A stable, engineered TL1A ligand co-stimulates T cells via specific binding to DR3.

TL1A (TNFSF15) is a TNF superfamily ligand which can bind the TNFRSF member death receptor 3 (DR3) on T cells and the soluble decoy receptor DcR3. Engagement of DR3 on CD4+ or CD8+ effector T cells by TL1A induces downstream signaling, leading to proliferation and an increase in secretion of inflammatory cytokines. We designed a stable recombinant TL1A molecule that (1) displays high monodispersity and stability, (2) displays the ability to activate T cells in vitro and in vivo, and (3) lacks binding to DcR3 while retaining functional activity via DR3. Together these results suggest the TL1A ligand can be amenable to therapeutic development on its own or paired with a tumor-targeting moiety.

INDI-integrated nanobody database for immunoinformatics.

Nanobodies, a subclass of antibodies found in camelids, are versatile molecular binding scaffolds composed of a single polypeptide chain. The small size of nanobodies bestows multiple therapeutic advantages (stability, tumor penetration) with the first therapeutic approval in 2018 cementing the clinical viability of this format. Structured data and sequence information of nanobodies will enable the accelerated clinical development of nanobody-based therapeutics. Though the nanobody sequence and structure data are deposited in the public domain at an accelerating pace, the heterogeneity of sources and lack of standardization hampers reliable harvesting of nanobody information. We address this issue by creating the Integrated Database of Nanobodies for Immunoinformatics (INDI, http://naturalantibody.com/nanobodies). INDI collates nanobodies from all the major public outlets of biological sequences: patents, GenBank, next-generation sequencing repositories, structures and scientific publications. We equip INDI with powerful nanobody-specific sequence and text search facilitating access to >11 million nanobody sequences. INDI should facilitate development of novel nanobody-specific computational protocols helping to deliver on the therapeutic promise of this drug format.

Conduit CAR: Redirecting CAR T-Cell Specificity with A Universal and Adaptable Bispecific Antibody Platform.

The success of chimeric antigen receptor (CAR) T-cell therapy against hematologic malignancies has altered the treatment paradigm for patients with these diseases. Nevertheless, the occurrence of relapse due to antigen escape or heterogeneous antigen expression on tumors remains a challenge for first-generation CAR T-cell therapies as only a single tumor antigen can be targeted. To address this limitation and to add a further level of tunability and control to CAR T-cell therapies, adapter or universal CAR T-cell approaches use a soluble mediator to bridge CAR T cells with tumor cells. Adapter CARs allow simultaneous or sequential targeting of multiple tumor antigens, control of immune synapse geometry, dose control, and the potential for improved safety. Herein, we described a novel CAR T-cell adapter platform that relies on a bispecific antibody (BsAb) targeting both a tumor antigen and the GGGGS (G4S) linker commonly used in single-chain Fv (ScFv) domains expressed on CAR T-cell surfaces. We demonstrated that the BsAb can bridge CAR T cells to tumor cells and potentiate CAR T-cell activation, proliferation, and tumor cell cytolysis. The cytolytic activity of CAR T-cells was redirected to different tumor antigens by changing the BsAb in a dose-dependent manner. This study highlights the potential of G4S-displaying CAR T cells to be redirected to engage alternative tumor-associated antigens (TAA). Significance: New approaches are needed to address relapsed/refractory disease and manage potential toxicities associated with CAR T-cell therapy. We describe an adapter CAR approach to redirect CAR T cells to engage novel TAA-expressing cells via a BsAb targeting a linker present on many clinical CAR T-cell therapeutics. We anticipate the use of such adapters could increase CAR T-cell efficacy and reduce potential CAR-associated toxicities.

A Novel Antagonistic CD73 Antibody for Inhibition of the Immunosuppressive Adenosine Pathway.

