Intracellular flow cytometry staining of antibody-secreting cells using phycoerythrin-conjugated antibodies: pitfalls and solutions

Antibody-secreting cells are terminally differentiated B cells that play a critical role in humoral immunity through immunoglobulin secretion along with possessing the potential to be long-lived. It is now appreciated that antibody-secreting cells regulate multiple aspects of biology through the secretion of various cytokines. In this regard, intracellular flow cytometry is a key tool used to assess the presence of intracellular proteins such as cytokines and transcription factors. Here, we showed that the use of phycoerythrin-containing antibody conjugates led to a false interpretation of antibody-secreting cell intracellular protein expression compared to other cell types. This was mainly due to the inappropriate retention of these antibodies specifically within antibody-secreting cells. Furthermore, we demonstrated how to reduce this retention which allowed for a more accurate comparison of intracellular protein expression between antibody-secreting cells and other cell types such as B lymphocytes. Using this methodology, our data revealed that spleen antibody-secreting cells expressed Toll-like receptor 7 as well as the pro-form of the inflammatory cytokine interleukin-1β.

Galactose Oxidase Enables Modular Assembly of Conjugates from Native Antibodies with High Drug-to-Antibody Ratios

The potential of antibody conjugates with high drug loading in anticancer therapy has recently been highlighted by the approval of Trastuzumab deruxtecan and Sacituzumab govitecan. These biopharmaceutical approaches have spurred interest in bioconjugation strategies with high and defined degrees antibody-to-drug (DAR) ratios, in particular on native antibodies. Here we report a glycoengineering methodology to generate antibody drug conjugates with DAR of up to eight, by combining highly selective enzymatic galactosylation and oxidation with biorthogonal tandem Knoevenagel-Michael addition chemistry. This three step approach offers a selective route to conjugates from native antibodies with high drug loading, and thus illustrates how biocatalysis can be used for the generation of biopharmaceuticals using mild reaction conditions.

784 BDC-2034: discovery of a CEA-targeting immune-stimulating antibody conjugate (ISAC) for solid tumors

BackgroundCEA (CEACAM5) is a well-validated cell-surface antigen that is highly expressed in multiple solid tumors. Bolt's immune-stimulating antibody conjugates (ISACs) direct a TLR7/8 agonist into tumors to activate tumor-infiltrating myeloid cells and initiate a broad innate and adaptive anti-tumor immune response.1 The favorable properties of CEA, including robust cell surface expression, low internalization rate, and limited normal tissue expression, suggest that the antigen may be a suitable ISAC target. We are evaluating an anti-CEA ISAC, BDC-2034, as a multi-functional approach to treat CEA-expressing cancers.MethodsAnti-CEA antibodies were tested for binding affinity and specificity, CEA-targeted antibody-dependent cellular phagocytosis (ADCP), and myeloid-mediated tumor cell killing. Selected antibodies were conjugated to proprietary TLR7/8 agonists, and the resulting CEA ISACs were evaluated for in vitro myeloid activation and in vivo efficacy against xenograft tumors.ResultsAntibody CEA1 binds to the CEA protein with high affinity (EC50 = 0.25 nM), binds selectively to CEA-positive tumor cell lines, and mediates ADCP more efficiently than a reference anti-CEA antibody, labetuzumab (figure 1). We generated BDC-2034 by conjugating a potent TLR7/8 agonist to CEA1. BDC-2034 tumor cell binding drives myeloid effector cell ADCP, agonist delivery to TLR7 and TLR8 in endosomes, and secretion of cytokines critical for innate and adaptive immunity (including IL-12p70, CXCL10, and TNFa). In the HPAF II + cDC co-culture model, IL-12p70 is induced with EC50 = 1.2 nM, and the level of induction is at least ten-fold higher than with ISACs using labetuzumab (figure 2). Potent cellular activity is strictly dependent on tumor cell CEA expression; in whole blood, in the absence of CEA-expressing tumor cells, cytokine induction was only observed at approximately 100-fold higher concentrations. BDC-2034 inhibits the growth of HPAF II xenograft tumors in SCID/beige mice with a minimal efficacious dose (MED) of 1 mg/kg, demonstrating anti-tumor activity solely through innate immune activation (figure 3). The TLR7/8 agonist in BDC-2034 has relatively poor activity in mice; a surrogate CEA1 ISAC with a mouse TLR7-activating agonist achieved MED = 0.5 mg/kg in the HPAF II model, with eradication of all tumors at the 5 mg/kg dose.ConclusionsThese pre-clinical data demonstrate the potential of BDC-2034 to treat CEA-expressing human cancers. Most importantly, the antigen-dependent induction of immune-stimulating cytokines promises a robust immune response that combines the activation of innate and adaptive arms.ReferenceAckerman S, Pearson C, Gregorio J. Immune-stimulating antibody conjugates elicit robust myeloid activation and durable antitumor immunity. Nature Cancer 2021;2:18–33. https://doi.org/10.1038/s43018-020-00136-xAbstract 784 Figure 1ADCP. Anti-CEA antibody CEA1 is an efficient inducer of ADCP of Raji/CEA cells by M-CSF differentiated monocyte-derived macrophagesAbstract 784 Figure 2Tumor-dependent dendritic cell activation. BDC-2034 induces IL-12p70 secretion from primary dendritic cells (cDC); native CEA1 antibody and reference anti-CEA ISAC are ineffectiveAbstract 784 Figure 3Efficacy against xenograft tumors. BDC-2034 inhibits the growth of HPAF II tumors in SCID/beige mice; native CEA1 antibody and isotype ISAC are ineffective

