Human mAb Research Articles

Abstract CT104: Bintrafusp alfa in combination with chemotherapy in patients with stage IV NSCLC: safety results of the INTR@PID LUNG 024 study

Abstract Background: Bintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor (a TGF-β “trap”) fused to a human IgG1 mAb blocking PD-L1. The global, phase 1b/2 INTR@PID LUNG 024 (NCT03840915) study evaluates bintrafusp alfa in combination with chemotherapy (CT) in patients with stage IV NSCLC. Here we report cumulative safety, which represents the first report of safety results from a clinical study with a bintrafusp alfa 2400-mg every-three-weeks (Q3W) regimen. Methods: Adults with stage IV nonsquamous or squamous NSCLC and an ECOG PS ≤1 are included. In cohorts A, B, and C, patients must not have received prior systemic therapy; in cohort D, patients must have had progression on previous anti-PD-(L)1 therapy. Patients received bintrafusp alfa 2400 mg Q3W intravenously in combination with CT for 4 cycles (A [nonsquamous only]: cisplatin or carboplatin + pemetrexed; B: carboplatin + nab-paclitaxel or paclitaxel; C: cisplatin or carboplatin + gemcitabine; D: docetaxel) followed by bintrafusp alfa maintenance (monotherapy or combination with pemetrexed in cohort A) for up to 31 cycles. The occurrence of dose-limiting toxicities (DLTs) was assessed during a 3-week observation period. The primary objective of this study was to evaluate the safety of bintrafusp alfa in combination with CT. Additionally, in cohort A, patients from the safety and expansion parts will be assessed for preliminary efficacy. Results: As of October 7, 2020, 64 patients received bintrafusp alfa in combination with CT. Of the 35 patients included in the DLT analysis, 4 experienced 1 DLT according to the safety monitoring committee (A: n=1/8; B: n=1/8; C: n=0/8; D: n=2/11). The cumulative safety is reported (Table). Conclusions: The combination of bintrafusp alfa 2400 Q3W in combination with CT was well tolerated. No new safety signals were identified. Cohort A (n=34)Cohort B (n=9)Cohort C (n=9)Cohort D (n=12)Treatment-emergent adverse events, n (%)Any*34 (100.0)9 (100.0)9 (100.0)12 (100.0)Grade ≥324 (70.6)6 (66.7)7 (77.8)12 (100.0)Grade ≥412 (35.3)3 (33.3)5 (55.6)6 (50.0)Serious22 (64.7)3 (33.3)6 (66.7)9 (75.0)Leading to death3 (8.8)1 (11.1)00Leading to dose reduction of CT9 (26.5)2 (22.2)5 (55.6)4 (33.3)Leading to permanent discontinuation of bintrafusp alfa12 (35.3)04 (44.4)4 (33.3)Leading to permanent discontinuation of CT13 (38.2)1 (11.1)4 (44.4)3 (25.0)Treatment-related adverse events, n (%)Any bintrafusp alfa-related adverse events28 (82.4)9 (100.0)9 (100.0)12 (100.0)Bintrafusp alfa-related serious adverse events9 (26.5)01 (11.1)3 (25.0)Bintrafusp alfa-related adverse events leading to death0000Any chemotherapy-related adverse events31 (91.2)8 (88.9)9 (100.0)12 (100.0)Chemotherapy-related serious adverse events10 (29.4)1 (11.1)4 (44.4)7 (58.3)Chemotherapy-related adverse events leading to death0000Adverse events of special interest, n (%)Skin lesions†2 (5.9)04 (44.4)1 (8.3)Immune-related adverse event15 (44.1)3 (33.3)5 (55.6)6 (50.0)Infusion-related reaction3 (8.8)1 (11.1)1 (11.1)1 (8.3)Anemia16 (47.1)5 (55.6)9 (100.0)9 (75.0)*Most common (≥50%) treatment-emergent adverse events in any cohort included anemia (cohort B: 55.6%; cohort C: 100.0%; cohort D: 75.0%), nausea (cohort A: 52.9%, cohort C: 66.7%), pruritus (cohort C: 66.7%), asthenia (cohort C: 55.6%; cohort D: 58.3%), neutropenia (cohort C: 55.6%), decreased appetite (cohort D: 50.0%), and diarrhea (cohort D: 50.0%).†Defined as actinic keratosis, basal cell carcinoma, Bowen's disease, hyperkeratosis, keratoacanthoma, lip squamous cell carcinoma, and squamous cell carcinoma of skin. Actinic keratosis, hyperkeratosis, and keratoacanthoma were reported in this study. Citation Format: Christian Rolfo, Laurent Greillier, Remi Veillon, Firas Badin, Francois Ghiringhelli, Nicolas Isambert, Astrid Paulus, Marc Lambrechts, Italia Grenga, Xiaoli You, Andre Koenig, Sandrine Hiret. Bintrafusp alfa in combination with chemotherapy in patients with stage IV NSCLC: safety results of the INTR@PID LUNG 024 study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT104.

Production and characterization of a human antisperm monoclonal antibody against CD52g for topical contraception in women.

BackgroundApproximately 40% of human pregnancies are unintended, indicating a need for more acceptable effective contraception methods. New antibody production systems make it possible to manufacture reagent-grade human monoclonal antibodies (mAbs) for clinical use. We used the Nicotiana platform to produce a human antisperm mAb and tested its efficacy for on-demand topical contraception.MethodsHeavy and light chain variable region DNA sequences of a human IgM antisperm antibody derived from an infertile woman were inserted with human IgG1 constant region sequences into an agrobacterium and transfected into Nicotiana benthamiana. The product, an IgG1 mAb [“Human Contraception Antibody” (HCA)], was purified on Protein A columns, and QC was performed using the LabChip GXII Touch protein characterization system and SEC-HPLC. HCA was tested for antigen specificity by immunofluorescence and western blot assays, antisperm activity by sperm agglutination and complement dependent sperm immobilization assays, and safety in a human vaginal tissue (EpiVaginal™) model.FindingsHCA was obtained at concentrations ranging from 0.4 to 4 mg/ml and consisted of > 90% IgG monomers. The mAb specifically reacted with a glycan epitope on CD52g, a glycoprotein produced in the male reproductive tract and found in abundance on sperm. HCA potently agglutinated sperm under a variety of relevant physiological conditions at concentrations ≥ 6.25 µg/ml, and mediated complement-dependent sperm immobilization at concentrations ≥ 1 µg/ml. HCA and its immune complexes did not induce inflammation in EpiVaginal™ tissue.InterpretationHCA, an IgG1 mAb with potent sperm agglutination and immobilization activity and a good safety profile, is a promising candidate for female contraception.FundingThis research was supported by grants R01 HD095630 and P50HD096957 from the National Institutes of Health.

