CD28 hinge used in chimeric antigen receptor (CAR) T-cells exhibits local structure and conformational exchange amidst global disorder.

T-cell therapies based on chimeric antigen receptor (CAR) targeting of a tumor-specific antigen offer hope for patients with relapsed or refractory cancers. CAR hinge and transmembrane regions link antigen recognition domains to intracellular signal transduction domains. Here, we apply biophysical methods to characterize the structure and dynamic properties of the CD28 CAR hinge (CD28H) used in an FDA-approved CD19 CAR for the treatment of B-lineage leukemia/lymphoma. By using nuclear Overhauser effect spectroscopy (NOESY), which detects even transiently occupied structural motifs, we observed otherwise elusive local structural elements amidst overall disorder in CD28H, including a conformational switch from a native ß-strand to a 310-helix and polyproline II helix-like structure. These local structural motifs contribute to an overall loosely formed extended geometry that could be captured by NOESY data. All FDA-approved CARs use prolines in the hinge region, which we find in CD28, and previously in CD8α, isomerize to promote structural plasticity and dynamics. These local structural elements may function in recognition and signaling events and constrain the spacing between the transmembrane and antigen recognition domains. Our study thus demonstrates a method for detecting local and transient structure within intrinsically disordered systems and moreover, our CD28H findings may inform future CAR design.

Engineering a natural ligand-based CAR: directed evolution of the stress-receptor NKp30.

B7H6, a stress-induced ligand which binds to the NK cell receptor NKp30, has recently emerged as a promising candidate for immunotherapy due to its tumor-specific expression on a broad array of human tumors. NKp30 can function as a chimeric antigen receptor (CAR) extracellular domain but exhibits weak binding with a fast on and off rate to B7H6 compared to the TZ47 anti-B7H6 single-chain variable fragment (scFv). Here, directed evolution using yeast display was employed to isolate novel NKp30 variants that bind to B7H6 with higher affinity compared to the native receptor but retain its fast association and dissociation profile. Two variants, CC3 and CC5, were selected for further characterization and were expressed as soluble Fc-fusion proteins and CARs containing CD28 and CD3ς intracellular domains. We observed that Fc-fusion protein forms of NKp30 and its variants were better able to bind tumor cells expressing low levels of B7H6 than TZ47, and that the novel variants generally exhibited improved in vitro tumor cell killing relative to NKp30. Interestingly, CAR T cells expressing the engineered variants produced unique cytokine signatures in response to multiple tumor types expressing B7H6 compared to both NKp30 and TZ47. These findings suggest that natural CAR receptors can be fine-tuned to produce more desirable signaling outputs while maintaining evolutionary advantages in ligand recognition relative to scFvs.

Cryptic association of B7-2 molecules and its implication for clustering.

T-cell co-stimulation through CD28/CTLA4:B7-1/B7-2 axis is one of the extensively studied pathways that resulted in the discovery of several FDA-approved drugs for autoimmunity and cancer. However, many aspects of the signaling mechanism remain elusive, including oligomeric association and clustering of B7-2 on the cell surface. Here, we describe the structure of the IgV domain of B7-2 and its cryptic association into 1D arrays that appear to represent the pre-signaling state of B7-2 on the cell membrane. Super-resolution microscopy experiments on heterologous cells expressing B7-2 and B7-1 suggest, B7-2 form relatively elongated and larger clusters compared to B7-1. The sequence and structural comparison of other B7 family members, B7-1:CTLA4 and B7-2:CTLA-4 complex structures, support our view that the observed B7-2 1D zipper array is physiologically important. This observed 1D zipper-like array also provides an explanation for its clustering, and upright orientation on the cell surface, and avoidance of spurious signaling.

NEDD4 regulates ubiquitination and stability of the cell adhesion molecule IGPR-1 via lysosomal pathway.