Introduction to Antibody-Drug Conjugates

Antibody-drug conjugates (ADCs) are innovative biopharmaceutical products in which a monoclonal antibody is linked to a small molecule drug with a stable linker. Most of the ADCs developed so far are for treating cancer, but there is enormous potential for using ADCs to treat other diseases. Currently, ten ADCs have been approved by the United States Food and Drug Administration (FDA), and more than 90 ADCs are under worldwide clinical development. Monoclonal antibodies have evolved from research tools to powerful therapeutics in the past 30 years. Tremendous strides have been made in antibody discovery, protein bioengineering, formulation, and delivery devices. This manuscript provides an overview of the biology, chemistry, and biophysical properties of each component of ADC design. This review summarizes the advances and challenges in the field to date, with an emphasis on antibody conjugation, linker-payload chemistry, novel payload classes, drug-antibody ratio (DAR), and product development. The review emphasizes the lessons learned in the development of oncology antibody conjugates and look towards future innovations enabling other therapeutic indications. The review discusses resistance mechanisms to ADCs, and give an opinion on future perspectives.

Antibody Conjugates for Sarcoma Therapy: How Far along Are We?

Sarcomas are one of the most difficult type of cancer to manage and treat because of their extremely heterogeneous molecular and morphological features. Despite the progress made over the years in the establishment of standard protocols for high and low grading/staging sarcoma patients, mostly with chemotherapy and/or radiotherapy, 50% of treated patients experience relapse episodes. Because of this, in the last 20 years, new therapeutic approaches for sarcoma treatment have been evaluated in preclinical and clinical studies. Among them, antibody-based therapies have been the most studied. Immunoconjugates consist of a carrier portion, frequently represented by an antibody, linked to a toxic moiety, i.e., a drug, toxin, or radionuclide. While the efficacy of immunoconjugates is well demonstrated in the therapy of hematological tumors and more recently also of epithelial ones, their potential as therapeutic agents against sarcomas is still not completely explored. In this paper, we summarize the results obtained with immunoconjugates targeting sarcoma surface antigens, considering both preclinical and clinical studies. To date, the encouraging results obtained in preclinical studies allowed nine immunoconjugates to enter clinical trials, demonstrating the validity of immunotherapy as a promising pharmacological tool also for sarcoma therapy.

Antibody Conjugates for Targeted Therapy Against HIV-1 as an Emerging Tool for HIV-1 Cure

Although advances in antiretroviral therapy (ART) have significantly improved the life expectancy of people living with HIV-1 (PLWH) by suppressing HIV-1 replication, a cure for HIV/AIDS remains elusive. Recent findings of the emergence of drug resistance against various ART have resulted in an increased number of treatment failures, thus the development of novel strategies for HIV-1 cure is of immediate need. Antibody-based therapy is a well-established tool in the treatment of various diseases and the engineering of new antibody derivatives is expanding the realms of its application. An antibody-based carrier of anti-HIV-1 molecules, or antibody conjugates (ACs), could address the limitations of current HIV-1 ART by decreasing possible off-target effects, reduce toxicity, increasing the therapeutic index, and lowering production costs. Broadly neutralizing antibodies (bNAbs) with exceptional breadth and potency against HIV-1 are currently being explored to prevent or treat HIV-1 infection in the clinic. Moreover, bNAbs can be engineered to deliver cytotoxic or immune regulating molecules as ACs, further increasing its therapeutic potential for HIV-1 cure. ACs are currently an important component of anticancer treatment with several FDA-approved constructs, however, to date, no ACs are approved to treat viral infections. This review aims to outline the development of AC for HIV-1 cure, examine the variety of carriers and payloads used, and discuss the potential of ACs in the current HIV-1 cure landscape.