-LALAPG variant for contraception in women could reduce antibody-mediated inflammation and antigen presentation but may have reduced contraceptive efficacy due to much weaker sperm trapping in mucus and complement-dependent sperm immobilization activity.

What Have We Learned From In Vitro Studies About Pig-to-primate Organ Transplantation?

Natural preformed and de novo antibodies against pig antigens are a major cause of pig xenograft rejection in nonhuman primates (NHPs). In vivo studies in pig-to-NHP models are time consuming. In vitro assays, for example, antibody binding to pig cells, complement-dependent cytotoxicity assays, provide valuable information quickly and inexpensively. Using in vitro assays for several years, it has been documented that (1) during the first year of life, humans and NHPs develop anti-wild-type pig antibodies, but humans develop no or minimal antibody to triple-knockout (TKO) pig cells. (2) Some adult humans have no or minimal antibodies to TKO pig cells and are therefore unlikely to rapidly reject a TKO organ, particularly if the organ also expresses human "protective" proteins. (3) There is good correlation between immunoglobulin (Ig)M (but no t IgG) binding and complement injury. (4) All Old World NHPs develop antibodies to TKO pig cells and are not optimal recipients of TKO organs. (5) galactosyltransferase gene-knockout/β4GalNT2KO pigs are preferred for Old World NHPs. (6) Humans develop anti-pig IgE and IgA antibodies against pig cells, but their role remains uncertain. (7) In a small percentage of allosensitized humans, antibodies that cross-react with swine leukocyte antigens may be detrimental to a pig organ xenograft. (8) Prior sensitization to pig antigens is unlikely to be detrimental to a subsequent allograft. (9) Deletion of expression of Gal and Neu5Gc is associated with a reduction in the T-cell response to pig cells. All of these valuable observations have largely predicted the results of in vivo studies.

Retreatment with Polatuzumab Vedotin in Combination with Rituximab Increases Antitumor Activity Against Polatuzumab Vedotin-Resistant DLBCL Cells

Introduction. Polatuzumab vedotin (Pola) is a monoclonal antibody against B-cell antigen CD79b conjugated to monomethyl auristatin E (MMAE), and is approved in combination with bendamustine and rituximab (Rit) in relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL) in various countries. Moreover, in the POLARIX study, Pola-R-CHP (cyclophosphamide, doxorubicin, and prednisone) achieved a significantly improved progression-free survival compared to R-CHOP (CHP plus vincristine) in previously untreated DLBCL. The treatment landscape of DLBCL have continued to change dynamically in recent years with the development of various agents. Although the detailed mechanism of resistance to Pola is not clear, understanding it is important for the development of next-line treatment to follow Pola-containing therapy, including retreatment with Pola. To address this, we established the Pola-resistant cells using Pola-sensitive human DLBCL and assessed the combination efficacy of Pola with Rit, the key component in the treatment of DLBCL to determine the efficacy of retreatment with Pola. Methods. We established Pola-resistant DLBCL cells from STR-428 and SU-DHL-4 cells through long-term exposure to Pola. The cell cytotoxicity to Pola was assessed by WST-8 assay. The in vitro combination efficacy of Pola plus Rit was assessed by measuring the sensitivity to complement-dependent cytotoxicity (CDC), rituximab's potent mechanism of action. In CDC assays, calcein-AM stained tumor cells were incubated with or without Rit in the medium containing 15% normal human serum. After incubating for 4 hours at 37℃, the fluorescence intensity was measured. Expression levels of protein were determined by flow cytometry or immunoblotting. For in vivo experiments, Pola-resistant STR-428 cells were injected subcutaneously into C.B-17/Icr-scid/scidJcl mice. Human IgG, Pola, or Rit was administered intravenously on Day 1. Results. Pola-resistant cells established from SU-DHL-4 cells (SU-DHL-4-Pola-R) showed a 2.3-fold higher IC50 value than parental cells. In SU-DHL-4-Pola-R cells, there was an increase in the expression of MDR1 (1.8-fold), a multidrug resistance pump mediating intracellular MMAE efflux. Interestingly, treatment with 1 μg/mL of Pola for 7 days further increased the IC50 value (16.8-fold) and the expression of MDR1 (3.6-fold）in SU-DHL-4-Pola-R cells.MDR1 inhibitor verapamil increased sensitivity to Pola, indicating that overexpression of MDR1 is one of the mechanisms of resistance to Pola in these cells. We then investigated the efficacy of combination therapy with Rit against Pola-resistant cells. Pretreatment with Pola or anti-CD79b antibody before the assays enhanced Rit-induced amount of membrane attack complex during complement activation and significantly increased CDC sensitivity in SU-DHL-4-Pola-R cells. Other Pola-resistant cells derived from STR-428 cells (STR-428-Pola-R) displayed an IC50 value 17.6-fold higher than the parental cells. STR-428-Pola-R cells did not exhibit Pola-resistant factors such as MDR1 upregulation or loss of CD79b expression, but showed decreased expression of Bim, an apoptosis-inducing factor. Stable knockdown of Bim reduced the sensitivity to Pola in parental STR-428 cells, indicating decreased Bim expression could be the resistant mechanism to Pola in these cells. In STR-428-Pola-R cells, 3 days pretreatment with Pola or MMAE significantly increased Rit-induced CDC sensitivity. Immunoblotting analysis revealed that pretreatment with Pola or MMAE downregulated the expression of Mcl-1, which is known to attenuate CDC sensitivity. Mcl-1 inhibitor AZD5991 significantly increased CDC sensitivity, suggesting that Pola-mediated downregulation of Mcl-1 could upregulate the sensitivity to CDC in these cells. In addition, treatment with Pola (1 mg/kg) plus Rit (10 mg/kg) significantly enhanced antitumor activity in mouse xenograft models derived from STR-428-Pola-R cells. Conclusion. Pretreatment with Pola enhanced Rit-induced CDC sensitivity in two Pola-resistant cells with distinct mechanisms of resistance: increased MDR1 or decreased Bim expression. Combination treatment of Pola with Rit enhanced antitumor activity in STR-428-Pola-R xenografted mice. These results indicate that retreatment with Pola in combination with Rit could be a therapeutic option in patients with Pola-resistant tumors.