BACKGROUND: The cell adhesion molecule IGPR-1 regulates various critical cellular processes including, cell-cell adhesion, mechanosensing and autophagy and plays important roles in angiogenesis and tumor growth; however, the molecular mechanism governing the cell surface levels of IGPR-1 remains unknown. RESULTS: In the present study, we used an in vitro ubiquitination assay and identified ubiquitin E3 ligase NEDD4 and the ubiquitin conjugating enzyme UbcH6 involved in the ubiquitination of IGPR-1. In vitro GST-pulldown and in vivo co-immunoprecipitation assays demonstrated that NEDD4 binds to IGPR-1. Over-expression of wild-type NEDD4 downregulated IGPR-1 and deletion of WW domains (1-4) of NEDD4 revoked its effects on IGPR-1. Knockdown of NEDD4 increased IGPR-1 levels in A375 melanoma cells. Deletion of 57 amino acids encompassing the polyproline rich (PPR) motifs on the C-terminus of IGPR-1 nullified its binding with NEDD4. Furthermore, we demonstrate that NEDD4 promotes K48- and K63-dependent polyubiquitination of IGPR-1. The NEDD4-mediated polyubiquitination of IGPR-1 stimulates lysosomal-dependent degradation of IGPR-1 as the treatment of cells with the lysosomal inhibitors, bafilomycine or ammonium chloride increased IGPR-1 levels ectopically expressed in HEK-293 cells and in multiple endogenously IGPR-1 expressing human skin melanoma cell lines. CONCLUSIONS: NEDD4 ubiquitin E3 ligase binds to and mediates polyubiquitination of IGPR-1 leading to its lysosomal-dependent degradation. NEDD4 is a key regulator of IGPR-1 expression with implication in the therapeutic targeting of IGPR-1 in human cancers.

Prediction of CD28-CD86 protein complex structure using different level of resolution approach.

Immune system plays essential role in functioning of higher organisms. Its hyperactivity can lead to autoimmune diseases or even anaphylactic shock while hypoactivity leads to proneness to infections or even cancer. T-cells play crucial role in immunity mechanisms and their activation and inhibition is strictly controlled by the regulatory proteins, such as CD28 and CTLA-4. Activity of these proteins is controlled by a pair of ligands, named CD80 and CD86, which can non-covalently bound to their receptors. While structure of human CTLA-4-CD86 complex in known, there is still no available structure for the CD28-CD86 system. To obtain the reliable structure of CD28-CD86 complex we first validated our methodology on the CTLA-4-CD86 system. Then coarse-grained UNRES-dock molecular docking simulation was performed followed by all-atom molecular dynamics simulations. As a result, we obtained a complete CD28-CD86 complex structure on atomistic level, in which interaction interface is consistent with available data. We also determined the kinetic properties for CTLA4-CD86 and CD28-CD86 complexes with use of coarse-grained model and determined the key residues for complex formation with use of Robetta, PPCheck and HawkDock servers. Our results not only verify high accuracy of the UNRES-dock method, but also provide a highly reliable model of the CD28-CD86 complex, which can be used in further studies and drug design.

Cell adhesion molecule IGPR-1 activates AMPK connecting cell adhesion to autophagy.

Autophagy plays critical roles in the maintenance of endothelial cells in response to cellular stress caused by blood flow. There is growing evidence that both cell adhesion and cell detachment can modulate autophagy, but the mechanisms responsible for this regulation remain unclear. Immunoglobulin and proline-rich receptor-1 (IGPR-1) is a cell adhesion molecule that regulates angiogenesis and endothelial barrier function. In this study, using various biochemical and cellular assays, we demonstrate that IGPR-1 is activated by autophagy-inducing stimuli, such as amino acid starvation, nutrient deprivation, rapamycin, and lipopolysaccharide. Manipulating the IκB kinase ß activity coupled with in vivo and in vitro kinase assays demonstrated that IκB kinase ß is a key serine/threonine kinase activated by autophagy stimuli and that it catalyzes phosphorylation of IGPR-1 at Ser220 The subsequent activation of IGPR-1, in turn, stimulates phosphorylation of AMP-activated protein kinase, which leads to phosphorylation of the major pro-autophagy proteins ULK1 and Beclin-1 (BECN1), increased LC3-II levels, and accumulation of LC3 punctum. Thus, our data demonstrate that IGPR-1 is activated by autophagy-inducing stimuli and in response regulates autophagy, connecting cell adhesion to autophagy. These findings may have important significance for autophagy-driven pathologies such cardiovascular diseases and cancer and suggest that IGPR-1 may serve as a promising therapeutic target.