Abstract 1827: Efficacy and characterization of a specific and potent monoclonal antibody, LYT-210, against immunosuppressive gd1 T cell in cancer

Abstract Background: Targeting and engineering γδ T cells has emerged as an orthogonal therapeutic approach in oncology with capacity to modulate both innate and adaptive immunity. Solid tumors (e.g. pancreatic ductal adenocarcinoma (PDAC), melanoma, glioblastoma, ovarian-, breast- cancer etc.), can be enriched for γδ1T cells, where they mediate a robust, multi-faceted immunosuppression. We verified the phenotypic and functional features, and the immunosuppressive potential of cancer patient γδ1T cells and have developed a therapeutic antibody (mAb), LYT-210, for selective depletion of these immune evasion culprits. LYT-210 is highly specific, fully human, species cross-reactive IgG1 antibody, aimed for IND enabling studies and clinical development. Methods: γδ1T cells were profiled and studied from blood of healthy and cancer donors, and from surgically excised primary or metastatic tumors. A proprietary, synthetic Fab-phage library was used to generate LYT-210, a fully human IgG1 mAb. Functional assays (ADCC/ADCP) confirmed an expected and desired, Fcγ-dependent LYT-210 mode of action. We used healthy and patient PBMCs to assess the dynamics, dose dependence and specificity of γδ1 T cell killing with LYT-210. Patient tumor organoids were used for ex vivo efficacy studies. Purified proteins and engineered cell lines served to determine specificity and affinity of LYT-210. Retrogenix protein array (>5500 cell surface and secreted proteins) was used to identify possible off-target binding of LYT-210. Results: Anti-δ1antibody was used to treat patient tumor organoids (n = 29) and showed potent intra-tumoral T cell activation across tumor types (e.g. colorectal and liver cancer). Additionally, LYT-210 exerted potent ADCP and ADCC on: i) stable cell lines expressing γδ1TCRs and ii) γδ1T cells from patient PBMCs and did so by sparing αβ T cells and γδ2 T cells. Furthermore, LYT-210 induced ADCP and ADCC were Fcγ receptor dependent, confirming LYT-210-specific and effector cell dependent mode of action. LYT-210 induced γδ1 T cell-specific death rapidly, i.e. within 30 minutes of treatment with an EC50 of 26 pM and with the effect sustained over 70 hours of culture. Cell surface proteomics microarray analysis showed very weak cross reaction of LYT-210 with CEACAM5 (a known human cancer cell surface antigen), but in confirmatory assays had minimal binding to CEACAM5 expressed onto HEK293 cells (~200 nM) and no binding at all to purified CEACAM5. The Retrogenix array and additional assays therefore confirm the profound specificity of LYT-210 to γδ1T cells. Conclusions: We have developed a novel, therapeutic immuno-oncology strategy specifically targeting imunosuppressive γδ1cells. Our data support that this therapeutic approach may have the potential to be transformative for the treatment of cancers where γδ1T cells drive a pro-tumorigenic, immunosuppressive environment. Citation Format: Tatyana Panchenko, Wei Wang, Eric Denbaum, Akiko Koide, Takamitsu Hattori, Aleksandra Filipovic, George Miller, Shohei Koide. Efficacy and characterization of a specific and potent monoclonal antibody, LYT-210, against immunosuppressive gd1 T cell in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1827.

#31: Children with Invasive S. aureus Infection Produce Broadly Neutralizing Antibodies Against Distantly Related Variants of the Cytotoxin LukAB

Abstract Background Staphylococcus aureus is the most common invasive bacterial pathogen in children, and novel targets of intervention are urgently needed. The two-component leukotoxin, LukAB, is critical for S. aureus targeting and killing of human neutrophils and is abundantly produced in the setting of invasive human infection. LukAB is unique among S. aureus cytotoxins in that it exists in variant form across distantly related strains of clinically relevant S. aureus. The broad diversity of circulating clinical S. aureus strains must be taken into account to successfully develop new anti-staphylococcal preventives or therapeutics. This diversity and ongoing S. aureus evolution may explain previous unsuccessful attempts to intervene against S. aureus at the virulence factor level. We have previously shown that LukAB is ubiquitous among clinical isolates and that children with invasive disease mount a high-titer neutralizing response, but the breadth of function of this response has not previously been explored. Methods B-cells were isolated from children with invasive S. aureus infections (e.g. bacteremia or acute hematogenous osteomyelitis). Following EBV-transformation, cell supernatants were screened for LukAB binding and selected for generation of monoclonal hybridomas. MAbs were assessed for binding by ELISA and neutralizing function by in vitro cytotoxicity assays, where neutrophil-like HL-60 cells were cultured in the presence of human mAbs and diverse allelic variants of LukAB. The infecting S. aureus isolates were typed using multilocus sequence typing (MLST) to determine clonal complexes. Results 34 distinct human anti-LukAB mAbs were generated from 3 children with invasive S. aureus disease. Of these, 22% were isolated following infection with a strain belonging to clonal complex 8 (CC8), consistent with the epidemic USA300 clone, and 78% were generated against CC5 strains. Within this panel, all mAbs potently neutralized LukAB from the same clonal complex as the corresponding infecting isolate, but 23 also demonstrated neutralization against other allelic variants of LukAB. 7 mAbs were capable of broad, potent neutralization against LukAB variants from all clinically relevant clonal complexes tested (CC8, CC30, CC45, CC75, CC1, CC5, and CC398). Conclusions A subset of human mAbs isolated from children with invasive S. aureus disease were capable of broad neutralization against distantly related variants of the important toxin LukAB. This has two important implications: First, we found strong evidence of a conserved target (or targets) for antibody-mediated toxin neutralization across diverse strains of S. aureus. Second, this provides additional support for this toxin as a target of intervention, as some previous vaccine attempts were likely unsuccessful due to activity against a narrow subset of circulating S. aureus strains.

Novel Human Anti-HLA-Bw4 and B61 Monoclonal Antibodies Kill Malignant B Cells Via CDC/ADCC While Sparing Normal Peripheral Blood Cells

Introduction: The development of monoclonal antibodies (mAbs), such as anti-CD20 mAb (rituximab) and anti-CD38 mAb (daratumumab), has revolutionized the treatment of lymphoid malignancies. The potential efficacy of anti-HLA mAbs in treating hematological malignancies has been reported in several studies. Previous attempts to develop mAbs specific for some HLA alleles (e.g., HLA-B61) by immunizing mice with recombinant HLA proteins have failed, likely because of a wide variety of HLA molecule polymorphisms. To overcome this problem, we recently created a novel method to develop mAbs using microarray technology and human peripheral blood (PB) B lymphocytes derived from donors who are positive for anti-HLA antibodies. The process takes approximately 1 month to produce human anti-HLA mAbs to treat myeloma and lymphoma.Methods: Approximately 20 ml of PB was collected from anti-HLA antibody-positive donors, and mononuclear cells were cultured in RPMI-1640 + 10% FCS containing a cytokine cocktail. CD138+ cells were then isolated using anti-CD138-antibody-conjugated microbeads. Microarray chips were coated with a PBS-containing purified HLA antigen (10 μg/ml). After removing the antigen solution, 100 μl of the CD138+ cell suspension was added to the chip. After washing with PBS, 2 μg/ml of anti-human IgG Fc-Cy3 solution was added to the microwells. Finally, antigen-specific antibody-secreting cells (ASCs) from individual microwells were isolated using a micromanipulator fitted with capillaries under the fluorescence microscope and were then expelled to microtubes for reverse transcription. The antibody cDNA was amplified and transfected into HEK293 cells to obtain a supernatant containing complete antibody molecules (Zaimoku et al., Blood 2017). The antigen specificity of the recombinant antibodies was examined using ELISA and flow cytometry. Next-generation sequencing (Adaptive Biotechnologies Corp) was used to quantify the ASC frequency in PB of donors. Complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) of mAbs were assessed using conventional methods. The following cell lines and PB mononuclear cells (PBMCs) from 5 healthy individuals who possessed Bw4 and/or B61 were used for CDC/ADCC assays: 5 B-lymphoblastoid cell lines (B-LCLs), 11 myeloma cell lines (AM01, IM-9, KMS-12BM, KMS-18, KMS-28PE, KMS-34, LP-1, NCI929, OPM-2, RPMI8226, and U266), 4 mantle cell lymphoma cell lines (JVM, Jeko, Mino, and Z138), and 2 diffuse-large B cell lymphoma cell lines (KPUM-UH1 and KPUM-MS3). Daratumumab, elotuzumab, and anti-influenza virus human mAbs were used as control mAbs.Results and Discussion: Two novel human mAbs specific for HLA-Bw4 or HLA-B61 were successfully generated. The ASC frequency that produced anti-Bw4 and B61 mAbs in the cytokine-stimulated PBMCs derived from the donors were 9.0 × 10−5 and 1.4 × 10−5, respectively. Flow cytometry showed that mAbs bound to all normal PB cells and malignant lymphoid cells with corresponding alleles, but the expression levels of the HLA alleles were lower in normal PBMCs than in malignant cells (HLA-Bw4-mean florescence intensity (MFI), 1098 ± 246 vs. 6556 ± 4045, P =0.02). mAbs showed CDC /ADCC activities against all 5 B-LCLs, 6 of 9 myeloma, and 2 of 2 lymphoma cell lines with corresponding alleles but not against 2 myeloma and 4 lymphoma cell lines without corresponding alleles; they showed very weak CDC activities against PBMCs derived from 5 healthy donors who have corresponding alleles (CDC, 39 ± 16 vs. 4 ± 3%, P =0.001). Four myeloma cell lines (IM-9, KMS-28PE, KMS-34, and U266) that had low CD38/high CD59 expression and were resistant to daratumumab were killed by anti-Bw4 and/or HLA-B61 mAbs. In keeping with a current report showing low CDC activities of daratumumab on myeloma cells that highly express CD55/CD59 (Nijhof et al., Blood 2016), some myeloma cell lines highly expressing CD59 were resistant to CDC induced by anti-Bw4/HLA-B61 mAbs despite their high expression levels of HLA alleles (Figure 1AB). Elotuzumab showed obvious CDC and ADCC activities against 0 of 11 and 2 (KMS-34 and U266) of 11 myeloma cell lines, respectively.Conclusions: NovelmAbs specific for HLA-Bw4 or B61 showed in vitro preferential killing of B-cell malignancies while sparing normal PBMCs, suggesting their potential usefulness in treating malignant lymphoma and myeloma. [Display omitted] DisclosuresTanaka:HLA Laboratory: Employment. Kawai:Wakunaga Pharmaceutical Co., Ltd.: Employment.