Novel Sirpα-Fc Fusion Protein IMM01 Exhibits Dual Anti-Tumor Activities By Targeting CD47/Sirpα Signal Pathway

Background: This study aims to evaluate the anti-tumor mechanism of IMM01, a novel recombinant SIRPα-Fc fusion protein targeting CD47-SIRPα pathway. Methods: IMM01 was a recombinant fusion protein of SIRPα (V2) extracellular segment domain 1 and human IgG1 Fc, with the N glycosylation site N80 mutated to A (Alanine) in D1 region. IMM01 fusion proteins were produced using in house developed CHO-K1 cell expression system. IMM01 blocking activities on Jurkat-CSR (Jurkat-CD47KO-CARSIRPα) cells, antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays were done by flow cytometry. Detection of cytokines of IL-1β, IL-2, IL-4, IL-5, IL-6, GM-CSF, IFN-γ, IL-10 and TNF production by PBMC was done CBA assay. In vivo mechanism of action study was done in different mouse models including HL-60 CB17-SCID, Daudi CB17-SCID, SNU-1 CB17-SCID, Raji CB17-SCID and CT26-hPDL1(Tg) hCD47(Tg) mPDL1(KO) mCD47(KO). In addition, human red blood cells binding and erythrocyte agglutination of IMM01 were also evaluated. Results:In vitro binding assay revealed that IMM01 has a strong binding affinity to CD47 with lowest EC50 at 0.4967 nM. The removal of N-glycosylation modification improves the consistence and purity of the IMM01 protein expression, and the potential mitigation of immunogenicity. Using a chimeric SIRPα receptor protein expressing cell line, IMM01 not only can block the CD47 at the protein level, but also block the signal transduction at the cellular levels. Compared to negative (hIgG1-Fc) controls, IMM01 stimulated secretion of minimal amount of IL-10 and TNF but inhibited IL-8 production by PBMC. IMM01 can induce strong ADCP and moderate ADCC, but not the CDC activities. The strong phagocytosis against tumor cells is dependent on interaction of Fc with activating FcγRs on the presence of macrophages. Mechanism of action studies demonstrated that IMM01 induced strong phagocytosis against tumor cells was attributed to dual activities of blocking "don't eat me" signal and activating "eat me" signal. IMM01 has strong and robust in vivo anti-tumor activities in different mouse tumor models either as monotherapy on hematological malignancies, or in combination therapy with PD-L1 mAb, PD-1 mAb, CD33 mAb, and HER-2 mAb, respectively. The in vivo therapeutic efficacy of IMM01 was done in HL-60 xenograft model showed that 100% of the mice achieved CR after administration of IMM01 at 5mg/kg for 2 weeks. Also, 100% of mice achieved CR after IMM01 combined with the above mentioned mAbs in different mouse models, respectively. This illustrated that IMM01 has a great therapeutic potential in combination with these commonly used immunotherapy or targeted therapy agents to be used in the treatment of solid tumors and hematological malignancies. Finally, IMM01 demonstrated a favorable safety profile with no human RBC binding, nor hemagglutination induction. Conclusion: Our study results demonstrated that, as a novel bi-functional SIRPα-Fc recombinant protein, IMM01 has very strong anti-tumor activities with a good curative effect on mouse xenograft tumor models with dual anti-tumor activities by blocking CD47 "don't eat me" signal and activating the phagocytosis "eat me" signal. Furthermore, IMM01 demonstrated a well synergistic effect when combined with different immunotherapeutic agents. Importantly, our results revealed that IMM01 has a favorable safety profile with no human RBC binding activity and no hemagglutination induction. Phase I clinical study is ongoing, and the clinical trial results will be reported soon. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Anti-rituximab antibodies demonstrate neutralizing capacity, associate with lower circulating drug levels and earlier relapse in lupus.