Engineering Cytoplasmic Signaling of CD28ζ CARs for Improved Therapeutic Functions.

Chimeric antigen receptor modified T cells (CAR-T) have yielded impressive clinical outcomes in treating hematopoietic malignancies. However, relapses have occurred in a substantial number of patients and limited the development of CAR-T therapy. Most underlying reasons for these relapses can be attributed to poor persistence and rapid exhaustion of CAR-T cells in vivo. Despite multiple strategies having been developed, how to improve CAR-T persistence or resist exhaustion while maintaining sufficient cytotoxic functions is still a great challenge. Here we discuss engineering cytoplasmic signaling as an important strategy for CAR optimization. This review summarizes recent advances showing that the anti-tumor function of CAR-T cells can be improved by optimizing the CD3ζ domain or downstream signaling of CD28ζ CAR.

Recent advances in CAR-T cell engineering.

Chimeric antigen receptor T (CAR-T) cell therapy is regarded as an effective solution for relapsed or refractory tumors, particularly for hematological malignancies. Although the initially approved anti-CD19 CAR-T therapy has produced impressive outcomes, setbacks such as high relapse rates and resistance were experienced, driving the need to discover engineered CAR-T cells that are more effective for therapeutic use. Innovations in the structure and manufacturing of CAR-T cells have resulted in significant improvements in efficacy and persistence, particularly with the development of fourth-generation CAR-T cells. Paired with an immune modifier, the use of fourth-generation and next-generation CAR-T cells will not be limited because of cytotoxic effects and will be an efficient tool for overcoming the tumor microenvironment. In this review, we summarize the recent transformations in the ectodomain, transmembrane domain, and endodomain of the CAR structure, which, together with innovative manufacturing technology and improved cell sources, improve the prospects for the future development of CAR-T cell therapy.

HIV-Resistant and HIV-Specific CAR-Modified CD4+ T Cells Mitigate HIV Disease Progression and Confer CD4+ T Cell Help In Vivo.

HIV infection preferentially depletes HIV-specific CD4+ T cells, thereby impairing antiviral immunity. In this study, we explored the therapeutic utility of adoptively transferred CD4+ T cells expressing an HIV-specific chimeric antigen receptor (CAR4) to restore CD4+ T cell function to the global HIV-specific immune response. We demonstrated that CAR4 T cells directly suppressed in vitro HIV replication and eliminated virus-infected cells. Notably, CAR4 T cells containing intracellular domains (ICDs) derived from the CD28 receptor family (ICOS and CD28) exhibited superior effector functions compared to the tumor necrosis factor receptor (TNFR) family ICDs (CD27, OX40, and 4-1BB). However, despite demonstrating limited in vitro efficacy, only HIV-resistant CAR4 T cells expressing the 4-1BBζ ICD exhibited profound expansion, concomitant with reduced rebound viremia after antiretroviral therapy (ART) cessation and protection of CD4+ T cells (CAR-) from HIV-induced depletion in humanized mice. Moreover, CAR4 T cells enhanced the in vivo persistence and efficacy of HIV-specific CAR-modified CD8+ T cells expressing the CD28ζ ICD, which alone exhibited poor survival. Collectively, these studies demonstrate that HIV-resistant CAR4 T cells can directly control HIV replication and augment the virus-specific CD8+ T cell response, highlighting the therapeutic potential of engineered CD4+ T cells to engender a functional HIV cure.