Humanized anti‑TLR4 monoclonal antibody ameliorates lipopolysaccharide‑related acute kidney injury by inhibiting TLR4/NF‑κB signaling.

A humanized anti-Toll-like receptor 4 (TLR4) monoclonal antibody (mAb) was previously produced using phage antibody library technology, and it was found that the mAb could effectively ameliorate lipopolysaccharide (LPS)-induced damage in macrophages. The present study investigated the protective effects exerted by the humanized anti-TLR4 mAb against LPS-induced acute kidney injury (AKI), as well as the underlying mechanisms. Female C57BL/6 mice were randomly divided into four groups (n=8 per group): i) Control; ii) LPS; iii) LPS + humanized anti-TLR4 mAb (1 µg/g); and iv) LPS + humanized anti-TLR4 mAb (10 µg/g). Serum creatinine, blood urea nitrogen, IL-6, TNFα and IL-1β levels were then examined, followed by renal pathology assessment, immunohistochemical staining, reverse transcription-quantitative PCR and western blotting to assess apoptosis/survival/inflammation-related molecules and kidney injury molecule (KIM)-1. The humanized anti-TLR4 mAb successfully ameliorated LPS-induced AKI and renal pathological damage. The humanized anti-TLR4 mAb also dose-dependently suppressed LPS-induced elevations in serum IL-6, TNFα and IL-1β, and decreased the renal expression levels of myeloid differentiation primary response 88 (MyD88), IKKα/β, IκB, p65 and KIM-1. Compared with the LPS group, renal Bax and KIM-1 expression levels were significantly downregulated, and Bcl-2 expression was notably upregulated by the humanized anti-TLR4 mAb. Moreover, the humanized anti-TLR4 mAb also significantly decreased the protein expression levels of MyD88, phosphorylated (p)-IKKα/β, p-IκB and p-p65 in the renal tissues compared with the LPS group. Therefore, the present study indicated that the anti-inflammatory effects of the humanized anti-TLR4 mAb against LPS-related AKI in mice were mediated via inhibition of the TLR4/NF-κB signaling pathway.

Pharmacokinetics and Pharmacodynamics of Tocilizumab for the Management of Cytokine Release Syndrome (CRS) in Pediatric and Young Adult Patients with Relapsed/Refractory (R/R) B-Cell Acute Lymphoblastic Leukemia (B-ALL) Treated with CAR T-Cell Therapy, CTL019

Background: ELIANA (NCT02435849) is a pivotal multicenter study to test the efficacy of CTL019 (tisagenlecleucel; anti-CD19 CAR-T) in children and young adult patients (pts) with relapsed/refractory B-ALL. Tocilizumab (toci) has been used for the management of moderate/severe (grade 3/4) CRS in ≈ 38% of pts treated with CTL019 (Buechner J et al, EHA 2017) at the same doses used in approved non-oncologic pediatric indications (<30 kg received 12 mg/kg and ≥30 kg received 8 mg/kg [800 mg max dose]). CRS onset generally occurred at a median of 3 days (range 1-22 days) after CTL019 infusion, requiring administration of 1-3 doses of toci in some patients per the Penn CRS treatment algorithm. Toci is a humanized mAb that binds to and blocks IL-6 receptor (IL-6R) signaling. The pharmacokinetics (PK) and pharmacodynamics (PD) of toci in pediatric B-ALL patients with CAR-associated CRS has not previously been described.Objectives: To characterize the PK/PD of toci for the management of CRS following CTL019 infusion in B-ALL in the ELIANA study. To describe the impact of toci on CTL019 cellular kinetics and expansion.Methods: Pharmacokinetics of toci and pharmacodynamics of soluble IL-6R (sIL-6R) concentrations (conc) were determined from serum and quantified using validated assays to provide mechanistic support for the use of toci in the management of CRS in B-ALL. Maximum toci conc (Cmax) was derived using non-compartmental methods and compared with levels in approved indications. sIL-6R, proinflammatory cytokines and time to CRS resolution were characterized to describe the pharmacodynamic effects of toci. Summary statistics and graphical analyses of CTL019 exposure by number of toci doses were performed for patients that responded to CTL019 infusion to describe the impact of toci on CTL019 expansion and persistence.Results: A total of 28 out of 58 pts with CRS received their first dose of toci at a median of 5 days (range 1-18) after CRS onset. 17 pts received 1 IV dose of toci; 8 pts 2 doses; 3 pts 3 doses. First dose of toci ranged from 6.9 to 12 mg/kg; second dose of toci ranged from 8 to 12 mg/kg. Following the first and second dose of toci, the mean Cmax (SD) were ≈ 111 (30.6) µg/mL and 265 (376) µg/mL, respectively (Table 1). Individual patient PK/PD concentration-time profiles in B-ALL, show increased sIL-6R after the first toci dose that remained elevated following the second dose (Figure 2). The median time to CRS resolution (including fever resolution) was 5 days (range, 2-29 days) after toci administration. CRS onset coincided with CTL019 expansion, and was followed by a peak in serum cytokines, including IL-6. Maximal expansion of the CTL019 transgene (determined by qPCR) was 159% higher in patients treated with toci (n=14) compared with patients that did not receive toci (n=28) demonstrating tocilizumab does not negatively impact expansion (Figure 3).Conclusions: Toci administration resulted in resolution of CRS symptoms within a median of 5 days after administration. Notably, levels of toci achieved in B-ALL were similar to levels published in pediatric non-oncologic indications [tocilizumab label], resulting in concentration- and time-dependent increases in sIL-6R. CTL019 transgene expanded and persisted following tocilizumab administration. Together these data support the use of toci for the management of CRS, showing the compound to be 1) pharmacologically active, 2) able to achieve levels consistent with those achieved in other disorders in spite of CRS and 3) able to be given without impairing CTL019 expansion. [Display omitted] DisclosuresLee:Novartis Pharmaceuticals Corporation: Other: Post-Doctoral Fellow; The State University of New Jersey: Other: Post doctoral fellow. Bittencourt:Novartis Pharmaceuticals Corporation: Consultancy; Amgen Inc.: Consultancy; Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Travel Grant. Rives:Novartis Pharmaceuticals Corporation: Consultancy; Jazz Pharmaceutical: Other: Travel expenses; Shire: Consultancy. Boyer:Novartis Pharmaceuticals Corporation: Honoraria. Pulsipher:Adaptive: Other: Advisory board 6/17; CSL Behring: Other: advisory board Feb 2017; Jazz Pharmaceuticals: Other: advisory board, education; Chimerix: Other: Advisory board Dec 2015; Novartis Pharmaceuticals Corporation: Consultancy, Other: Phase II steering committee. Jaitner:Novartis Pharma AG: Employment. Yi:Novartis Pharmaceuticals Corporation: Employment. Lund:Novartis Pharmaceuticals Corporation: Employment. Leung:Novartis Pharmaceuticals Corporation: Employment. Awasthi:Novartis Pharmaceuticals Corporation: Employment. Wood:Novartis Pharmaceuticals Corporation: Employment, Equity Ownership. Maude:Novartis Pharmaceuticals: Consultancy, Other: Medical Advisory Boards. Grupp:Novartis Pharmaceuticals Corporation: Consultancy, Other: grant; Adaptimmune: Consultancy; Jazz Pharmaceuticals: Consultancy; University of Pennsylvania: Patents & Royalties. Mueller:Novartis Pharmaceuticals Corporation: Employment.