High rates of anti-drug antibodies (ADA) to rituximab have been demonstrated in patients undergoing treatment for SLE. However, little is known with regard to their long-term dynamics, impact on drug kinetics and subsequent implications for treatment response. In this study, we aimed to evaluate ADA persistence over time, impact on circulating drug levels, assess clinical outcomes and whether they are capable of neutralizing rituximab. Patients with SLE undergoing treatment with rituximab were recruited to this study (n = 35). Serum samples were collected across a follow-up period of 36 months following treatment (n = 114). Clinical and laboratory data were collected pre-treatment and throughout follow-up. ADA were detected via electrochemiluminescent immunoassays. A complement dependent cytotoxicity assay was used to determine neutralizing capacity of ADA in a sub-cohort of positive samples (n = 38). ADA persisted over the 36-month study period in 64.3% of patients undergoing treatment and titres peaked earlier and remained higher in those who had previously been treated with rituximab when compared with than those who were previously treatment naive. ADA-positive samples had a significantly lower median drug level until six months post rituximab infusion (P = 0.0018). Patients with persistent ADA positivity showed a significant early improvement in disease activity followed by increased rates of relapse. In vitro analysis confirmed the neutralizing capacity of ADA to rituximab. ADA to rituximab were common and persisted over the 36-month period of this study. They associated with earlier drug elimination, an increased rate of relapse and demonstrated neutralizing capacity in vitro.

National Control Laboratory Assessment of Quality of Rituximab Biosimilars in India.

In past few years many rituximab (RTX) biosimilars have been launched in India. Biosimilars are products that are similar in terms of quality, safety, and efficacy to its innovator product and are expected to offer improved affordability. The less clinical examination is a significant source of reduction in the cost of development of a biosimilar. However, this clinical relief is predicated on the assumption that there is analytical similarity between the biosimilar and the innovator product. Therefore, the role of National Control Laboratory become very important to ensure the quality of these drugs by carrying out analytical characterization at the point of drug product release level as when referred by National Regulatory Authority for quality evaluation. To assess the similarity between innovator and biosimilars, different physicochemical and biological quality attributes were assessed. A multitude of state-of-the-art analysis of N = 3 RTX biosimilars marketed in India revealed that the impurity profiles of these biosimilars measured by charge variant analysis (cation exchange chromatography-high performance liquid chromatography [HPLC], capillary zone electrophoresis, and capillary isoelectric focusing), aggregates profiling (size exclusion chromatography-HPLC), fragments analysis (capillary electrophoresis-sodium dodecyl sulfate) were found to be significantly varying as compared with the innovator product. There were significant variations in acidic variants (p = 0.023) and basic variants (p = 0.0005), isoelectric point value (p < 0.0001), aggregates (p = 0.0231), and fragments (p < 0.0001) of biosimilars were found as that of innovator product. However, these differences were not affecting the biological activity in the cell-based potency analysis by complement-dependent cytotoxicity (CDC) assay (p = 0.1026), antibody-dependent cell-mediated cytotoxicity (ADCC) (p = 0.3736), and binding assay by flow cytometer fluorescence-activated cell sorting (p = 0.4005) of these biosimilars as compared with the innovator product.

Will previous palliative surgery for congenital heart disease be detrimental to subsequent pig heart xenotransplantation?

