De novo protein design has been successful in expanding the natural protein repertoire. However, most de novo proteins lack biological function, presenting a major methodological challenge. In vaccinology, the induction of precise antibody responses remains a cornerstone for next-generation vaccines. Here, we present a protein design algorithm called TopoBuilder, with which we engineered epitope-focused immunogens displaying complex structural motifs. In both mice and nonhuman primates, cocktails of three de novo–designed immunogens induced robust neutralizing responses against the respiratory syncytial virus. Furthermore, the immunogens refocused preexisting antibody responses toward defined neutralization epitopes. Overall, our design approach opens the possibility of targeting specific epitopes for the development of vaccines and therapeutic antibodies and, more generally, will be applicable to the design of de novo proteins displaying complex functional motifs.

CD47 is a ubiquitously expressed immune checkpoint receptor that is often upregulated in cancer. CD47 interacts with its counter-receptor SIRPα on macrophages and other myeloid cells to inhibit cancer cell phagocytosis and drive immune evasion. To overcome tolerability and “antigen sink” issues arising from widespread CD47 expression, we generated dual-targeting bispecific antibodies that selectively block the CD47-SIRPα interaction on malignant cells expressing a specific tumor-associated antigen; e.g., CD19 or mesothelin. These bispecific κλ bodies are fully human, native IgG1 molecules, combining tumor targeting and selective CD47 blockade with immune activating mechanisms mediated by the Fc portion of the antibody. CD47-neutralizing κλ bodies efficiently kill cancer cells in vitro and in vivo but interact only weakly with healthy cells expressing physiological levels of CD47. Accordingly, a κλ body administered to non-human primates showed a typical IgG pharmacokinetic profile and was well tolerated. Importantly, κλ bodies preserve their tumoricidal capabilities in the presence of a CD47 antigen sink. Thus, dual-targeting κλ bodies allow for efficacious yet safe targeting of CD47 in cancer. Such a bispecific design could be applied to limit the extent of neutralization of other ubiquitously expressed therapeutic targets.

Bispecific antibodies enable unique therapeutic approaches but it remains a challenge to produce them at the industrial scale, and the modifications introduced to achieve bispecificity often have an impact on stability and risk of immunogenicity. Here we describe a fully human bispecific IgG devoid of any modification, which can be produced at the industrial scale, using a platform process. This format, referred to as a κλ-body, is assembled by co-expressing one heavy chain and two different light chains, one κ and one λ. Using ten different targets, we demonstrate that light chains can play a dominant role in mediating specificity and high affinity. The κλ-bodies support multiple modes of action, and their stability and pharmacokinetic properties are indistinguishable from therapeutic antibodies. Thus, the κλ-body represents a unique, fully human format that exploits light-chain variable domains for antigen binding and light-chain constant domains for robust downstream processing, to realize the potential of bispecific antibodies.

Cancer evolution determines molecular and morphological intra-tumor heterogeneity and challenges the design of effective treatments. In lung adenocarcinoma, disease progression and prognosis are associated with the appearance of morphologically diverse tumor regions, termed histologic patterns. However, the link between molecular and histological features remains elusive. Here, we generated multi-omics and spatially resolved molecular profiles of histologic patterns from primary lung adenocarcinoma, which we integrated with molecular data from >2,000 patients. The transition from indolent to aggressive patterns was not driven by genetic alterations but by epigenetic and transcriptional reprogramming reshaping cancer cell identity. A signature quantifying this transition was an independent predictor of patient prognosis in multiple human cohorts. Within individual tumors, highly multiplexed protein spatial profiling revealed coexistence of immune desert, inflamed, and excluded regions, which matched histologic pattern composition. Our results provide a detailed molecular map of lung adenocarcinoma intra-tumor spatial heterogeneity, tracing non-genetic routes of cancer evolution. Statement of significance Lung adenocarcinomas are classified based on histologic pattern prevalence. However, individual tumors exhibit multiple patterns with unknown molecular features. We characterized non-genetic mechanisms underlying intra-tumor patterns and molecular markers predicting patient prognosis. Intra-tumor patterns determined diverse immune microenvironments warranting their study in the context of current immunotherapies.

Highlights d CTSS is overexpressed and mutated (Y132D) in follicular lymphoma d CTSS regulates antigen processing and communication with CD4 + Tfh cells d Loss of CTSS activity promotes CD8 + T cell infiltration d Alteration of CTSS-mediated antigen processing contributes to antigen diversification

In diffuse large B-cell lymphoma (DLBCL), activation of the B-cell receptor (BCR) promotes multiple oncogenic signals, which are essential for tumor proliferation. Inhibition of the Bruton's tyrosine kinase (BTK), a BCR downstream target, is therapeutically effective only in a subgroup of patients with DLBCL. Here, we used lymphoma cells isolated from patients with DLBCL to measure the effects of targeted therapies on BCR signaling and to anticipate response. In lymphomas resistant to BTK inhibition, we show that blocking BTK activity enhanced tumor dependencies from alternative oncogenic signals downstream of the BCR, converging on MYC upregulation. To completely ablate the activity of the BCR, we genetically and pharmacologically repressed the activity of the SRC kinases LYN, FYN, and BLK, which are responsible for the propagation of the BCR signal. Inhibition of these kinases strongly reduced tumor growth in xenografts and cell lines derived from patients with DLBCL independent of their molecular subtype, advancing the possibility to be relevant therapeutic targets in broad and diverse groups of DLBCL patients.