A Prospective Phase II of Daratumumab in Previously-Treated Systemic Light-Chain (AL) Amyloidosis

Background: Daratumumab (DARA) is a novel, high-affinity, therapeutic, IgG1ĸ human mAb that specifically recognizes CD38. It has emerged as a breakthrough targeted therapy for patients with multiple myeloma. Plasma cells in AL amyloidosis are similar to plasma cells in myeloma and always express CD38 (Matsuda, M. et al., Amyloid, 2003. 10(2): p. 110-6.). We report here the preliminary results of a prospective multi-center, phase II study of DARA in AL amyloidosis (NCT02816476).Methods:Forty patients will be recruited in this trial. Patients aged ≥18 years with evaluable AL amyloidosis, who have received ≥1 prior therapy and are not in very good partial response (VGPR) or better with a measurable plasma cell dyscrasia with dFLC > 50 mg/L (difference between involved and uninvolved free light chain levels), with at least one major vital organ involvement, with ECOG performance status 0,1 or 2, no chronic atrial fibrillation, a supine blood pressure > 100 mmHg and NT-proBNP < 8500 ng/L are eligible. They receive DARA intravenously in a standard schedule and dose: 16 mg/kg weekly during the first two 28-day cycles and every other week during cycles 3 through 6 for a total of six 28-day cycles. Hematologic responses are measured after 1 injection of DARA and at each cycle. The objectives are to assess hematologic responses, organ responses and safety.Results:At data cut-off (July 31, 2017), 30 of the 40 planned patients have been enrolled in 9 French centers. The median age is 69 years (range, 45-81), and median number of prior therapies is 2.5 (range, 1-5): 13 patients (43%) have received Melphalan and Dexamethasone, 28 patients (93%) bortezomib, and 14 patients (46%) lenalidomide. The median time from diagnosis to enrollment is 23 months (range, 3.5-116). The median number of organ system involvement is 2 (range, 1-5), 8 patients (27%) have >2 organ system involvement, 18 patients (60%) have cardiac and 16 patients (53%) renal involvement, 14 patients (47%) have cardiac biomarker stage II and 4 patients (13%) stage III disease. There was one on-study death due to cardiac progression. Four patients have discontinued study treatment before 6 cycles due to disease progression (n=2), death (n=1) or lung cancer (n=1). Nine patients have received 6 cycles and 17 patients are on therapy. Six patients (20 %) experienced at least one grade ≥3 AE (any cause) and only one was considered as drug related (lymphopenia). The most common drug-related AEs were infusion reaction in 10 patients (33%), all grade I or II. Hematologic complete response (CR) was observed in 4 of 24 evaluable (completing at least 1 cycle) patients (17%), VGPR in 7 patients (29%) and partial response in 4 patients (17%). The overall response rate is 63%. The responses were usually very rapid (Figure 1). After a single DARA injection all 15 responding patients had a decrease in dFLC of more than 30 % with a median dFLC decrease after 1 injection in these 15 responding patients of 57% (range 31-96).Conclusion: Daratumumab demonstrates encouraging efficacy in previously-treated patients with AL amyloidosis with deep and rapid responses. The administration of DARA in these patients is associated with a good safety profile and non-severe adverse events mostly after the first infusion. Further studies on DARA-based associations in AL amyloidosis patients are warranted. The data will be updated at the meeting. [Display omitted] DisclosuresRoussel:JANSSEN: Honoraria, Research Funding. Perrot:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Sanofi: Honoraria; Amgen: Honoraria. Karlin:Janssen: Honoraria, Other: Travel expenses. Macro:JANSSEN: Honoraria. Frenzel:Shire: Research Funding. Palladini:Jannsen-Cilag: Membership on an entity's Board of Directors or advisory committees; Celgene: Other: Travel grants; Prothena: Honoraria, Other: Travel grant. Jaccard:Celgene: Honoraria, Other: Travel expenses, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses, Research Funding; Amgen: Honoraria.

Wnt5a Induces ROR1 to Complex with DOCK2 and Promote Proliferation of Chronic Lymphocytic Leukemia Cells

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic protein expressed on chronic lymphocytic leukemia (CLL) B cells, but not on virtually all normal adult tissues. Studies have found that high-level leukemia-cell expression of ROR1 associated with enhanced leukemia-cell proliferation, aggressive disease, and shorter overall survival compared to that of patients with CLL cells that had low-to-negligible expression of ROR1 (Cui B. et al., Blood, 128:2931, 2016). Analysis of ROR1-immune precipitates of CLL-cell lysates via mass spectrometry and immunoblot analyses revealed that ROR1 was associated with DOCK2 (Dedicator of Cytokinesis 2) in freshly isolated CLL cells. DOCK2 is a member of the DOCK-A subfamily of guanine exchange factors (GEFs) specific for Rac1 and Rac2 (Rac1/2) that is expressed primarily in leukocytes, including CLL B cells. DOCK proteins differ from other GEFs in that they do not possess the canonical tandem Dbl-homology (DH) and Pleckstrin-homology (PH) domains that elicit nucleotide exchange. Instead DOCK2 possess a DOCK homology region 2 (DHR2) domain, which mediates Rac activation by stabilizing Rac in its nucleotide-free state. DOCK2 also contains a N-terminal SH3 domain. We found that ROR1 dissociated from DOCK2 in CLL cells cultured in serum-free medium, unless the cells were stimulated with exogenous Wnt5a, which we found present at high-levels in the plasma of CLL patients relative to that of age-matched healthy adults. Wnt5a could induce ROR1 to complex with DOCK2 and cause activation of Rac1/2; this effect could be inhibited by silencing DOCK2 in primary CLL cells. Furthermore, these effects of Wnt5a on CLL cells also could be completely blocked by treatment of the CLL cells with cirmtuzumab, a humanized anti-ROR1 mAb, which is undergoing clinical evaluation in patients with CLL. We find that silencing DOCK2 specifically impaired the capacity of Wnt5a to enhance CXCL12-directed migration or CLL-cell proliferation in vitro. We corroborated these findings using the CLL-cell-line MEC1, which also expresses DOCK2, but does not express ROR1. MEC1 cells can be transduced to express ROR1 or various mutant forms of ROR1, allowing us to examine the structure-function relationships required for ROR1-DOCK2 interactions. We confirmed that DOCK2 complexes with ROR1 in response to Wnt5a in MEC1-ROR1 cells. We generated truncated forms of ROR1 and found the cytoplastmic proline-rich domain (PRD) of ROR1 was required for ROR1 to complex with DOCK2 and activate Rac1/2 upon stimulation with Wnt5a. We next introduced single amino-acid substitutions of proline (P) to alanine (A) in the ROR1-PRD at positions 784, 808, 826, or 841 in potential SH3-binding sites. In contrast to wild-type ROR1, or other ROR1P==>A mutants, ROR1P(808)A had impaired capacity to recruit DOCK2 to ROR1 in response to Wnt5a. Moreover, unlike MEC1 cells transfected with wild-type ROR1 or ROR1 with P->A substitutions at positions 784, 826, or 841, MEC1 cells transfected to express ROR1P(808)A did not have a growth advantage over that of MEC1 cells that do not express ROR1. This study reveals that the recruitment of DOCK2 may be critical for the capacity of Wnt5a to enhance CLL proliferation, which may contribute to the observed increased tendency for disease progression in CLL patients who have leukemia cells that express high-levels of ROR1. DisclosuresKipps:Oncternal: Research Funding; Gilead: Consultancy, Honoraria, Speakers Bureau; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria.

