IgV Domain Research Articles

VSTM2A reverses immunosuppression in colorectal cancer by antagonizing the PD-L1/PD-1 interaction

Immunoglobulin (Ig) VSTM2A (V-set and transmembrane domain containing 2A) is a top-ranked secretory protein frequently silenced during colorectal carcinogenesis; however, its role in immune modulation remains largely unknown. Bioinformatic and immunohistochemistry analysis of human colorectal specimens and Vstm2a+/- knockout mice indicated that VSTM2A positively correlated with CD8a and immune infiltration in both physiological and pathological conditions. We then utilized liquid chromatography-mass spectrometry to pinpoint programmed death ligand 1 (PD-L1) as a membrane receptor of VSTM2A. A series of invitro biochemistry assays further revealed the binding pattern and kinetics between VSTM2A and PD-L1 proteins through their IgV domains ata dissociation constant of 0.7-2.5nM. Recombinant VSTM2A protein inhibited the PD-1/PD-L1 interaction and induced NFAT response element (RE) luciferase activity dose dependently. Furthermore, interleukin (IL)-2 production from DO11.10 Tcells upon co-culture with mouse non-T splenocytes was upregulated in the presence of VSTM2A conditioned medium. Finally, tumor killing assay and exvivo data from human peripheral blood mononuclear cells and autologous dendritic cell-T cell co-culture demonstrated that VSTM2A significantly enhanced immune activation via the release of granzyme B and interferon (IFN)-γ cytokines. In conclusion, our study demonstrates the tumor-extrinsic role of VSTM2A in sterically blocking the PD-L1/PD-1 interaction at a picomole to nanomole affinity, which leads to the enhanced anti-tumor effect of cytotoxic Tcells.

The constant domain of CRTAM is essential for high-affinity interaction with Nectin-like 2

CRTAM (Class-I MHC restricted T cell-associated molecule) is a member of the Nectin-like family, composed of two extracellular domains, one constant domain (IgC) and another variable domain (IgV), expressed in activated CD8 T cells, epithelial cells, natural killer (NK) cells, and in a subpopulation of CD4 T cells. CRTAM recognizes the ligand Nectin-like 2 (Necl2) through the IgV domain. However, the role of the IgC domain during this ligand recognition has yet to be understood. In this study, we show the purification of soluble-folded Ig domains of CRTAM, and we demonstrate that the IgC domain forms a homodimer in solution via hydrophobic interactions. By surface plasmon resonance (SPR) analysis, we also demonstrate that CRTAM binds to Necl2 with an affinity of 2.16 nM. In conclusion, CRTAM's IgC is essential for a high-affinity interaction with Necl-2.

Dual-Action Protein-siRNA Conjugates for Targeted Disruption of CD47-Signal Regulatory Protein α Axis in Cancer Therapy.

A series of successes in RNA interference (RNAi) therapies for liver diseases using lipid nanoparticles and N-acetylgalactosamine have heralded a current era of RNA therapeutics. However, alternative delivery strategies are required to take RNAi out of the comfort zone of hepatocytes. Here we report SIRPα IgV/anti-CD47 siRNA (vS-siCD47) conjugates that selectively and persistently disrupt the antiphagocytic CD47/SIRPα axis in solid tumors. Conjugation of the SIRPα IgV domain protein to siRNAs enables tumor dash through CD47-mediated erythrocyte piggyback, primarily blocking the physical interaction between CD47 on cancer cells and SIRPα on phagocytes. After internalization of the vS-siCD47 conjugates within cancer cells, the detached free-standing anti-CD47 siRNAs subsequently attack CD47 through the RNAi mechanism. The dual-action approach of the vS-siCD47 conjugate effectively overcomes the "don't eat me" barrier and stimulates phagocyte-mediated tumor destruction, demonstrating a highly selective and potent CD47-blocking immunotherapy. This delivery strategy, employing IgV domain protein-siRNA conjugates with a dual mode of target suppression, holds promise for expanding RNAi applications beyond hepatocytes and advancing RNAi-based cancer immunotherapies for solid tumors.

TOP CAR with TMIGD2 as a safe and effective costimulatory domain in CAR cells treating human solid tumors.