T cell costimulation, checkpoint inhibitors and anti-tumor therapy.

The hallmarks of the adaptive immune response are specificity and memory. The cellular response is mediated by T cells which express cell surface T cell receptors (TCRs) that recognize peptide antigens in complex with major histocompatibility complex (MHC) molecules on antigen presenting cells (APCs). However, binding of cognate TCRs with MHC-peptide complexes alone (signal 1) does not trigger optimal T cell activation. In addition to signal 1, the binding of positive and negative costimulatory receptors to their ligands modulates T cell activation. This complex signaling network prevents aberrant activation of T cells. CD28 is the main positive costimulatory receptor on nai¨ve T cells; upon activation, CTLA4 is induced but reduces T cell activation. Further studies led to the identification of additional negative costimulatory receptors known as checkpoints, e.g. PD1. This review chronicles the basic studies in T cell costimulation that led to the discovery of checkpoint inhibitors, i.e. antibodies to negative costimulatory receptors (e.g. CTLA4 and PD1) which reduce tumor growth. This discovery has been recognized with the award of the 2018 Nobel prize in Physiology/Medicine. This review highlights the structural and functional roles of costimulatory receptors, the mechanisms by which checkpoint inhibitors work, the challenges encountered and future prospects.

Structures of Immune Checkpoints: An Overview on the CD28-B7 Family.

The co-stimulation and co-inhibition signal pathways, immune checkpoints, are among the central mechanisms to regulate the T-cell immunity. Optimal signals involve intricate interactions of numerous ligands and receptors. Manipulation of these signals offers great clinical opportunities and has revolutionized the cancer treatment therapies. The 2018 Nobel Prize in Physiology or Medicine was awarded to James P. Allison and Tasuku Honjo in recognition of their discovery of cancer immunotherapy by inhibition of immune checkpoint molecules. Despite the landmark discovery in cancer immunotherapy, the efforts to harness immunity against cancer are also restricted by the limited knowledge on the co-stimulation and co-inhibition signaling networks. Understanding the structures of these molecules, in particular, tackling the interaction paradigms from the structural perspective, help to provide more accurate insights into the signaling mechanisms, which may further facilitate the development of novel biologics and improve the efficacy of the existing biologics against these targets. Here we review our current understanding on the structures of these co-stimulatory and co-inhibitory molecules. Specifically, we focus on the structural basis of several checkpoint molecules among the CD28-B7 family and discuss the therapeutic drugs against these targets for the treatment of human cancers, autoimmune disorders, and transplantation.

Revealing the atomistic details behind the binding of B7-1 to CD28 and CTLA-4: A comprehensive protein-protein modelling study.

BACKGROUND: CD28 and CTLA-4 are homologous T-cell receptors that bind with B7-1 and produce two opposing immunological signals required for T-cell activation and inactivation, respectively. It has been clinically proven that specific blockade of these key protein-protein interactions at the synapse can offer immunotherapeutic benefits for cancers and autoimmune treatments. Hence, there is a growing interest towards developing anti-CD28 and anti-CTLA-4 small molecule inhibitors. To achieve this goal, it is important to understand unique molecular level fingerprint interactions that stabilize CTLA-4/B7-1 and CD28/B7-1 complexes. However, until recently, the structure of the human CD28/B7-1 complex has not been resolved experimentally, which remains a significant setback in achieving specific inhibitors against CTLA-4 or CD28. METHODS: Here, we employed a combination of advanced molecular modelling and extensive molecular dynamics (MD) simulations to model the CD28/B7-1 complex and characterize the key interactions that stabilize the complex. RESULTS: Ensemble protein-protein docking and MD-based binding-free energy calculations were used to obtain a comprehensive structural model of the CD28/B7-1 complex, which was validated with various mutation-based experimental data from literature. Our CD28/B7-1 model has much weaker binding affinity than the CTLA-4/B7-1 complex, which is in agreement with the results from our binding assay experiments and previous studies. CONCLUSIONS: Per-residue energy decomposition of the binding affinities of the two complexes revealed the unique fingerprint hot-spot sites in CTLA-4/B7-1 and CD28/B7-1 complexes. GENERAL SIGNIFICANCE: The results presented in this work will, on a long-run, be useful to develop new generation of specific CD28 and CTLA-4 inhibitors for targeted immunotherapy.