Despite some impressive clinical results with immune checkpoint inhibitors, the majority of patients with cancer do not respond to these agents, in part due to immunosuppressive mechanisms in the tumor microenvironment. High levels of adenosine in tumors can suppress immune cell function, and strategies to target the pathway involved in its production have emerged. CD73 is a key enzyme involved in adenosine production. This led us to identify a novel humanized antagonistic CD73 antibody, mAb19, with distinct binding properties. mAb19 potently inhibits the enzymatic activity of CD73 in vitro, resulting in an inhibition of adenosine formation and enhanced T-cell activation. We then investigated the therapeutic potential of combining CD73 antagonism with other immune modulatory and chemotherapeutic agents. Combination of mAb19 with a PD-1 inhibitor increased T-cell activation in vitro Interestingly, this effect could be further enhanced with an agonist of the adenosine receptor ADORA3. Adenosine levels were found to be elevated upon doxorubicin treatment in vivo, which could be blocked by CD73 inhibition. Combining CD73 antagonism with doxorubicin resulted in superior responses in vivo Furthermore, a retrospective analysis of rectal cancer patient samples demonstrated an upregulation of the adenosine pathway upon chemoradiation, providing further rationale for combining CD73 inhibition with chemotherapeutic agents.This study demonstrates the ability of a novel CD73 antibody to enhance T-cell function through the potent suppression of adenosine levels. In addition, the data highlight combination opportunities with standard of care therapies as well as with an ADORA3 receptor agonist to treat patients with solid tumors.

Selective recruitment of γδ T cells by a bispecific antibody for the treatment of acute myeloid leukemia.

Despite significant progress over the last few decades in the treatment of acute myeloid leukemia (AML), there still remains a major unmet medical need for this disease. Immunotherapy approaches for redirecting pan CD3+ T cells to target leukemia blasts have shown limited efficacy in clinical trials and often accompanied with severe toxicity in AML patients. We designed an alternative engager molecule (Anti-TRGV9/anti-CD123), a bispecific antibody that can simultaneously bind to the Vγ9 chain of the Vγ9Vδ2+ γδ T cell receptor and to AML target antigen, CD123, to selectively recruit Vγ9+ γδ T cells rather than pan T cells to target AML blasts. Our results suggest that prototypic bispecific antibodies (a) selectively activate Vγ9+ γδ T cells as judged by CD69 and CD25 surface expression, and intracellular Granzyme B expression, (b) selectively recruit Vγ9+ γδ T cells into cell-cell conjugate formation of γδ T cells with tumor cells indicating selective and effective engagement of effector and target tumor cells, and (c) mediate γδ T cell cytotoxicity (in vitro and in vivo) against tumor antigen-expressing cells. Collectively, these findings suggest that selectively redirecting Vγ9+ γδ T cells to target AML blasts has a potential for immunotherapy for AML patients and favors further exploration of this concept.

Bispecific Antibodies for Triple Negative Breast Cancer.

Triple negative breast cancer (TNBC), an aggressive breast cancer subtype lacking estrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor 2 (HER2) expression, is associated with heightened metastatic potential and poor prognosis. While systemic chemotherapy, radiation, and surgical excision remain the current treatment modalities for patients with TNBC, the immunogenic nature of this aggressive disease has presented opportunity for the development of TNBC-targeting immunotherapies. Bispecific antibody-based therapeutics for the treatment of TNBC have gained recent attention in the scientific community. Clinical precedent has been previously established for the FDA-approved bispecific T cell engager, blinatumomab, for acute lymphoblastic leukemia. The present review discusses novel bispecific antibodies for TNBC and emerging TNBC targets for future bispecific antibody development.

Regulatory T cell targeting in cancer: Emerging strategies in immunotherapy.

The adaptive immune system is modulated by an important subset of CD4+ T lymphocytes called Treg cells that function in maintaining immune homeostasis by preventing excessive immune activation. Both deficiency and overactivation of Treg cell function can result in disease pathology. While loss of Treg function can lead to autoimmunity, an overabundance of Treg activity can promote tumorigenesis. Blocking and/or depleting Tregs has emerged as a viable strategy to enhance antitumor immunity. A major limitation underlying the limited efficacy observed with Treg therapies in the clinic is lack of selective targeting, often attributed to concurrent depletion of antitumor effector T-cell populations. Novel approaches to improve the specificity of Treg targeting in the context of cancer include the use of T-cell receptor mimic antibodies, bispecific antibodies, and near-infrared photoimmunotherapy. Next-generation technology platforms and transcriptomic/computational-based screening methods have been recently developed to identify preferential Treg targets. Herein, we highlight key advancements and challenges pertaining to the development of novel Treg targeting cancer therapeutics and discuss ongoing clinical trials evaluating next-generation Treg therapies for solid tumors.