Alemtuzumab for Treatment-Naïve and Relapsed-Refractory Severe Acquired Aplastic Anemia

Introduction: Antithymocyte globulin (ATG) + cyclosporine is effective in restoring hematopoiesis in severe aplastic anemia (SAA). Horse ATG appears to be better than rabbit ATG. Limitations to ATG use in developing countries include cost, horse ATG availability and the reduced number of hospital beds in hematology treatment centers are frequent. Alemtuzumab, a humanized anti-CD52 mAb, might be active in SAA because of its lymphocytotoxic properties. Subcutaneous alemtuzumab have about ORR 66% and is available in Brazil through a compassionate use program. Methods: Fourteen consecutive transplant-ineligible SAA patients were treated. Alemtuzumab was administered subcutaneously in a single at escalating dose of 3-10-30-(30)-(30) mg. Premedication included intravenous steroid, antihistamine and oral acetaminophen. The total dose was 103 mg for 14 patients, and 43 mg for one elderly frail patient. Low dose (1 mg/kg) oral cyclosporine was started on day 7, and then adjusted according to plasma levels (targeting 100-200 ng/ml using a monoclonal assay) at the time of immune reconstitution. Anti-infectious prophylaxis included oral acyclovir and trimethoprim-sulphametoxazole, weekly monitoring of CMV viraemia, and oral quinolone and fluconazole in case of severe neutropenia (<0.5x 109/L). Results: Fifteen SAA patients were treated. Primary outcome was hematologic response at 3months. Median age was 48 years, and 9 (60%) were male. Eight patients were treatment-naive, and 7 (46%) were relapsed-refractory. Eight patients presented PNH clone at the initial investigation, with a size clone between 0.001 to 55 in granulocytes and 0.001 to 98 in monocytes. Median time between SAA diagnosis and alemtuzumab treatment was 9 months (range 1-300). No treatment-related serious adverse events due were observed. Infectious complications were rare. The overall response rate was 66% (60% with PR, 40% CR). The median follow-up is about 29 months (range 5-69). The 3-year overall survival was 73%. Conclusion: The combination of subcutaneous alemtuzumab + cyclosporine appears to be effective and sufficiently safe in transplant-ineligible SAA patients. Moreover, it is feasible in a scenario of limited resources and low accessibility to ATG. DisclosuresNo relevant conflicts of interest to declare.

Platelets from Donors with Consistent Low HLA-B8, B12 or B35 Expression Do Not Undergo Antibody-Mediated Internalization

Introduction Patients refractory to platelet (PLT) transfusions due to HLA specific alloantibodies require HLA-matched PLTs. However, even with large HLA-typed donor pools patients with rare HLA haplotypes may not have suitable donors. In order to improve the efficiency of HLA-matching, PLTs with low HLA class I expression may be an alternative for fully HLA matched transfusions. This study established the proportion of donors with consistent low expression of HLA-B8, B12, and B35 on PLTs and studied the effect of this low HLA expression on antibody-mediated internalization of PLTs by macrophages.Methods HLA-B8, B12, or B35 expression on PLTs of HLA typed blood donors was determined using AlexaFluor488 labeled human monoclonal antibodies (mAbs) specifically targeted to either one of these antigens. Donors were tested several times for their specific HLA class I expression. HLA typed PLTs with low (<2SDs above background) or high (>median expression) expression were opsonized with saturating concentrations of human IgG1 mAbs targeting either HLA-B8, B12 or B35. In addition, PLTs were opsonized with increasing concentrations of anti-HPA1a humanized IgG1 or anti-pan HLA class I mouse IgG2a. Subsequently, these opsonized PLTs were incubated with macrophages and antibody-mediated internalization was determined using imaging flow cytometry.Results The expression of HLA-B8 (n=113), B12 (n=98) or B35 (n=66) on PLTs was extremely variable between individuals (coefficients of variation: 47.6, 73.6% and 41.4, respectively). For HLA-B8, but not for B12 or B35, this variation was in part explained by hetero- and homozygosity. The observed variations were most pronounced in, but not exclusive to PLTs. In contrast, expression within one donor was consistent over time. Remarkably, 32% of HLA-B8, 34% of HLA-B12 and 9% of HLA-B35 donors showed PLT antigen expression that was not or only minimally (<2SDs) above background. Opsonization of PLTs with low HLA expression did not affect internalization, whereas PLTs with high HLA expression did show significant antibody-mediated internalization. Furthermore, this internalization of PLTs by macrophages correlated with the level of antibody opsonization and PLT antigen expression.Discussion Our findings show high inter-individual variation for HLA-B8, B12 and B35 expression on PLTs, with 9 to 34% of donors showing reproducibly undetectable or low expression. Because of this consistency, donors can be marked as ‘low expressors’ in the HLA-typed donor file which might greatly facilitate HLA-matching efficiency. In line with this hypothesis, antibody-mediated internalization showed a strong correlation with mAb opsonization and PLT antigen expression and was not observed in PLTs with low antigen expression. As in vitro antibody-mediated internalization has been correlated to in vivo clearance, this suggest that PLTs from ‘low expressors’ may be used to treat refractory patients despite HLA mismatches. DisclosuresNo relevant conflicts of interest to declare.

Wnt5a Can Induce Activation of Rac1 and Proliferation in Mantle Cell Lymphoma Via a ROR1-Dependent, but BTK-Independent, Signaling Pathway

Signaling via the B cell receptor (BCR) plays an important role in the pathogenesis and progression of chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL). This is underscored by the clinical effectiveness of an inhibitor of Bruton's tyrosine kinase (BTK), ibrutinib, which can block BCR-signaling. However, ibrutinib cannot induce complete responses (CR) or durable remissions without continued therapy, particularly in patients with MCL. This suggests that ancillary pathways contribute to the growth/survival of MCL/CLL that are independent of BCR-signaling and/or inhibition of BTK. Studies have found that Wnt5a can promote activation of Rho GTPase Rac1 to enhance CLL-cell proliferation and survival via a ROR1-dependent,but BTK-independent pathway (Yu J, et al., Leukemia 31:1333, 2017). Furthermore, Wnt5a-induced Rac1 activation could be blocked by cirmtuzumab, a humanized anti-ROR1 mAb, which significantly could enhance the capacity of ibrutinib to clear CLL cells in vivo . MCL cells also express ROR1. Moreover, we find that the median level of ROR1 on primary MCL cells is higher than that on CLL cells; the median absolute mean fluorescence intensity of ROR1 (ROR1 DMFI) on MCL (87.5 ± 18.5 (S.D.), N = 8) was significantly higher than the median ROR1 DMFI on CLL cells (38.5 ± 18.5 (S.D.), N = 1567; (Cui B, et al., Blood 128:2931, 2016)). We also found that the plasma of patients with MCL had high levels of Wnt5a that were comparable to those found in patients with CLL; in contrast Wnt5a was virtually undetectable in the plasma of age-matched healthy adults. We cultured primary MCL cells of different patients with ibrutinib, cirmtuzumab, or both ibrutinib and cirmtuzumab for 2 h, and then stimulated the cells with exogenous Wnt5a for 30 min. In parallel, the same MCL cells were cultured without exogenous Wnt5a. We found that Wnt5a induced Rac1 activation in the primary MCL cells. Cirmtuzumab, but not ibrutinib, could inhibit the capacity of Wnt5a to induce primary MCL cells to activate Rac1. We induced proliferation of MCL cells by co-culturing MCL cells with HeLa cells expressing CD154 (HeLaCD154) and recombinant interleukin (IL)-4 and IL-10. Addition of exogenous Wnt5a to co-cultures of MCL cells with HeLaCD154 cells and IL-4/10 significantly enhanced the proportion of dividing MCL entering S/G2 and the numbers of cell divisions over time. Treatment of the MCL cells with cirmtuzumab, but not ibrutinib, blocked Wnt5a-enhanced survival and proliferation of MCL cells from each of 3 different patients. This study demonstrates that Wnt5a can induce Rac1 activation, which leads to enhanced proliferation and survival of primary MCL cells via a ROR1-dependent signaling pathway that can be blocked by cirmtuzumab. On the other hand, this signaling pathway cannot be blocked by ibrutinib, even at concentrations that completely inhibit BTK or BCR-signaling. This implies that cirmtuzumab and ibrutinib may have additive, if not synergistic, activity in the treatment of patients with MCL. DisclosuresKipps:Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Speakers Bureau; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Oncternal: Research Funding.

