Blocking CD47 using SIRPα-Fc or anti-CD47 antibodies (Ab) has emerged as a promising strategy to neutralize CD47 and promote tumor eradication. However, CD47 is also expressed on red blood cells (RBC) and platelets (PLT) which can act as a large Ab sink. Targeting CD47 also led to anemia and thrombocytopenia in animal studies and phase I trials, which is of serious concern. Here we report the discovery of a new CD47 Ab (TJ-C4) with a novel epitope that endows it with enhanced phagocytic and RBC-sparing properties, thus differentiating itself from current CD47-targeting therapies.

A naïve human single chain variable fragment (ScFv) library was subjected for the binders to human CD47-extracellular domain (ECD). All the binders with unique sequences were converted to full antibodies and screened against human RBCs and tumor cells, leading to the discovery of TJC4. A series of head to head experiments have been performed with other CD47 antibodies to compare the in vitro RBC binding and hemagglutination, ability to block the CD47-SIRPa interaction and enhance the macrophage mediated phagocytosis of tumor cells. Different in vivo tumor models were employed to evaluate the anti-tumor efficacy of TJ-C4 either by mono or combination treatment. In addition, a comprehensive analysis of the hematological parameters was assessed in cynomolgus monkeys receiving a single intravenous infusion or weekly repeated injections.

To explore the underlined mechanism of the RBC sparing properties of TJC4, the binding pose and epitope were identified by X-ray crystallography and the influence of CD47 glycosylation in RBCs were further examined.

TJC4 is a fully human anti-CD47 IgG4 antibody that shares a similar binding affinity to human and cynomolgus monkey CD47. Like other anti-CD47 antibodies, TJC4 blocks the interaction of CD47 and SIRPa, leading to the enhanced macrophage phagocytosis of various CD47+ tumor cell lines and primary AML cells. Mono-treatment of TJC4 completely eradicated tumor cells in a Raji cell xenograft model and significantly extended the overall survival of treated mice in an AML model. When combined with Rituximab, TJC4 showed a superior efficacy in a DLBCL model over the mono-treatment group.

TJC4 has unique RBC sparing properties as evident by the negligible binding to healthy human RBCs and platelets respectively. Single dose or repeat dose treatment of TJC4 minimally and transiently impacts RBCs in cynomolgus monkeys and no other safety findings were observed up to the highest dose (100 mg/kg). No impact was observed in platelets. The unique functional properties of TJC4 can be explained in part by its structure when in complex with CD47, which reveals an almost straight head-to-head binding and a novel conformational epitope that is distinct from other CD47 antibodies. Upon the structural analysis of the binding epitope, we identified a potential N-linked glycosylation site located nearby the two critical epitopes on the CD47 protein. Due to the nature of the high glycosylation degree of membrane proteins by RBCs, the N-linked glycan is hypothesized to function as a “shield” to block the exposure of the epitopes and prevent the TJC4 binding to human RBCs. This hypothesis is validated by the restoration of TJC4 binding to the deglycosylated RBCs after the PNGase treatment.

In summary, TJC4 is a next generation therapeutic anti-CD47 antibody that is devoid of the hematological liabilities while maintaining anti-tumor efficacy. These attributes of TJC4 differentiate it from other CD47 targeting agents currently in clinical evaluation.

This phase 1 study (NCT03934814) is comprised of 2 parts. Part 1 consists of lemzoparlimab monotherapy dose escalation and 2 separate dose escalations of combination therapy with pembrolizumab or rituximab. The study is a standard 3+3 design. Part 2 is a dose expansion study. During monotherapy dose escalation, patients with relapsed/refractory solid tumors were administered an intravenous weekly dose (1 to 30 mg/kg) of lemzoparlimab to determine tolerability, safety, pharmacokinetics (PK), pharmacodynamics (PD) and anti-tumor activity based on Response Evaluation Criteria in Solid Tumors (RECIST v1.1) and iRECIST. Preliminary data from fully enrolled monotherapy cohorts in Part 1 are reported as of 17 July 2020.

Twenty patients with relapsed/refractory solid tumors were enrolled to monotherapy dose escalation cohorts (1, 3, 10, 20 and 30 mg/kg). Lemzoparlimab toxicity was manageable up to 30 mg/kg without a dose-limiting toxicity (DLT) observed. The most common treatment-related adverse events (TRAEs) were anemia (30.0%, n=6), fatigue (25.0%, n=5), infusion-related reactions (20.0%, n=4), and diarrhea (15.0%, n=3). All TRAEs were Grade 1 or 2. A transient, non-dose-dependent average reduction of 1.5 mg/dL (range: 0.4–2.6 mg/dL) in hemoglobin during the first cycle was observed across all cohorts consistent with the results of pre-clinical good laboratory practice toxicity studies. Laboratory or clinical evidence of hemolysis was not observed in any cohort. Preliminary results indicate the PK of lemzoparlimab appears to be linear at mid- to high dose levels following a single dose. CD47 receptor occupancy shows complete saturation on peripheral T cells at peak concentrations of 20 mg/kg and above.