Emerging CAR-T Cell Therapy for the Treatment of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC), a highly aggressive breast cancer subtype that lacks estrogen receptor, progesterone receptor, and HER2 expression, does not respond to traditional endocrine and anti-HER2-targeted therapies. Current treatment options for patients with TNBC include a combination of surgery, radiotherapy, and/or systemic chemotherapy. FDA-approved therapies that target DNA damage repair mechanisms in TNBC, such as PARP inhibitors, only provide marginal clinical benefit. The immunogenic nature of TNBC has prompted researchers to harness the body's natural immune system to treat this aggressive breast cancer. Clinical precedent has been recently established with the FDA approval of two TNBC immunotherapies, including an antibody-drug conjugate and an anti-programmed death-ligand 1 monoclonal antibody. Chimeric antigen receptor (CAR)-T cell therapy, a type of adoptive cell therapy that combines the antigen specificity of an antibody with the effector functions of a T cell, has emerged as a promising immunotherapeutic strategy to improve the survival rates of patients with TNBC. Unlike the remarkable clinical success of CAR-T cell therapies in hematologic cancers with Kymriah and Yescarta, the development of CAR-T cell therapies for solid tumors has been much slower and is associated with unique challenges, including a hostile tumor microenvironment. The aim of the present review is to discuss novel approaches and inherent challenges pertaining to CAR-T cell therapy for the treatment of TNBC.

In Pursuit of Stability Enhancement of a Prostate Cancer Targeting Antibody Derived from a Transgenic Animal Platform.

Accelerated timelines necessitate the discovery of fully human antibodies as biotherapeutics using transgenic animals with a notion that such mAbs bypass humanization. A transgenic animal derived mAb (PCa75) targeted against a prostate cancer antigen had several 'unusual residues' (rare somatic hypermutations, rSHM, with positional frequency of <1%) that resulted in compromised biophysical properties (Tm = 61 °C and intrinsic stability ΔGu = 24.3 kJ/mol) and a sub-optimal immunogenicity profile. In our quest for quality medicine, we pursued antibody engineering strategies to enhance the stability of PCa75. PCa62, an engineered variant of PCa75, retained function while significantly improving the drug-like attributes of the molecule (Tm = 75 °C and intrinsic stability ΔGu = 63.5 kJ/mol). rSHM is rather prevalent, 18 out the 21 approved transgenic animal-derived antibodies have at least one 'unusual residue'. Thus, engineering of rSHM remains critical to enhance the stability and minimize immunogenicity risk of biotherapeutics.

Preclinical efficacy of humanized, non-FcγR-binding anti-CD3 antibodies in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that accounts for ∼20% of ALL cases. Intensive chemotherapy regimens result in cure rates >85% in children and <50% in adults, warranting a search of novel therapeutic strategies. Although immune-based therapies have tremendously improved the treatment of B-ALL and other B-cell malignancies, they are not yet available for T-ALL. We report here that humanized, non-Fcγ receptor (FcγR)-binding monoclonal antibodies (mAbs) to CD3 have antileukemic properties in xenograft (PDX) models of CD3+ T-ALL, resulting in prolonged host survival. We also report that these antibodies cooperate with chemotherapy to enhance antileukemic effects and host survival. Because these antibodies show only minor, manageable adverse effects in humans, they offer a new therapeutic option for the treatment of T-ALL. Our results also show that the antileukemic properties of anti-CD3 mAbs are largely independent of FcγR-mediated pathways in T-ALL PDXs.