Wnt5a Induces Interleukin-6 Via a ROR1-Dependent Pathway in Chronic Lymphocytic Leukemia, Leading to Leukemia-Cell Activation of STAT3

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic cell surface protein, which is expressed on the neoplastic B cells of patients with chronic lymphocytic leukemia (CLL), but not on virtually all normal post-partem tissues. ROR1 functions as a receptor for Wnt5a, which induces activation of Rho-GTPases, AKT, and HS-1 and promotes migration, proliferation, and survival of CLL B cells. We observed that freshly isolated CLL cells also had high levels of pSTAT3 (Y705), which attenuated over time in culture in serum-free media unless the CLL cells were stimulated with exogenous Wnt5a. This effect of Wnt5a could be blocked by cirmtuzumab, a humanized anti-ROR1 mAb that currently is undergoing clinical evaluation in patients with CLL. We examined the capacity of recombinant Wnt5a (rWnt5a) to induce activation of STAT3 in isolated CLL cells by performing a time-course study, evaluating for pSTAT3 in serum-starved CLL cells at time 0, 5', 15', 30' and 1, 2, 3, 4, and 24 hours after stimulation with exogenous Wnt5a. Surprisingly, activation of STAT3 in CLL cells was delayed, appearing first at 3 hours after addition of rWnt5a, and peaking at 24 hours, the last time point examined. In contrast, activation of CLL cells via treatment of CLL cells with interleukin (IL)-6, induced activation of STAT3 within 15' of the addition of this cytokine to the cultures. We hypothesized that the activation of STAT3 in CLL cells by Wnt5a was indirect, being caused by a factor(s) made by isolated CLL cells in response to stimulation with Wnt5a. To test this hypothesis, we examined CLL cells, and harvested supernatants of cultured CLL cells, at various times after stimulation with rWnt5a. RNA isolated from CLL cells was examined via real-time PCR for cytokine transcripts. This revealed a prominent, time-dependent increase in transcripts encoding IL-6 following stimulation of CLL cells with rWnt5a. Consistent with this observation we found Wnt5a induced CLL cells to secrete IL-6, which was detected via ELISA at increasing concentrations over time in culture-supernatants of CLL cells treated with rWnt5a, but not in the culture supernatants of unstimulated CLL cells. Culture supernatants collected from CLL cells stimulated for 24 hours with rWnt5a could induce activation of STAT3 within 15' in serum-starved CLL cells; this effect could be inhibited significantly by the anti-IL-6-receptor mAb, tocilizumab, but not by cirmtuzumab, even when used at concentrations that could block the capacity of concomitantly-added Wnt5a to induce latent STAT3 activation at 24 hours, suggesting that cirmtuzumab could inhibit Wnt5a-induced CLL-cell production of cytokines, such as IL-6. We examined for IL-6 in cultures of CLL cells treated for 24 hours with rWnt5a and cirmtuzumab, or rWnt5a and an antibody of irrelevant specificity. We found the concentration of IL-6 in the supernatants of CLL cells treated with rWnt5a and cirmtuzumab (110.4 ± 18.9 pg/ml) was significantly lower than that found in the supernatants of CLL cells treated with rWnt5a and control antibody (416.2 ± 53.7 pg/ml, p = 0.0007, Students t test). This study reveals that Wnt5a can induce CLL to secrete cytokines, such as IL-6, via a ROR1-dependent pathway, leading to latent, autocrine activation of STAT3. DisclosuresKipps:Gilead: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding; Oncternal: Research Funding; Roche: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria.

Nurse-like Cells Express High-Levels of Wnt5a, Which Induces ROR1-Dependent Signaling That Promotes Migration and Survival of Neoplastic Cells in Chronic Lymphocytic Leukemia

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic antigen expressed on chronic lymphocytic leukemia (CLL) cells, but not on normal postpartum tissues. The ligand that binds to ROR1 is Wnt5a, which we find present at high levels in the plasma of patients with CLL relative to that of age-matched healthy adults. Wnt5a can induce CLL-cell activation of Rho-GTPases in a ROR1-dependent manner, thereby enhancing leukemia-cell proliferation, migration, and survival. Currently, the cell-source(s) of Wnt5a in CLL is not known. Good candidates include Nurse-Like cells (NLCs), which are monocyte-derived accessory cells that develop in the context of CLL cells and that are found within the leukemia-cell microenvironment in lymphoid tissues. Already NLCs have been shown to elaborate factors that can promote leukemia-cell activation/survival, including chemokines (e.g. CXCL12, CXCL13) and members of the tumor-necrosis-factor family (e.g. BAFF and APRIL). Because activated monocytes can produce Wnt5a, we hypothesize that NLC also may produce this non-canonical Wnt factor. For this study, we generated NLCs from blood mononuclear cells of CLL patients through co-culture with leukemia B cells using established methods. NLCs, which stained positive for CD163 and CD68, were isolated free of leukemia cells via adherence. We isolated RNA from purified CLL cells or purified NLC of the same patient for real-time PCR to measure the levels of WNT5A transcripts. WNT5A transcripts were abundant in the RNA isolated from NLC, but were negligible in RNA isolated from CLL cells each patient tested (N = 5). We evaluated for Wnt5a via ELISA in the culture-medium of isolated NLCs of five different patients. The cultured medium of NLCs from each patient had detectable Wnt5a that increased in concentration over time. Furthermore, ultra-high resolution confocal fluorescent microscopy of NLC-CLL co-cultures showed Wnt5a was expressed at high-levels in NLCs, but was undetectable in CLL cells of each patient tested (N = 3). We performed co-culture studies and trans-well assays to assess the survival and migration of CLL cells when cultured with or without NLCs, and with or without a neutralizing mAb specific for Wnt5a or cirmtuzumab, which is a humanized anti-ROR1 mAb that blocks Wnt5a-induced, ROR1-dependent signaling. We found that anti-Wnt5a or cirmtuzumab, but not a mAb of irrelevant specificity, each could comparably and significantly inhibit the protective effects of NLCs for CLL cells in vitro (p= 0.008, N = 3 and p= 0.0032, N = 3, respectively by paired student's t-test). Moreover, anti-Wnt5a or cirmtuzumab each could comparably and significantly inhibit the migration of CLL cells enhanced by co-culture with NLCs in vitro (p < 0.0001, N = 3, by paired student's t-test). We also examined for Rho-GTPase activation in CLL cells co-cultured with NLCs. CLL cells co-cultured with NLCs, but not CLL cells cultured alone, had high-level activation of Rac1 and RhoA. The activation of RhoA and Rac1 in co-cultured CLL cells could be blocked by neutralizing anti-Wnt5a or cirmtuzumab, but not by control mAb of irrelevant specificity. We conclude that NLCs express Wnt5a, which can enhance CLL-cell migration and survival via a ROR1-dependent pathway. We speculate that the high-level Wnt5a noted in the plasma of patients with CLL is produced primarily by NLCs, which reside in lymphoid tissues. If so, then the high-level Wnt5a present in the plasma of CLL patients may extend the protective effects of NLCs beyond the CLL microenvironment in which NLCs reside. Treatment with cirmtuzumab to block ROR1-dependent Wnt5a-signaling may mitigate such effects, potentially enhancing the clearance of leukemia cells when used alone or in combination with drugs that target other survival-signaling pathways in CLL. DisclosuresKipps:AbbVie: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Speakers Bureau; Oncternal: Research Funding; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