Chimeric antigen receptor (CAR)-T cell therapy shows impressive efficacy treating hematologic malignancies but requires further optimization in solid tumors. Here, we developed a TMIGD2 optimized potent/persistent (TOP) CAR that incorporated the costimulatory domain of TMIGD2, a T and NK cell costimulator, and monoclonal antibodies targeting the IgV domain of B7-H3, an immune checkpoint expressed on solid tumors and tumor vasculature. Comparing second- and third-generation B7-H3 CARs containing TMIGD2, CD28, and/or 4-1BB costimulatory domains revealed superior antitumor responses in B7-H3.TMIGD2 and B7-H3.CD28.4-1BB CAR-T cells in vitro. Comparing these two constructs using in vivo orthotopic human cancer models demonstrated that B7-H3.TMIGD2 CAR-T cells had equivalent or superior antitumor activity, survival, expansion, and persistence. Mechanistically, B7-H3.TMIGD2 CAR-T cells maintained mitochondrial metabolism; produced less cytokines; and established fewer exhausted cells, more central memory cells, and a larger CD8/CD4 T cell ratio. These studies demonstrate that the TOP CAR with TMIGD2 costimulation offered distinct benefits from CD28.41BB costimulation and is effective against solid tumors.

Abstract P18: Splicing polymorphism rs12459419 modulates antibody-bound internalization of CD33 isoforms

Abstract P18: Splicing polymorphism rs12459419 modulates antibody-bound internalization of CD33 isoforms

Identification, expression and molecular polymorphism of T-cell receptors α and β from the glacial relict Hucho bleekeri

The T-cell receptor (TCR) is a specific molecule on the surface of all T cells that mediates cellular adaptive immune responses to antigens. Hucho bleekeri is a critically endangered species and is regarded as a glacial relict that has the lowest-latitude distribution compared with any Eurasian salmonid. In the present study, two TCR genes, namely, TCR α and β, were identified and characterized in H. bleekeri. Both TCR α and TCR β have typical TCR structures, including the IgV domain, IgC domain, connecting peptide, transmembrane and cytoplasmic domains. The two TCR genes were constitutionally expressed in various tissues, with the highest expression found in the spleen for TCR α and in the trunk kidney for TCR β. Challenge of H. bleekeri with LPS or poly(I:C) resulted in significant upregulation of both TCR α and β expression in headkidney and spleen primary cells, indicating their potential roles in the immune response. Molecular polymorphism analysis of the whole ORF regions of TCR α and β in different individuals revealed high diversity of IgV domains of these two genes, especially in complementarity-determining region (CDR) 3. The ratio of nonsynonymous substitution occurred at a significantly higher frequency than synonymous substitution in the CDR of TCR α and β, demonstrating the existence of positive selection. The results obtained in the present study enhance our understanding of TCR roles in regulating immune mechanisms and provide new information for the study of TCR lineage diversity in fish.

An Enzyme‐Cleavable Solubilizing‐Tag Facilitates the Chemical Synthesis of Mirror‐Image Proteins

AbstractMirror‐image proteins (D‐proteins) are useful in biomedical research for purposes such as mirror‐image screening for D‐peptide drug discovery, but the chemical synthesis of many D‐proteins is often low yielding due to the poor solubility or aggregation of their constituent peptide segments. Here, we report a Lys‐C protease‐cleavable solubilizing tag and its use to synthesize difficult‐to‐obtain D‐proteins. Our tag is easily installed onto multiple amino acids such as DLys, DSer, DThr, and/or the N‐terminal amino acid of hydrophobic D‐peptides, is impervious to various reaction conditions, such as peptide synthesis, ligation, desulfurization, and transition metal‐mediated deprotection, and yet can be completely removed by Lys‐C protease under denaturing conditions to give the desired D‐protein. The efficacy and practicality of the new method were exemplified in the synthesis of two challenging D‐proteins: D‐enantiomers of programmed cell death protein 1 IgV domain and SARS‐CoV‐2 envelope protein, in high yield. This work demonstrates that the enzymatic cleavage of solubilizing tags under denaturing conditions is feasible, thus paving the way for the production of more D‐proteins.

An Enzyme-Cleavable Solubilizing-Tag Facilitates the Chemical Synthesis of Mirror-Image Proteins.