Lemzoparlimab appears safe up to 30 mg/kg with favorable PK and PD characteristics in patients with relapsed/refractory solid tumors to date. No TRAEs greater than Grade 2 have been observed. Results will be updated at presentation including available tumor response data.

Clinical studies showed striking synergy between LAG3 and PD1 in multiple settings, highlighting the therapeutic potential of LAG3. Here we reported the discovery of a novel LAG3 antibody (TJ-A3) with sub-nanomolar binding affinity. Upon binding, it blocked the interaction of LAG3 to its receptor MHCII, leading to increased production of IL-2 in Jurkat cells overexpressing LAG3 and in activated human primary T cells. TJ-A3 can significantly inhibit tumor growth in combination with a PD-L1 blocking antibody in a syngeneic mouse tumor model by using LAG3-humanized mice. Together with favorable cynomolgus monkey PK and cell line development profile, these studies support further clinical development of TJ-A3.

Previous reports showed strong efficacy of anti-TIGIT antibodies only in combination with anti-PD(L)1 agents in multiple tumor models, indicating a potential for TIGIT blocking antibodies as a combination partner for cancer treatment. Here we report discovery of a novel, humanized, Fc-enabled, subnanomolar anti-TIGIT antibody with significantly enhanced tumor inhibition as a monotherapy, thus differentiating it from current anti-TIGIT molecules.

TJT6 was generated by traditional hybridoma technology using extracellular domain (ECD) of human TIGIT as immunogen and then humanized through complementarity determining region (CDR) grafting. The TIGIT antigen binding and CD155 receptor blocking activities of TJT6 were evaluated for through both protein-based and cell-based assays. The immunological function of TJT6 was further studied in a TIGIT- and CD226-overexpressed Jurkat cell activation assay. The in vivo efficacy of TJT6 was investigated in MC38 tumor model using human TIGIT knock-in mice. Percentages of tumor infiltrating CD4+ T and CD8+ T cells and the production of interferon g were analyzed by FACS.

TJT6, an Fc-enabled human IgG1 antibody, binds human and cynomolgus TIGIT with similar sub-nanomolar binding. It blocked the interaction of TIGIT to its ligand CD155, leading to increased production of interleukin 2 (IL-2) in a Jurkat cell line which overexpressed TIGIT. Mono-treatment of TJT6 significantly suppressed tumor growth in a dose-dependent manner in a syngeneic MC38 tumor model compared to vehicle, achieving maximal 80% tumor growth inhibition at 10 mg/kg. Percentages of tumor infiltrating CD4+ and CD8+ T cells and the production of IFN-g in CD8+ T cells were significantly enhanced after anti-TIGIT antibody treatment.

We have successfully discovered a TIGIT-blocking therapeutic antibody with strong efficacy in the inhibition of tumor growth and reinvigoration of tumor-infiltrating CD8+ T cells. TJT6 represents potentially a best-in-class molecule with superior potency as a single agent and is undergoing preclinical development with an aim to enter clinical studies in 2019.

Therefore, reprogramming of M2-type macrophages into M1-type macrophages has been considered as a better potential therapy to boost the antitumoral effects by macrophages. The key myeloid cytokine GM-CSF is approved as an adjunctive agent in the treatment of cancer by virtue of its ability to stimulate hematopoiesis and particularly induce the differentiation of pro-inflammatory M1 macrophages. Here we report the development of a novel immunocytokine composed of a newly discovered CD47 IgG1 fused with GMCSF at the C-terminus (1F8-GMCSF). In macrophage-tumor cell co-culture, treatment with 1F8-GMCSF fusion molecule led to significantly higher levels of tumor cell phagocytosis and M1 characteristic cytokine production than that with anti-CD47 alone. 1F8-GMCSF demonstrated superior efficacy in suppression of tumor growth as well as shift of tumor associated macrophages from M2 to M1 phenotype compared to 1F8 or GM-CSF treatment alone or in combination. In vivo tracking of injected anti-CD47 antibodies by live imaging confirmed the enriched localization of antibodies in the CD47+ tumor site. Taken together, our data showed that 1F8-GMCSF fusion exhibited enhanced phagocytosis and M1 macrophage activation, leading to a superior antitumor efficacy.