HMBD004, a Novel Anti-CD47xCD33 Bispecific Antibody Displays Potent Anti-Tumor Effects in Pre-Clinical Models of AML

CD47 is the “don't-eat-me” signal and is ubiquitously expressed on normal cells where binding to SIRPalpha on macrophages inhibits phagocytosis. CD47 is commonly over-expressed in tumors where it correlates with immune evasion and poor prognosis. Blocking CD47-SIRPalpha interaction restores macrophage phagocytosis of tumor cells and anti-CD47 mAbs have shown anti-tumor efficacy in mouse models of solid tumors and hematological malignancies. Nevertheless, the widespread expression of CD47 decreases the bioavailability of anti-CD47 mAbs at the tumor due to antigen sink effects, and also risks off-target toxicities, most significantly from cross-linking of erythrocytes that show high expression of CD47. Therefore, increasing the specificity for binding CD47, and inhibiting the interaction with SIRPalpha, to tumor cells alone should increase the efficacy and safety of anti-CD47 therapy.CD33 is expressed on myeloid cells, but is highly over-expressed in 90% of AML patients, where it marks leukemic blast cells. The safety of CD33 as an antibody target for directing effector molecules to tumor cells has been convincingly demonstrated by the efficacy of gemtuzumab ozogamicin (Mylotarg), an antibody-drug conjugate (ADC) that binds CD33 with high affinity, in the treatment of relapsed AML. However, ADCs are notable for their significant risk of toxicities, and gemtuzumab ozogmicin was withdrawn for this reason in 2010. Still, other companies continue development on next generation anti-CD33 ADCs, e.g. vadastuximab talirine.Hummingbird Bioscience has used its proprietary Rational Antibody Development Platform for the design and development of novel therapeutic antibodies against CD47. Computational sequence and structural analyses predicted a highly specific surface epitope, with strong antigenicity and good safety profile, where antibody binding would inhibit binding to SIRPalpha. Subsequently, an in-house mouse immunization and screening strategy efficiently yielded a monoclonal antibody (mAb, 11a1) that bound with low nM (KD 5 nM) affinity and high specificity to the desired epitope on human CD47. In vitro, 11a1 demonstrated 100% inhibition of SIRPalpha binding at 1 nM, and potently induced phagocytosis of Raji cells by isolated macrophages.11a1 was subsequently humanized by CDR grafting and back-mutation, and the resulting humanized mAb maintained high affinity binding to CD47. Hummingbird Bioscience then constructed a bispecific anti-CD47xCD33 antibody (bsAb), based on a standard 1+1 IgG format, with a humanized anti-CD47 variable domain as an effector arm and the gemtuzumab variable domain as an anti-CD33 specificity arm. This bsAb (HMBD004) maintained the potent inhibition of SIRPalpha binding and induction of phagocytosis but, unlike many CD47 mAbs, caused negligible hemagglutination (i.e. cross-linking) of erythrocytes in vitro . Furthermore, HMBD004 showed preferential sorting of CD47+ CD33+ cells during FACS of mixed populations of CD47+ cells. Finally, in vivo CDX mouse models of AML were established by engrafting NCr nude mice with the CD47+ CD33+ AML cell line, HL-60, via subcutaneous implantation. Treatment of these mice with HMBD004 showed a significant decrease in tumor burden and increased PFS.Together, these data highlight the benefit of using CD33 binding to enhance the specificity, efficacy and safety of anti-CD47 antibody therapy in AML. Hummingbird Bioscience is currently advancing HMBD004 towards the clinic in 2018. DisclosuresNo relevant conflicts of interest to declare.

Co-Stimulatory versus Cell Death Aspects of Agonistic CD40 Monoclonal Antibody Selicrelumab in Chronic Lymphocytic Leukemia.

Simple SummaryPrevious observations have shown that CD40 activation of CLL cells via coculture with CD40L-expressing fibroblasts increases sensitivity to cell death by CD20 mAbs rituximab and obinutuzumab. We studied the activity of the fully human-agonistic CD40 mAb selicrelumab in primary CLL cells in relation to cell activation, induced pro-survival profile and sensitization for cell death by aCD20 mAbs. We found that the pro-survival effect of selicrelumab is minimal, while cell death by combined selicrelumab plus anti-CD20 antibody treatment is maintained. Thus, further investigation of applying selicrelumab combined with anti-CD20 mAbs in a therapeutic setting might be considered.Objectives: Chronic lymphocytic leukemia (CLL) is a common form of leukemia with a heterogeneous clinical course that remains incurable due to the development of therapy resistance. In lymph node proliferation centers, signals from the microenvironment such as CD40 ligation through interaction with follicular T helper cells shield CLL cells from apoptosis. Previous observations have shown that, despite CD40-induced changes in apoptotic mediators resulting in cell survival, CD40 activation also increases sensitivity to cell death by CD20 mAbs rituximab and obinutuzumab. To further investigate these observations, we here studied the activity of the fully human agonistic CD40 mAb selicrelumab in primary CLL cells in relation to cell activation, induced pro-survival profile, and sensitization for cell death by aCD20 mAbs, in vitro. Methods: CLL cells from peripheral blood were isolated by the Ficoll density method. The expression of activation markers and cytokine production following CD40 stimulation was quantified by flow cytometry and ELISA. The anti-apoptotic profile of CLL induced by stimulation was evaluated by the expression of BCL-2 proteins with Western blot, and resistance to venetoclax with flow cytometry. Cell death induced by the combination of selicrelumab and aCD20 mAbs was quantified by flow cytometry. Results: CLL cells treated with selicrelumab upregulated co-stimulatory molecules such as CD86, TNF-α and death receptor CD95/Fas. In contrast to the CD40 ligand-transfected NIH3T3 cells, induction of resistance to venetoclax by selicrelumab was very moderate. Importantly, selicrelumab stimulation positively sensitized CLL cells to CD20-induced cell death, comparable to CD40 ligand-transfected NIH3T3 cells. Conclusions: Taken together, these novel insights into selicrelumab-stimulatory effects in CLL may be considered for developing new therapeutic strategies, particularly in combination with obinutuzumab.

Blocking Borrelia burgdorferi transmission from infected ticks to nonhuman primates with a human monoclonal antibody.

Disrupting transmission of Borrelia burgdorferi sensu lato complex (B. burgdorferi) from infected ticks to humans is one strategy to prevent the significant morbidity from Lyme disease. We have previously shown that an anti-OspA human mAb, 2217, prevents transmission of B. burgdorferi from infected ticks in animal models. Maintenance of a protective plasma concentration of a human mAb for tick season presents a significant challenge for a preexposure prophylaxis strategy. Here, we describe the optimization of mAb 2217 by amino acid substitutions (2217LS: M428L and N434S) in the Fc domain. The LS mutation led to a 2-fold increase in half-life in cynomolgus monkeys. In a rhesus macaque model, 2217LS protected animals from tick transmission of spirochetes at a dose of 3 mg/kg. Crystallographic analysis of Fab in complex with OspA revealed that 2217 bound an epitope that was highly conserved among the B. burgdorferi, B. garinii, and B. afzelii species. Unlike most vaccines that may require boosters to achieve protection, our work supports the development of 2217LS as an effective preexposure prophylaxis in Lyme-endemic regions, with a single dose at the beginning of tick season offering immediate protection that remains for the duration of exposure risk.