High-Throughput Generation of Bipod (Fab × scFv) Bispecific Antibodies Exploits Differential Chain Expression and Affinity Capture.

Generation of bispecific antibodies (BsAbs) having two unique Fab domains requires heterodimerization of the two heavy chains and pairing of each heavy chain with its cognate light chain. An alternative bispecific scaffold (Bipod) comprising an scFv and a Fab on a heterodimeric Fc eliminates the possibility of light chain mispairing. However, unpredictable levels of chain expression and scFv-induced aggregation can complicate purification and reduce the yield of desired Bipod. Here, we describe a high-throughput method for generation of Bipods based on protein A and CH1 domain affinity capture. This method exploits over-expression of the scFv chain to maximize heterodimer yield. Bipods purified by this method have purity suitable for cell-based functional assays and in vivo studies.

An allosteric anti-hepsin antibody derived from a constrained phage display library.

The serine protease hepsin is highly upregulated in prostate cancer and is implicated in tumor progression. Therefore, specific inhibition of hepsin enzymatic activity by an antibody constitutes an attractive therapeutic approach. Here, we report the identification of the anti-hepsin antibody Fab25 by screening of a Fab phage display library with a restricted chemical diversity at the complementary determining regions. Hepsin with its S1 pocket occupied by 3,4-dichloro-isocoumarin was used as the 'bait' for library screening. Fab25 was highly specific and it potently inhibited hepsin activity toward a panel of synthetic and macromolecular substrates. Biochemical and enzymatic studies with synthetic substrates of variable length suggested that Fab25 acts as an allosteric inhibitor based on non-competitive inhibition kinetics. Isothermal titration calorimetric experiments showed that the high-affinity (K(D) 6.1 nM) binding of Fab25 with hepsin is enthalpically driven. Despite an unusually long CDR-H3 loop with several potential hepsin cleavage sites (Lys, Arg residues), Fab25 was not processed by hepsin. Antibody-25 should be valuable for investigating hepsin's role in cancer progression and for potential therapeutic applications. Furthermore, the herein presented phage display strategy using an active site-modified protease should be widely applicable for identifying potential allosteric anti-protease antibodies.

Proteolytic activation of pro-macrophage-stimulating protein by hepsin.

Macrophage-stimulating protein (MSP) is a plasminogen-related growth factor and ligand for the receptor tyrosine kinase RON. The MSP/RON system promotes wound healing and invasive tumor growth and suppresses proinflammatory immune response. MSP binding to RON requires proteolytic conversion of the inactive single-chain form (pro-MSP) into the disulfide-linked α/ß heterodimer. The pro-MSP cleavage sequence (Ser-Lys-Leu-Arg(483)↓Val(484)) closely matches the substrate recognition sequences of hepsin, a type II transmembrane serine protease, that is overexpressed in several cancers. Here, we show that recombinant hepsin cleaves pro-MSP at the consensus site Arg(483)-Val(484) with superior efficiency compared with the known activators MT-SP1 and hepatocyte growth factor activator (HGFA). At least 50% of pro-MSP was processed within 1 hour at a hepsin concentration of 2.4 nmol/L and at a molar enzyme to substrate ratio of 1:500. An uncleavable single-chain variant of MSP weakly bound to a RON-Fc fusion protein, whereas hepsin-cleaved MSP bound with a K(D) of 10.3 nmol/L, suggesting that the high-affinity binding site in MSP ß-chain was properly formed. LNCaP prostate cancer cells overexpressing hepsin on the cell surface efficiently activated pro-MSP, which was blocked by a specific anti-hepsin antibody. Incubation of pro-MSP with hepsin led to robust RON-mediated phosphorylation of mitogen-activated protein kinase, ribosomal S6 protein, and Akt in human A2780 ovarian carcinoma cells stably expressing RON protein. In macrophages, pro-MSP with hepsin induced chemotaxis and attenuated lipopolysaccharide-dependent production of nitric oxide. These findings suggest that the MSP/RON signaling pathway may be regulated by hepsin in tissue homeostasis and in disease pathologies, such as in cancer and immune disorders.

