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The host immune system generally serves as a barrier against tumor formation. Programmed death-ligand 1 (PD-L1) is a critical “don't find me” signal to the adaptive immune system, whereas CD47 transmits an anti-phagocytic signal, known as the “don't eat me” signal, to the innate immune system. These and similar immune checkpoints are often overexpressed on human tumors. Thus, dual targeting both innate and adaptive immune checkpoints would likely maximize anti-tumor therapeutic effect and elicit more durable responses. Herein, based on the variable region of atezolizumab and consensus variant 1 (CV1) monomer, we constructed a dual-targeting fusion protein targeting both CD47 and PD-L1 using “Knobs-into-holes” technology, denoted as IAB. It was effective in inducing phagocytosis of tumor cells, stimulating T-cell activation and mediating antibody-dependent cell-mediated cytotoxicity in vitro. No obvious sign of hematological toxicity was observed in mice administered IAB at a dose of 100 mg/kg, and IAB exhibited potent antitumor activity in an immune-competent mouse model of MC38. Additionally, the anti-tumor effect of IAB was impaired by anti-CD8 antibody or clodronate liposomes, which implied that both CD8+ T cells and macrophages were required for the anti-tumor efficacy of IAB and IAB plays an essential role in the engagement of innate and adaptive immune responses. Collectively, these results demonstrate the capacity of an elicited endogenous immune response against tumors and elucidate essential characteristics of synergistic innate and adaptive immune response, and indicate dual blockade of CD47 and PD-L1 by IAB may be a synergistic therapy that activates both innate and adaptive immune response against tumors.

Although rituximab has revolutionized the treatment of hematological malignancies, the acquired resistance is one of the prime obstacles for cancer treatment, and development of novel CD20-targeting antibodies with potent anti-tumor activities and specificities is urgently needed. Emerging evidence has indicated that lysosomes can be considered as an "Achilles heel" for cancer cells, and might serve as an effective way to kill resistant cancer cells. HLA-DR antibody L243 has been recently reported to elicit potent lysosome-mediated cell death in lymphoma and leukemia cells, suggesting that HLA-DR could be used as a potential target against lymphoma. In this study, we generated a bispecific immunoglobulin G-like antibody targeting both CD20 and HLA-DR (CD20-243 CrossMab) through CrossMab technology. We found that the CrossMab could induce remarkably high levels of complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and anti-proliferative activity. Notably, although HLA-DR is expressed on normal and malignant cells, the CrossMab exhibited highly anti-tumor specificity, showing efficient eradication of hematological malignancies both in vitro and in vivo. Our data indicated that combined targeting of CD20 and HLA-DR could be an effective approach against malignancies, suggesting that CD20-243 CrossMab would be a promising therapeutic agent against lymphoma.

Antibody-drug conjugates (ADCs) have exhibited potent clinical benefits in cancer therapy. However, development of ADCs against epidermal growth factor receptor (EGFR) has limitations because of wide expression of EGFR in both normal and tumor tissues. Previously, we developed an anti-EGFR protease-activated antibody (pro-antibody), termed as PanP, which remains inert against EGFR until activated by tumor-specific protease. Herein, we for the first time report a new class of pro-antibody-drug conjugate (PDC) against EGFR, denoted as PanP-DM1. It has been designed to selectively target the EGFR-overexpressing tumor cells and exert greater anti-tumor activity compared with PanP. Our data showed that PanP-DM1 also could be selectively activated by tumor-specific protease 'uPA'. Furthermore, activated PanP-DM1 was potently cytotoxic against EGFR-overexpressing tumor cell lines in vitro. Crucially, our data indicated that PanP-DM1 was significantly more effective in eradicating EGFR-overexpressing tumors in vivo. Additionally, toxicity was preliminarily evaluated in mice as measured by body weight loss. In summary, our study suggests that PanP-DM1, a novel pro-antibody-drug conjugate, has cancer-selectivity, efficacy and safety profile that supports its potential use for EGFR-overexpressing tumors.

The goal of the studies presented here was to determine the tolerability, pharmacokinetic and pharmacodynamic profiles of CMAB007, a biosimilar of omalizumab (Xolair; a humanized anti-immunoglobulin E monoclonal antibody), in healthy, male Chinese subjects. Thirty-six healthy Chinese men participated in two open-label, dose-escalation studies: 27 in a single-dose study (150, 300 or 600 mg) and 9 in a multiple-dose study (150 or 300 mg every 4 weeks for 20 weeks). The safety profiles of both studies were generally unremarkable. No drug-related adverse event was observed. CMAB007 exhibited a linear PK profile over the dose range of 150-600 mg. In the single-dose study, maximum concentration (Cmax) was reached within 6-8 d, and Cmax and area under concentration-time curve (AUC) increased linearly with the dose. In the multiple-dose study, steady-state appeared to have been achieved after the third dose. Css-max and AUCτ also showed dose-linearity. A dose-dependent suppression of free IgE was observed during treatment, as a median percentage change from baseline, 91.9-98.8%, in the three single-dose groups. No anti-CMAB007 antibodies were detected after dosing in any subject. Subcutaneous administration of CMAB007 was well-tolerated and seemed to be effective in reducing free IgE in healthy Chinese volunteers, which provides important information for further clinical studies.