Computational Model of Chimeric Antigen Receptors Explains Site-Specific Phosphorylation Kinetics.

Chimeric antigen receptors (CARs) have recently been approved for the treatment of hematological malignancies, but our lack of understanding of the basic mechanisms that activate these proteins has made it difficult to optimize and control CAR-based therapies. In this study, we use phosphoproteomic mass spectrometry and mechanistic computational modeling to quantify the in vitro kinetics of individual tyrosine phosphorylation on a variety of CARs. We show that each of the 10 tyrosine sites on the CD28-CD3ζ CAR is phosphorylated by lymphocyte-specific protein-tyrosine kinase (LCK) with distinct kinetics. The addition of CD28 at the N-terminal of CD3ζ increases the overall rate of CD3ζ phosphorylation. Our computational model identifies that LCK phosphorylates CD3ζ through a mechanism of competitive inhibition. This model agrees with previously published data in the literature and predicts that phosphatases in this system interact with CD3ζ through a similar mechanism of competitive inhibition. This quantitative modeling framework can be used to better understand CAR signaling and T cell activation.

Clinical and immunological responses after CD30-specific chimeric antigen receptor-redirected lymphocytes.

BACKGROUND: Targeting CD30 with monoclonal antibodies in Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) has had profound clinical success. However, adverse events, mainly mediated by the toxin component of the conjugated antibodies, cause treatment discontinuation in many patients. Targeting CD30 with T cells expressing a CD30-specific chimeric antigen receptor (CAR) may reduce the side effects and augment antitumor activity. METHODS: We conducted a phase I dose escalation study in which 9 patients with relapsed/refractory HL or ALCL were infused with autologous T cells that were gene-modified with a retroviral vector to express the CD30-specific CAR (CD30.CAR-Ts) encoding the CD28 costimulatory endodomain. Three dose levels, from 0.2 × 108 to 2 × 108 CD30.CAR-Ts/m2, were infused without a conditioning regimen. All other therapy for malignancy was discontinued at least 4 weeks before CD30.CAR-T infusion. Seven patients had previously experienced disease progression while being treated with brentuximab. RESULTS: No toxicities attributable to CD30.CAR-Ts were observed. Of 7 patients with relapsed HL, 1 entered complete response (CR) lasting more than 2.5 years after the second infusion of CD30.CAR-Ts, 1 remained in continued CR for almost 2 years, and 3 had transient stable disease. Of 2 patients with ALCL, 1 had a CR that persisted 9 months after the fourth infusion of CD30.CAR-Ts. CD30.CAR-T expansion in peripheral blood peaked 1 week after infusion, and CD30.CAR-Ts remained detectable for over 6 weeks. Although CD30 may also be expressed by normal activated T cells, no patients developed impaired virus-specific immunity. CONCLUSION: CD30.CAR-Ts are safe and can lead to clinical responses in patients with HL and ALCL, indicating that further assessment of this therapy is warranted. TRIAL REGISTRATION: ClinicalTrials.gov NCT01316146. FUNDING: National Cancer Institute (3P50CA126752, R01CA131027 and P30CA125123), National Heart, Lung, and Blood Institute (R01HL114564), and Leukemia and Lymphoma Society (LLSTR 6227-08).

Chemical proteomic map of dimethyl fumarate-sensitive cysteines in primary human T cells.