Local protease signaling contributes to neural tube closure in the mouse embryo.

We report an unexpected role for protease signaling in neural tube closure and the formation of the central nervous system. Mouse embryos lacking protease-activated receptors 1 and 2 showed defective hindbrain and posterior neuropore closure and developed exencephaly and spina bifida, important human congenital anomalies. Par1 and Par2 were expressed in surface ectoderm, and Par2 was expressed selectively along the line of closure. Ablation of G(i/z) and Rac1 function in these Par2-expressing cells disrupted neural tube closure, further implicating G protein-coupled receptors and identifying a likely effector pathway. Cluster analysis of protease and Par2 expression patterns revealed a group of membrane-tethered proteases often coexpressed with Par2. Among these, matriptase activated Par2 with picomolar potency, and hepsin and prostasin activated matriptase. Together, our results suggest a role for protease-activated receptor signaling in neural tube closure and identify a local protease network that may trigger Par2 signaling and monitor and regulate epithelial integrity in this context.

Pegylated kunitz domain inhibitor suppresses hepsin-mediated invasive tumor growth and metastasis.

The transmembrane serine protease hepsin is one of the most highly upregulated genes in prostate cancer. Here, we investigated its tumor-promoting activity by use of a mouse orthotopic prostate cancer model. First, we compared the tumor growth of low hepsin-expressing LnCaP-17 cells with hepsin-overexpressing LnCaP-34 cells. After implantation of cells into the left anterior prostate lobe, LnCaP-34 tumors not only grew faster based on increased serum prostate-specific antigen levels but also metastasized to local lymph nodes and, most remarkably, invaded the contralateral side of the prostate at a rate of 100% compared with only 18% for LnCaP-17 tumors. The increased tumor growth was not due to nonspecific gene expression changes and was not predicted from the unaltered in vitro growth and invasion of LnCaP-34 cells. A likely explanation is that the in vivo effects of hepsin were mediated by specific hepsin substrates present in the tumor stroma. In a second study, mice bearing LnCaP-34 tumors were treated with a PEGylated form of Kunitz domain-1, a potent hepsin active site inhibitor derived from hepatocyte growth factor activator inhibitor-1 (K(i)(app) 0.30 +/- 0.02 nmol/L). Treatment of established tumors with PEGylated Kunitz domain-1 decreased contralateral prostate invasion (46% weight reduction) and lymph node metastasis (50% inhibition). Moreover, serum prostate-specific antigen level remained reduced during the entire treatment period, reaching a maximal reduction of 76% after 5 weeks of dosing. The findings show that hepsin promotes invasive prostate tumor growth and metastasis and suggest that active site-directed hepsin inhibition could be effective in prostate cancer therapy.

Laminin-332 is a substrate for hepsin, a protease associated with prostate cancer progression.

Hepsin, a cell surface protease, is widely reported to be overexpressed in more than 90% of human prostate tumors. Hepsin expression correlates with tumor progression, making it a significant marker and target for prostate cancer. Recently, it was reported that in a prostate cancer mouse model, hepsin up-regulation in tumor tissue promotes progression and metastasis. The underlying mechanisms, however, remain largely uncharacterized. Hepsin transgenic mice displayed reduced laminin-332 (Ln-332) expression in prostate tumors. This is an intriguing cue, since proteolytic processing of extracellular matrix macromolecules, such as Ln-332, is believed to be involved in cancer progression, and Ln-332 expression is lost during human prostate cancer progression. In this study, we provide the first direct evidence that hepsin cleaves Ln-332. Cleavage is specific, since it is both inhibited in a dose-dependent manner by a hepsin inhibitor (Kunitz domain-1) and does not occur when catalytically inactive hepsin is used. By Western blotting and mass spectrometry, we determined that hepsin cleaves the beta3 chain of Ln-332. N-terminal sequencing identified the cleavage site at beta3 Arg(245), in a sequence context (SQLR(245) LQGSCFC) conserved among species and in remarkable agreement with reported consensus target sequences for hepsin activity. In vitro cell migration assays showed that hepsin-cleaved Ln-332 enhanced motility of DU145 prostate cancer cells, which was inhibited by Kunitz domain-1. Further, hepsin-overexpressing LNCaP prostate cancer cells also exhibited increased migration on Ln-332. Direct cleavage of Ln-332 may be one mechanism by which hepsin promotes prostate tumor progression and metastasis, possibly by up-regulating prostate cancer cell motility.