CD47 serves as an anti-phagocytic receptor that is upregulated by cancer to promote immune escape. As such, CD47 is the focus of intense immuno-oncology drug development efforts. However, as CD47 is expressed ubiquitously, clinical development of conventional drugs, e.g., monoclonal antibodies, is confronted with patient safety issues and poor pharmacology due to the widespread CD47 "antigen sink". A potential solution is tumor-directed blockade of CD47, which can be achieved with bispecific antibodies (biAbs). Using mouse CD47-blocking biAbs in a syngeneic tumor model allowed us to evaluate the efficacy of tumor-directed blockade of CD47 in the presence of the CD47 antigen sink and a functional adaptive immune system. We show here that CD47-targeting biAbs inhibited tumor growth in vivo, promoting durable antitumor responses and stimulating CD8 + T cell activation in vitro. In vivo efficacy of the biAbs could be further enhanced when combined with chemotherapy or PD-1/PD-L1 immune checkpoint blockade. We also show that selectivity and pharmacological properties of the biAb are dependent on the affinity of the anti-CD47 arm. Taken together, our study validates the approach to use CD47-blocking biAbs either as a monotherapy or part of a multi-drug approach to enhance antitumor immunity.

Upregulation of the immune checkpoint receptor, CD47, on cancer cells promotes immune evasion and is correlated with poor clinical outcome. CD47 is therefore an attractive immuno-oncology target but also a challenging one, given its ubiquitous distribution in healthy tissues. Bispecific antibodies (biAbs) offer superior selectivity as compared to mAbs, as they combine two antigen specificities in one molecule allowing the simultaneous targeting of two cell surface receptors. We used such a dual targeting design to create NI-1701, a biAb that pairs an anti CD47 arm with a high-affinity arm specific for CD19, a clinically validated target expressed by B leukemias and lymphomas. The target cell selectivity of NI-1701 relies principally on the binding affinity of the biAbs anti-CD19 arm. Thus, NI-1701 binds weakly to CD19-negative healthy cells expressing physiological levels of CD47, such as erythrocytes, platelets or T cells. In contrast, NI-1701 binds strongly to CD19-positive cells and blocks CD47 upon concurrent engagement of the two targets at the cell surface. As shown by numerous experiments involving CD19-positive human cancer cell lines and patients cells, NI-1701 effectively kills B cell tumors through antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cell-mediated cytotoxicity (ADCC). Furthermore, NI-1701 controls the growth of sub-cutaneous Raji cell tumors, in a way that was dependent on the co-ligation of both CD19 and CD47 antigens. Examination of the excised tumors revealed that NI-1701 actively reshaped the tumors microenvironment by enhancing the phagocytic activity of macrophages and by reducing the proportion of CD11b+Gr1+myeloid-derived suppressor cells (MDSCs) infiltrating the xenograft tumors. In disseminated mouse models of B-ALL, using both leukemia cell lines and patient-derived xenografts (PDX), NI-1701 was able to reduce tumor burden in peripheral blood and to block the spread of tumor cells to the bone marrow. The therapeutic potential of NI-1701 was also expanded to Diffuse Large B-Cell Lymphoma (DLBCL) using a PDX model, in which the tumor burden was abrogated with significantly higher efficacy than the BTK inhibitor, ibrutinib. In vitro and in vivo studies demonstrated a favorable pharmacokinetic (PK) and tolerability profile of NI-1701. Single and multiple dose studies in non-human primates showed typical IgG PK and no effects on hematological parameters (e.g., red blood cell and platelet counts) up to 100mg/kg, the highest dose tested. Accordingly, in vitro safety testing with human blood showed no evidence of platelet activation or aggregation, hemagglutination or hemolysis event at high antibody concentrations. We also show that NI-1701 target cell selectivity is important for the preservation of tumor cell killing efficacy in the presence of CD47 antigen sink. ADCP and ADCC experiments performed with an excess of bystander CD47-positive cells demonstrate that NI-1701-induced tumor cell killing is not affected by the presence of such antigen sink, in contrast to anti-CD47 mAbs, which loose potency in this situation. We conclude that the dual targeting biAb approach allows a safe yet effective blockade of CD47 due to selectivity for a B cell associated antigen, resulting in impressive tumor cell killing in a range of preclinical models. Thus, dual-targeting biAb open the way to the safe and efficacious therapeutic neutralization of CD47, an immune checkpoint receptor hijacked by cancer cells. NI-1701 is in preclinical enabling studies in preparation for a Phase I clinical study in patients with B cell malignancies, planned for 2017. Citation Format: Krzysztof Masternak, Xavier Chauchet, Vanessa Buatois, Susana Salgado-Pires, Limin Shang, Zoë Johnson, Elie Dheilly, Valéry Moine, Walter G. Ferlin, Marie H. Kosco-Vilbois, Nicolas Fischer. NI-1701, a bispecific antibody for selective neutralization of CD47 in B cell malignancies. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B37.