Treatment with recombinant human IL7 preferentially expands recent thymic emigrants, naïve and central memory T cells, but spares regulatory T cells. Here we constructed a series of immunocytokine L1I7 comprised of a PDL1 blocking mAb fused with an IL7 molecule with varying degrees of attenuated potency. The aim of the study was to identify the right molecule that achieves enhanced biodistribution at the tumor site and an optimal balance between synergistic anti-tumor activity and an acceptable safety profile.

PDL1 binding affinity and antagonist function of L1I7 were evaluated by BiaCore and PD1/PDL1 cell-based functional assay, respectively. IL7 receptor (IL7R) binding and internalization were evaluated in human primary CD4+ T cells by flow cytometry (FACS). IL7 potency was evaluated by p-STAT5 signaling assay and human CD4+ T cell proliferation assay. The in vitro synergistic effect of L1I7 was evaluated in the human mixed lymphocyte reaction (MLR) assay. The in vivo tracking assay was conducted by injecting fluorescence-labeled L1I7 into HCC827-transplanted CD34+ hematopoietic stem cells humanized mice. The in vivo efficacy of L1I7 was investigated in B16F10 melanoma syngeneic mice model with surrogate L1I7. Absolute numbers of tumor-infiltrating CD4+ T and CD8+ T cells were analyzed by FACS.

L1I7 series of immunocytokine maintained PDL1 binding and antagonist function when compared with PDL1 mAb. As compared with wildtype L1I7, L1I7 variants showed attenuated IL7 activity as evidenced by reduced IL7R binding/internalization, p-STAT5 activation and CD4+ T cell proliferation. Both wildtype and the attenuated variants were more effective in promoting CD4 T cell activation than anti-PDL1 or IL7 monotreatment in MLR. Similar with PDL1 mAb, L1I7 was enriched in the tumor site whereas IL7 was widely distributed in in vivo tracking experiment. In an anti-PDL1-resistant B16F10 tumor model, L1I7 showed superior efficacy as compared with either anti-PDL1, IL7 alone or in combination, which was correlated with an increased number of tumor-infiltrating CD4+ T and CD8+ T cells.

The novel immunocytokine L1I7 is designed to target poor and non-responders to PD-(L)1 treatment and even PD-(L)1 treatment relapsed patients. L1I7 represents potentially a best-in-class molecule with enhanced local immunostimulation achieved by concurrent PD-(L)1 blockade and IL7 stimulation. L1I7 is undergoing preclinical development with an aim to enter clinical studies in 2021.

TJ107 could play a pivotal role in reconstitution and reinvigoration of T cell immunity in cancer patients, providing unique opportunities for immuno-oncology combination strategies. The aim of this study (NCT04001075) is to determine the safety, tolerability and PKPD profile of TJ107 in Chinese cancer patients.

This ongoing study is to evaluate the safety, tolerability, PK profile, and anti-tumor activity of TJ107 in patients with advanced solid tumors who failed standard therapy. Patients receive TJ107 every 4 weeks by intramuscular (IM) injection. Dose escalation is aided by a 3+3 scheme from 240μg/kg to 1200μg/kg. A dose expansion cohort is being planned after the RP2D is determined. Safety is assessed by monitoring AEs and the associated grades per NCI CTCAE v5.0. Tumor response will be assessed per RECIST v1.1. Samples will be collected for PK, PD, ADA, immunophenotyping and TCR repertoire analysis.

Three patients with colorectal cancer were enrolled in the first cohort(240μg/kg).TJ107 was well tolerated and no DLTs were reported during the first cycle at this dose level. The preliminary PK results shows that TJ107 was rapidly absorbed and reached serum peak concentration around 24 hours post-dose. TJ107 was slowly cleared from the body and remained detectable in serum until Day 14 post-dose. A substantial increase in absolute lymphocyte count (ALC) from baseline was observed and peaked around 3 to 4 weeks post first dose. FACS analysis revealed increases in CD3+, CD4+ and CD8+ T cells. The numeric increase in T cells is consistent with increased Ki67 expression on Day 8 post first dose. There were no notable changes in B cells, monocytes, NK cells, neutrophils, nor Tregs, as expected.

Preliminary results from this trial suggest that TJ107 activated IL-7 pathway and expanded T cells in cancer patients in a similar way to data previously reported in healthy subjects. TJ107 exhibits a promising safety and tolerability profile in cancer patients under current dose. These findings support further clinical investigation.