IntroductionPig heart xenotransplantation might act as a bridge in infants with complex congenital heart disease (CHD) until a deceased human donor heart becomes available. Infants develop antibodies to wild-type (WT, i.e., genetically-unmodified) pig cells, but rarely to cells in which expression of the 3 known carbohydrate xenoantigens has been deleted by genetic engineering (triple-knockout [TKO] pigs). Our objective was to test sera from children who had undergone palliative surgery for complex CHD (and who potentially might need a pig heart transplant) to determine whether they had serum cytotoxic antibodies against TKO pig cells. MethodsSera were obtained from children with CHD undergoing Glenn or Fontan operation (n = 14) and healthy adults (n = 8, as controls). All of the children had complex CHD and had undergone some form of cardiac surgery. Seven had received human blood transfusions and 3 bovine pericardial patch grafts. IgM and IgG binding to WT and TKO pig red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs) were measured by flow cytometry, and killing of PBMCs by a complement-dependent cytotoxicity assay. ResultsAlmost all children and adults demonstrated relatively high IgM/IgG binding to WT RBCs, but minimal binding to TKO RBCs (p < 0.0001 vs WT), although IgG binding was greater in children than adults (p < 0.01). All sera showed IgM/IgG binding to WT PBMCs, but this was much lower to TKO PBMCs (p < 0.0001 vs WT) and was greater in children than in adults (p < 0.05). Binding to both WT and TKO PBMCs was greater than to RBCs. Mean serum cytotoxicity to WT PBMCs was 90% in both children and adults, whereas to TKO PBMCs it was only 20% and < 5%, respectively. The sera from 6/14 (43%) children were cytotoxic to TKO PBMCs, but no adult sera were cytotoxic. ConclusionsAlthough no children had high levels of antibodies to TKO RBCs, 13/14 demonstrated antibodies to TKO PBMCs, in 6 of these showed mild cytotoxicity. As no adults had cytotoxic antibodies to TKO PBMCs, the higher incidence in children may possibly be associated with their exposure to previous cardiac surgery and biological products. However, the numbers were too small to determine the influence of such past exposures. Before considering pig heart xenotransplantation for children with CHD, testing for antibody binding may be warranted.

P871: ISB 1442, A FIRST-IN-CLASS CD38 AND CD47 BISPECIFIC ANTIBODY INNATE CELL MODULATOR FOR THE TREATMENT OF CD38 POSITIVE HEMATOLOGICAL MALIGNANCIES

Background: ISB 1442 is a first in class 2 + 1 biparatopic bispecific antibody targeting CD38 x CD47 using the BEAT® 2.0 (Bispecific Engagement by Antibodies based on the TCR) platform. The CD38 binding arm consists of bi-paratopic Fabs that strongly bind to CD38 in two different epitopes on tumor cells and do not functionally compete with daratumumab. The anti-CD47 arm comprises a single Fab arm designed to block the interaction between CD47 and the signal-regulatory protein alpha (SIRPα) receptor present on phagocytes (including macrophages, monocytes and dendritic cells). With this design, the high affinity anti-CD38 Fab arms preferentially drive binding to tumor cells and enables blocking of proximal CD47 receptors on the same cell via avidity-induced binding. The Fc portion of ISB 1442 is engineered to enhance antibody dependent cell phagocytosis (ADCP), antibody dependent cell cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). Aims: ISB 1442 was developed for the treatment of hematologic malignancies that express CD38, including multiple myeloma (MM), acute myeloid leukemia (AML), and T-acute lymphoblastic leukemia (T-ALL), with the intention of overcoming mechanisms of resistance to CD38-targeted therapies such as daratumumab and isatuximab, and minimizing hemagglutination/hemolysis on red blood cell (RBC) observed with anti-CD47 monoclonal antibodies (mAb) such as magrolimab. Methods: ISB 1442 was tested for its capacity in vitro to induce ADCP, ADCC and CDC relative to daratumumab and magrolimab across a broad range of MM, AML and T-ALL cell lines expressing different levels of CD38 and CD47. To assess the complex mechanisms of action of ISB 1442 in a single system, a multiple mode of action of killing (MMoAK) assay was established to allow for simultaneous killing by natural killer cells (ADCC), autologous macrophages (ADCP), and complement from human serum (CDC). In vivo, ISB 1442 was assessed in a therapeutic model of subcutaneously established Raji tumor xenograft in CB17/SCID mice compared to daratumumab and magrolimab. On-target specificity was evaluated in vitro in human and monkey whole blood assays. Results: In vitro, ISB 1442 exhibited higher killing potency compared to daratumumab across a range of CD38-expressing MM, AML and T-ALL tumor cells. Additionally, ISB 1442 showed in vitro tumor killing potency through phagocytosis comparable to magrolimab, acting mostly through ADCP. In the CDC, ADCC and MMoAK assays, ISB 1442 exhibited tumor cell killing that was twice as high as daratumumab in MM cell lines. In vivo, ISB 1442 induced higher tumor growth inhibition than daratumumab and comparable tumor regression to magrolimab. ISB 1442 did not cause any detectable hemolysis, RBC depletion or platelet aggregation and showed markedly lower hemagglutination relative to magrolimab, suggesting a more favorable on-target specificity profile in humans. In monkeys, ISB 1442 showed more pronounced binding on RBC than magrolimab due to higher expression of CD38, suggesting that monkey is a more sensitive species than human for toxicological evaluation of CD38-targeted therapies. Summary/Conclusion: In summary, we report a novel approach for the treatment for CD38 positive hematologic malignancies by co-targeting CD38 and CD47 using a first in class multispecific antibody. Based on its unique design and multiple mechanisms of action, ISB 1442 is anticipated to enhance antitumor activity in patients relative to anti-CD38 mAbs by overcoming primary and acquired tumor escape mechanisms of resistance.