Mirror-image proteins (D-proteins) are useful in biomedical research for purposes such as mirror-image screening for D-peptide drug discovery, but the chemical synthesis of many D-proteins is often low yielding due to the poor solubility or aggregation of their constituent peptide segments. Here, we report a Lys-C protease-cleavable solubilizing tag and its use to synthesize difficult-to-obtain D-proteins. Our tag is easily installed onto multiple amino acids such as DLys, DSer, DThr, and/or the N-terminal amino acid of hydrophobic D-peptides, is impervious to various reaction conditions, such as peptide synthesis, ligation, desulfurization, and transition metal-mediated deprotection, and yet can be completely removed by Lys-C protease under denaturing conditions to give the desired D-protein. The efficacy and practicality of the new method were exemplified in the synthesis of two challenging D-proteins: D-enantiomers of programmed cell death protein 1 IgV domain and SARS-CoV-2 envelope protein, in high yield. This work demonstrates that the enzymatic cleavage of solubilizing tags under denaturing conditions is feasible, thus paving the way for the production of more D-proteins.

N-terminal ectodomain of BTNL2 inhibits T cell activation via a non-canonical interaction with its putative receptor that results in a delayed progression of DSS-induced ulcerative colitis

Butyrophilin-like 2 (BTNL2) is a T cell inhibitory molecule that interacts with unknown binding partners to modulate the immune response in a number of inflammatory and autoimmune diseases. In this study, we found that the inhibitory effects of BTNL2 on T cell activation and effector functions can be executed by its N-terminal IgV domain (BTNL2 IgV1) alone. Structure-guided mutation of key residues on BTNL2 IgV1 based on known receptor-ligand interfaces involving immunoglobulin superfamily members revealed that BTNL2 uses a non-canonical binding interface with its putative receptor. A high avidity BTNL2 IgV1 probe revealed that in an inducible model of ulcerative colitis, severe colitis was accompanied by a selective enrichment of BTNL2-receptor expressing effector-memory CD4+ and CD8+ T cells in the Peyer’s patches. Intraperitoneal administration of BTNL2 IgV1 resulted in a significant delay in the progression of DSS-induced colitis and also showed reduced activation of the BTNL2-receptor-expressing T cells in the Peyer’s patches. Thus, this study demonstrates that the BTNL2-receptor-expressing T cells in the Peyer’s patches participate in the disease pathogenesis and can serve as a novel therapeutic target in ulcerative colitis, which can be modulated by BTNL2 IgV1.

Splicing Polymorphism rs12459419 Modulates Antibody-Bound Internalization of CD33 Isoforms