Novel human neutralizing mAbs specific for Spike-RBD of SARS-CoV-2

Among the therapies against the pandemic SARS-CoV-2 virus, monoclonal Antibodies (mAbs) targeting the Spike glycoprotein represent good candidates to interfere in the Spike/ACE2 interaction, preventing virus cell entry. Since anti-spike mAbs, used individually, might be unable to block the virus entry in the case of resistant mutations, we designed an innovative strategy for the isolation of multiple novel human scFvs specific for the binding domain (RBD) of Spike. By panning a large phage display antibody library on immobilized RBD, we obtained specific binders by eluting with ACE2 in order to identify those scFvs recognizing the epitope of Spike interacting with its receptor. We converted the novel scFvs into full size IgG4, differently from the previously isolated IgG1 mAbs, to avoid unwanted potential side effects of IgG1 potent effector functions on immune system. The novel antibodies specifically bind to RBD in a nanomolar range and interfere in the interaction of Spike with ACE2 receptor, either used as purified protein or when expressed on cells in its native conformation. Furthermore, some of them have neutralizing activity for virus infection in cell cultures by using two different SARS-CoV-2 isolates including the highly contagious VOC 202012/01 variant and could become useful therapeutic tools to fight against the SARS-CoV-2 virus.

AdvanTIG-302: Anti-TIGIT monoclonal antibody (mAb) ociperlimab (OCI) plus tislelizumab (TIS) versus pembrolizumab (PEM) in programmed death ligand-1 (PD-L1) selected, previously untreated, locally advanced, unresectable or metastatic non-small cell lung cancer (NSCLC).

TPS9128 Background: Monotherapy with programmed death 1 (PD-1)/PD-L1 antibodies has improved clinical outcomes for patients (pts) with non-oncogenic driven NSCLC but clinical responses are limited by primary and secondary resistance, and improvements in durability of response are required. T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domain (TIGIT) is a co-inhibitory, immune checkpoint receptor upregulated on T-cells and natural killer cells in multiple solid tumors, which can inhibit anticancer immune responses. OCI (BGB-A1217) is a novel, humanized mAb that binds to TIGIT with high affinity and specificity, which has demonstrated competent binding with C1q and all Fcγ receptors while inducing antibody-dependent cellular cytotoxicity. Preclinical and clinical studies suggest that dual targeting with anti-TIGIT and anti-PD-1 antibodies produces synergistic immune cell activation and enhanced antitumor activity. Methods: AdvanTIG-302 is a Phase 3, multicenter, international, randomized, double-blind study (NCT04746924) investigating OCI in combination with TIS compared with PEM in adult pts (≥ 18 years of age) with PD-L1 selected, previously untreated, locally advanced, unresectable or metastatic NSCLC without oncogenic EGFR or ALK mutation. Approximately 605 pts will be randomized 5:5:1 to receive: OCI 900 mg intravenously (IV) plus TIS 200 mg IV every three weeks (Q3W; Arm A), PEM 200 mg IV plus placebo IV Q3W (Arm B) or TIS 200 mg IV plus placebo IV Q3W (Arm C). Pts will be treated until disease progression, loss of clinical benefit, intolerable toxicity or withdrawal of consent. Stratification factors include histology (squamous vs non-squamous) and region (Asian vs non-Asian). Cross-over is not permitted. Key eligibility criteria include histologically confirmed disease, PD-L1 expression ≥ 50%, no known EGFR or ALK mutations and no prior checkpoint inhibitor therapy. Dual primary endpoints are investigator-assessed progression-free survival (PFS; RECIST v1.1) and overall survival (Arms A and B) in the Intention-to-Treat population. Secondary endpoints include PFS (assessed by Blinded Independent Review Committee), investigator-assessed overall response rate and duration of response, safety and tolerability, and patient-reported health-related quality of life (EORTC-QLQ-C30, QLQ-LC13 and EQ-5D-5L; Arms A and B). Exploratory endpoints include disease control rate, clinical benefit rate and time to response. This study will also evaluate the association between biomarkers and response or resistance. Study enrollment has begun and recruitment is ongoing. Clinical trial information: NCT04746924.

Safety and PK/PD of ALLO-647, an anti-CD52 antibody, with fludarabine (Flu)/cyclophosphamide (Cy) for lymphodepletion in the setting of allogeneic CAR-T cell therapy.

2527 Background: Allogeneic chimeric antigen receptor (CAR) T cell therapy holds promise in addressing logistical/manufacturing challenges of autologous CAR T cell therapy. ALLO-501 (anti-CD19; uses Cellectis technologies) and ALLO-715 (anti-BCMA) are allogeneic CAR T cell products whose a) disrupted TCRα constant gene may reduce GvHD risk, and b) edited CD52 gene may permit use of ALLO-647 (a humanized anti-CD52 mAb) to selectively deplete host T cells. Methods: The ongoing ALPHA (ALLO-501) and UNIVERSAL (ALLO-715) trials include patients (pts) with relapsed/refractory large B-cell or follicular lymphoma and multiple myeloma, respectively. The lymphodepletion regimen included ALLO-647 (39 mg [low dose, LD; n = 33], 60 mg [n = 12], or 90 mg [high dose, HD; n = 27]) with Flu 30 mg/m2/d x 3d +/- Cy 300 mg/m2/d x 3d (Flu+Cy, n = 66; Cy, n = 6) (ALLO-647/Flu +/- Cy) before CAR T infusion. A mixed-effects population pharmacokinetic (PK) model was fit to ALLO-647 concentration vs. time data. Pharmacodynamic (PD) effects on host T cells, IL15, and CAR T cell expansion were also studied. Results: As of the data cut, 72 pts were treated. Common Grade ≥3 AEs were neutropenia (71%), thrombocytopenia (42%), anemia (39%), and lymphopenia (28%). Neutrophil, hemoglobin, and platelet counts did not differ by ALLO-647 dose, suggesting these dim-CD52+ cells were unaffected. Gr ≥3 infections were seen in 21% pts; 33 had infusion-related reactions (IRR; Gr 1: 13%; Gr 2: 32%; Gr 3: 1.4%). IRR incidence and severity were higher with HD ALLO-647. The optimal PK model included a saturable (concentration-dependent) elimination pathway; clearance varied as a function of baseline lymphocyte count (LC). Serum ALLO-647 levels increased with dose; median modelled Cmax was 4,224 and 14,139 ng/mL for LD and HD, respectively. With ALLO-647/Flu +/- Cy, all but 2 pts reached a LC nadir &lt; 0.05x109 cells/L, typically by D-3. Duration of lymphodepletion was typically longer with HD ALLO-647. Median duration of T-cell suppression ( &lt; 10 cells/µl) was ̃ 8.5 and 13.6 days from CAR T infusion, respectively, for LD and HD ALLO-647. With HD, T cell counts were &lt; 10 through D+28 in 17 pts and &gt; 10 in 2. In 68 evaluable pts, compared to LD, HD ALLO-647 was associated with higher D0 serum IL15 levels, which have been linked to improved clinical response (eg, Kochenderfer JCO 2017). Higher CAR T expansion was also observed post D+14 with HD ALLO-647 compared to LD, creating an opportunity for clinical response. Conclusions: ALLO-647/Flu +/- Cy had a tolerable safety profile and produced a deep and durable window of lymphocyte depletion. ALLO-647 exhibited target-mediated drug disposition; clearance increased with higher baseline LC. HD was associated with higher IL15 levels and better CAR T expansion, suggesting dose responses. Enrollment in both studies is ongoing; updated safety and PK/PD data will be presented. Clinical trial information: NCT04093596.