Dimethyl fumarate (DMF) is an electrophilic drug that is used to treat autoimmune conditions, including multiple sclerosis and psoriasis. The mechanism of action of DMF is unclear but may involve the covalent modification of proteins or DMF serving as a prodrug that is converted to monomethyl fumarate (MMF). We found that DMF, but not MMF, blocked the activation of primary human and mouse T cells. Using a quantitative, site-specific chemical proteomic platform, we determined the DMF sensitivity of >2400 cysteine residues in human T cells. Cysteines sensitive to DMF, but not MMF, were identified in several proteins with established biochemical or genetic links to T cell function, including protein kinase Cθ (PKCθ). DMF blocked the association of PKCθ with the costimulatory receptor CD28 by perturbing a CXXC motif in the C2 domain of this kinase. Mutation of these DMF-sensitive cysteines also impaired PKCθ-CD28 interactions and T cell activation, designating the C2 domain of PKCθ as a key functional, electrophile-sensing module important for T cell biology.

Expression, Clinical Significance, and Receptor Identification of the Newest B7 Family Member HHLA2 Protein.

PURPOSE: HHLA2 (B7H7/B7-H5/B7y) is a newly identified B7 family member that regulates human T-cell functions. However, its protein expression in human organs and significance in human diseases are unknown. The objective of this study was to analyze HHLA2 protein expression in normal human tissues and cancers, as well as its prognostic significance, to explore mechanisms regulating HHLA2 expression, and to identify candidate HHLA2 receptors. EXPERIMENTAL DESIGN: An immunohistochemistry protocol and a flow cytometry assay with newly generated monoclonal antibodies were developed to examine HHLA2 protein. HHLA2 gene copy-number variation was analyzed from cancer genomic data. The combination of bioinformatics analysis and immunologic approaches was established to explore HHLA2 receptors. RESULTS: HHLA2 protein was detected in trophoblastic cells of the placenta and the epithelium of gut, kidney, gallbladder, and breast, but not in most other organs. In contrast, HHLA2 protein was widely expressed in human cancers from the breast, lung, thyroid, melanoma, pancreas, ovary, liver, bladder, colon, prostate, kidney, and esophagus. In a cohort of 50 patients with stage I-III triple-negative breast cancer, 56% of patients had aberrant expression of HHLA2 on their tumors, and high HHLA2 expression was significantly associated with regional lymph node metastasis and stage. The Cancer Genome Atlas revealed that HHLA2 copy-number gains were present in 29% of basal breast cancers, providing a potential mechanism for increased HHLA2 protein expression in breast cancer. Finally, Transmembrane and Immunoglobulin Domain Containing 2 (TMIGD2) was identified as one of the receptors for HHLA2. CONCLUSIONS: Wide expression of HHLA2 in human malignancies, together with its association with poor prognostic factors and its T-cell coinhibitory capability, suggests that the HHLA2 pathway represents a novel immunosuppressive mechanism within the tumor microenvironment and an attractive target for human cancer therapy.

Phosphatidylinositol 4-phosphate 5-kinase α and Vav1 mutual cooperation in CD28-mediated actin remodeling and signaling functions.

Phosphatidylinositol 4,5-biphosphate (PIP2) is a cell membrane phosphoinositide crucial for cell signaling and activation. Indeed, PIP2 is a pivotal source for second messenger generation and controlling the activity of several proteins regulating cytoskeleton reorganization. Despite its critical role in T cell activation, the molecular mechanisms regulating PIP2 turnover remain largely unknown. In human primary CD4(+) T lymphocytes, we have recently demonstrated that CD28 costimulatory receptor is crucial for regulating PIP2 turnover by allowing the recruitment and activation of the lipid kinase phosphatidylinositol 4-phosphate 5-kinase (PIP5Kα). We also identified PIP5Kα as a key modulator of CD28 costimulatory signals leading to the efficient T cell activation. In this study, we extend these data by demonstrating that PIP5Kα recruitment and activation is essential for CD28-mediated cytoskeleton rearrangement necessary for organizing a complete signaling compartment leading to downstream signaling functions. We also identified Vav1 as the linker molecule that couples the C-terminal proline-rich motif of CD28 to the recruitment and activation of PIP5Kα, which in turn cooperates with Vav1 in regulating actin polymerization and CD28 signaling functions.