Design, synthesis, and evaluation of aza-peptide Michael acceptors as selective and potent inhibitors of caspases-2, -3, -6, -7, -8, -9, and -10.

Aza-peptide Michael acceptors are a novel class of inhibitors that are potent and specific for caspases-2, -3, -6, -7, -8, -9, and -10. The second-order rate constants are in the order of 10(6) M(-1) s(-1). The aza-peptide Michael acceptor inhibitor 18t (Cbz-Asp-Glu-Val-AAsp-trans-CH=CH-CON(CH(2)-1-Naphth)(2) is the most potent compound and it inhibits caspase-3 with a k(2) value of 5620000 M(-1) s(-1). The inhibitor 18t is 13700, 190, 6.4, 594, 37500, and 173-fold more selective for caspase-3 over caspases-2, -6, -7, -8, -9, and -10, respectively. Aza-peptide Michael acceptors designed with caspase specific sequences are selective and do not show any cross reactivity with clan CA cysteine proteases such as papain, cathepsin B, and calpains. High-resolution crystal structures of caspase-3 and caspase-8 in complex with aza-peptide Michael acceptor inhibitors demonstrate the nucleophilic attack on C2 and provide insight into the selectivity and potency of the inhibitors with respect to the P1' moiety.

Exploring the S4 and S1 prime subsite specificities in caspase-3 with aza-peptide epoxide inhibitors.

Caspase-3 is a prototypic executioner caspase that plays a central role in apoptosis. Aza-peptide epoxides are a novel class of irreversible inhibitors that are highly specific for clan CD cysteine proteases. The five crystal structures of caspase-3-aza-peptide epoxide inhibitor complexes reported here reveal the structural basis for the mechanism of inhibition and the specificities at the S1' and the S4 subsites. Unlike the clan CA cysteine proteases, the catalytic histidine in caspase-3 plays a critical role during protonation and subsequent ring opening of the epoxide moiety and facilitates the nucleophilic attack by the active site cysteine. The nucleophilic attack takes place on the C3 carbon atom of the epoxide and results in an irreversible alkylation of the active site cysteine residue. A favorable network of hydrogen bonds involving the oxyanion hole, catalytic histidine, and the atoms in the prime site of the inhibitor enhance the binding affinity and specificity of the aza-peptide epoxide inhibitors toward caspase-3. The studies also reveal that subtle movements of the N-terminal loop of the beta-subunit occur when the P4 Asp is replaced by a P4 Ile, whereas the N-terminal loop and the safety catch Asp179 are completely disordered when the P4 Asp is replaced by P4 Cbz group.

Extended substrate recognition in caspase-3 revealed by high resolution X-ray structure analysis.

Caspases are cysteine proteases involved in the signalling cascades of programmed cell death in which caspase-3 plays a central role, since it propagates death signals from intrinsic and extrinsic stimuli to downstream targets. The atomic resolution (1.06 Angstroms) crystal structure of the caspase-3 DEVD-cmk complex reveals the structural basis for substrate selectivity in the S4 pocket. A low-barrier hydrogen bond is observed between the side-chains of the P4 inhibitor aspartic acid and Asp179 of the N-terminal tail of the symmetry related p12 subunit. Site-directed mutagenesis of Asp179 confirmed the significance of this residue in substrate recognition. In the 1.06 Angstroms crystal structure, a radiation damage induced rearrangement of the inhibitor methylketone moiety was observed. The carbon atom that in a substrate would represent the scissile peptide bond carbonyl carbon clearly shows a tetrahedral coordination and resembles the postulated tetrahedral intermediate of the acylation reaction.