In the present study, we conducted a Phase 1 study of a recombinant anti-EGFR monoclonal antibody (CMAB009) that has the same amino acid sequence as cetuximab. The purpose of this study was to evaluate the safety, pharmacokinetics, and potential benefit of CMAB009 in Chinese patients with advanced chemotherapy-resistant epithelial malignancies. In this study, 18 patients were treated with two successive treatment schedules comprising a single-dose escalation phase followed by a weekly, multiple-dose extension phase. No dose-limiting toxicity was reported during the evaluation period. CMAB009-associated toxicity was minimal, and the most commonly reported adverse events were fever, asthenia, transaminase elevation, nausea, and skin toxicities. CMAB009 exhibited a non-linear PK profile over the dose range of 100 to 400 mg/m2. In the single-dose phase, CMAB009 reached peak serum concentrations at the end of the infusion and then declined slowly with a Tl/2 of 77.15 ±13.96 h, 79.79 ±6.91 h, and 86.25 ±9.93 h after infusion of 100, 250, and 400 mg/m ( 2) based on a two compartmental model analysis. Mean Cmax increased roughly dose-proportional while AUC0-∞ showed a greater than dose-proportionate increase from 100 to 400 mg/m ( 2) . After multiple infusions, serum concentrations dropped slowly and the Tl/2 was 102.25 ± 33.54 h and 118.91 ± 29.12 h based on a two compartmental model analysis. No neutralizing anti-antibody antibodies were detectable. Two patients achieved partial remissions. The study results suggest that CMAB009 shows acceptable tolerance and primary efficacy and should be studied as a treatment in patients with advanced chemotherapy-resistant epithelial malignancies.

Hu12F6mu is an Fc-mutated, humanized anti-CD3 antibody developed in our lab. The aim of this study was to assess single dose escalation pharmacokinetics (PK) and safety profile of hu12F6mu and to measure the effects of the antibody on levels of circulating T cells over time. Twenty-seven patients receiving renal allografts were randomized to receive hu12F6mu intravenously at a single-dose of 2.5, 5 or 10 mg. The concentration-time data obtained by a validated ELISA method were subjected to non-compartmental PK analysis by DAS 2.1 software. Subgroups of CD2(+), CD3(+), CD4(+) and CD8(+) lymphocytes were monitored periodically by flow cytometry. Our results showed that hu12F6mu exhibited linear PK over the dose range of 2.5 to 10 mg. A significant decline in the proportion of T cells was observed immediately after the infusion, followed by a progressive increase occurring over the ensuing days of therapy. A significant negative correlation was observed between serum concentration of hu12F6mu and CD3(+) cell proportion. Intravenous infusion of hu12F6mu was well-tolerated in patients receiving renal allografts. These results suggest that hu12F6mu may have potential as a therapeutic agent, although further studies are needed.

Panitumumab, as a commercially available antibody, is an effective anticancer therapeutic against epidermal growth factor receptor (EGFR), although it exerts weak antibody-dependent cell-mediated cytotoxicity (ADCC) activity owing to its IgG2 nature. Here, we firstly engineered panitumumab by grafting its variable region into an IgG1 backbone. The engineered panitumumab (denoted as Pan) retained binding activity identical to the parental antibody while exhibiting stronger ADCC activity in vitro and more potent antitumor effect in vivo. To further enhance the target selectivity of Pan, we generated Pan-P by tethering an epitope-blocking peptide to Pan via a tumor-specific protease selective linker. Pan-P showed almost 40-fold weaker affinity compared with Pan, but functional activity was restored to a similar extent as Pan when Pan-P was selectively activated by urokinase-type plasminogen activator (uPA). More importantly, targeted localization of Pan-P was observed in tumor samples from colorectal cancer (CRC) patients and tumor-bearing nude mice, strongly indicating that specific activation also existed ex vivo and in vivo. Furthermore, Pan-P also exhibited effective in vivo antitumor potency similar to Pan. Taken together, our data evidence the enhanced antitumor potency and excellent target selectivity of Pan-P, suggesting its potential use for minimizing on-target toxicity in anti-EGFR therapy.