Polymer-based synthetic dendritic cells for tailoring robust and multifunctional T cell responses.

Dendritic cells (DCs) are antigen-presenting cells that play an essential role in T cell activation. Recent efforts in cancer immunotherapy have been directed at the development of artificial antigen presenting cells (aAPCs) loaded with tumor antigens. These aAPCs are designed to mimic DCs with the goal of triggering an efficient and specific T cell response directed against the tumor. We have designed a novel synthetic dendritic cell (sDC) that possesses the essential features of natural DCs. Our sDC is based on a semiflexible poly(isocyano peptide) polymer and carries anti-CD3 antibodies (αCD3) for triggering the T cell receptor/CD3 complex as well as anti-CD28 antibodies (αCD28) as a co-stimulatory signal. Multiple copies of both antibodies facilitate multivalent binding similar to natural DCs. The high mobility of these polymer-bound antibodies, reminiscent of protein motility in a natural plasma membrane, enables receptor rearrangements to occur during T cell activation. We show that our bifunctional αCD3/αCD28-sDC triggers T cell activation at significantly lower antibody concentrations than freely soluble antibodies. This superior performance is further demonstrated in comparison to a mixture of monofunctional αCD3-sDC and αCD28-sDC. The presence of both antibodies on the same polymer not only reduces the threshold for T cell activation but, more importantly, critically shapes the specificity of the T cell response. αCD3/αCD28-sDC is a far more efficient activator of multifunctional killer cells. These findings demonstrate the potential of multifunctional polymers for mimicking natural DCs, paving the way for their exploitation in immunotherapeutic strategies.

CD28 homodimer interface mimetic peptide acts as a preventive and therapeutic agent in models of severe bacterial sepsis and gram-negative bacterial peritonitis.

BACKGROUND: Severe gram-negative bacterial infections and sepsis are major causes of morbidity and mortality. Dysregulated, excessive proinflammatory cytokine expression contributes to the pathogenesis of sepsis. A CD28 mimetic peptide (AB103; previously known as p2TA) that attenuates CD28 signaling and T-helper type 1 cytokine responses was tested for its ability to increase survival in models of polymicrobial infection and gram-negative sepsis. METHODS: Mice received AB103, followed by an injection of Escherichia coli 0111:B4 lipopolysaccharide (LPS); underwent induction E. coli 018:K1 peritonitis induction, followed by treatment with AB103; or underwent cecal ligation and puncture (CLP), followed by treatment with AB103. The effects of AB103 on factors associated with and the lethality of challenge infections were analyzed. RESULTS: AB103 strongly attenuated induction of tumor necrosis factor α and interleukin 6 (IL-6) by LPS in human peripheral blood mononuclear cells. Receipt of AB103 following intraperitoneal injection of LPS resulted in survival among 73% of CD1 mice (11 of 15), compared with 20% of controls (3 of 15). Suboptimal doses of antibiotic alone protected 20% of mice (1 of 5) from E. coli peritonitis, whereas 100% (15 of 15) survived when AB103 was added 4 hours following infection. Survival among mice treated with AB103 12 hours after CLP was 100% (8 of 8), compared with 17% among untreated mice (1 of 6). In addition, receipt of AB103 12 hours after CLP attenuated inflammatory cytokine responses and neutrophil influx into tissues and promoted bacterial clearance. Receipt of AB103 24 hours after CLP still protected 63% of mice (5 of 8). CONCLUSIONS: Single-dose AB103 reduces mortality in experimental models of polymicrobial and gram-negative bacterial infection and sepsis, warranting further studies of this agent in clinical trials.