Background: While the standard regimen for AML has been intense chemotherapy followed by stem-cell transplantation (SCT), addition of immunotherapeutics such as gemtuzumab ozogamicin (GO) to this has shown to improve outcomes in multiple clinical trials. Since these trials, GO has been added to the National Comprehensive Cancer Network (NCCN) practice guidelines for use in AML therapy (NCCN., 2018). GO is a humanized monoclonal antibody conjugated to calicheamicin and targets the cell surface antigen CD33, observed on most AML blasts. Upon binding to GO, CD33 is internalized and the subsequent release of calicheamicin mediates cytotoxicity. CD33 contains two extracellular domains: IgV and IgC, the IgV domain is coded by CD33 exon 2 and is the binding site for GO. A splicing SNP, rs12459419 (C>T; A14V) in exon 2 results in a shorter isoform (CD33-D2) and has been previously associated with CD33 abundance on cell surface as well as clinical benefit from GO addition to standard chemotherapy. Patients with CC genotype (~50% of the cohort) expressed full length CD33 (CD33-FL) and benefited significantly from GO. However, heterozygous CT and homozygous TT genotype groups did not derive the same benefit. These results were perplexing as CT genotype group had intermediate levels of CD33-FL and CD33-D2 isoforms (Lamba et al., 2017). It is hypothesized that lack of IgV domain in CD33-D2 and putative CD33-FL/CD33-D2 dimerization prevents GO binding and subsequent internalization in rs12459419 CT/TT genotype cells, however this has never been tested. We have generated novel antibodies directed against the IgC domain that recognizes CD33-D2 in AML cell lines as well as primary AML cells (Gbadamosi et al., 2021) thus facilitating testing the above indicated hypothesis. Objective: In this study, we first tested the ability of antibody-bound CD33-D2 to internalize, followed by impact of the internalization status of CD33-FL and CD33-D2 in cells co-expressing both isoforms along with the concomitant impact on GO induced cytotoxicity. Methods: Using pMXs retroviral particles encoding CD33-FL or CD33-D2, we engineered CRISPR/Cas9 mediated CD33 KO in AML cell lines to overexpress either one or both CD33 isoforms to mimic cells expression varying proportions of CD33-FL/CD33-D2. Internalization of either CD33-FL or CD33-D2 over a duration of 4 hours in the presence of antibodies recognizing either of the two isoforms was performed. Cells were labeled with unconjugated mouse anti-CD33-FL P67.6 or anti-CD33-D2 HL2541 antibody on ice and incubated at 37°C for 0-4 hours, followed by staining with mouse anti-mouse IgG antibody conjugated with biotin. Surface CD33-FL or CD33-D2 was then detected using streptavidin-FITC conjugate. Percentage antibody-bound CD33-FL or CD33-D2 internalization was expressed as change in FITC MFI between each time point incubated and cells kept at 0°C post CD33 antibody labeling. Results: While P67.6-bound-CD33-FL internalized as expected in AML cell lines expressing CD33-FL only (Fig. 1A), we show for the first time that HL2541-bound-CD33-D2 also internalizes upon antibody binding in cell lines expressing CD33-D2 isoform only (Fig. 1B). In AML cell lines co-expressing both CD33-FL and CD33-D2 isoforms, a >2.5-3 fold decrease in this antibody-bound internalization was observed for each isoform (Figs. 1C and D). In response to GO exposure for 48 hours, cells co-expressing both CD33-FL and CD33-D2 isoforms had 7-9.5-fold higher IC50 values (233 nmol and 277 nmol) compared to cells expressing only CD33-FL (33.19 nmol and 29 nmol) (Figs. 1E and F). As expected, cells expressing CD33-D2 only did respond to GO. Discussion: Our finding that antibody-bound CD33-D2 internalizes like CD33-FL adds a novel piece of evidence in understanding the biology of this variant isoform and opens opportunities to develop antibody drug conjugate (ADC) such as GO using CD33-D2 antibody. This new CD33-D2 targeting ADC will provide a treatment option in patients with TT genotypes. Our results also provide a mechanistic insight on the previous clinical observation showing lack of benefit from GO in patients heterozygous for the splicing SNP. Our ongoing studies are focused on validating this result using live cell imaging and efforts towards design of new ADC or T cell engagers using CD33-D2 antibody. The study was supported by Leukemia Lymphoma Society, UFHealth Cancer Center, and the Frank A. Duckworth Endowment.

Safety and Preliminary Efficacy of Sabestomig (AZD7789), an Anti-PD-1 and Anti-TIM-3 Bispecific Antibody, in Patients with Relapsed or Refractory Classical Hodgkin Lymphoma Previously Treated with Anti-PD-(L)1 Therapy

Background Immunotherapies targeting the PD-1/PD-L1 pathway have been shown to induce high response rates in patients with relapsed/refractory (r/r) classical Hodgkin lymphoma (cHL). However, most responses are partial and not durable. T cell immunoglobulin and mucin domain-containing protein-3 (TIM-3) is implicated as a resistance mechanism following anti-PD-1 therapy, and increased TIM-3 expression has been observed on T cells of patients with cHL post anti-PD-1 therapy. Therefore, we hypothesized that dual targeting of PD-1 and TIM-3 may reinvigorate immune responses and lead to more durable antitumor activity. Sabestomig is a monovalent, bispecific, humanized IgG1 monoclonal antibody that binds PD-1 and a unique epitope in the TIM-3 IgV domain without blocking phosphatidylserine binding, eliciting differential functionality. In preclinical mouse models, sabestomig was more effective at inhibiting growth of solid tumors than treatment targeting only PD-1, and sequential sabestomig after anti-PD-1 therapy increased antitumor responses. Here we report preliminary data from the ongoing dose escalation portion of a Phase I/II, open-label, multicenter study to assess the safety and preliminary efficacy of sabestomig in patients with r/r cHL (NCT05216835). Methods Eligible patients are aged ≥16 years with r/r cHL, an Eastern Cooperative Oncology Group performance status 0-1, at least one positron emission tomography-avid measurable lesion according to modified Lugano Criteria, and exposure to at least 2 prior lines of systemic therapy including a minimum of 3 cycles of an anti-PD-(L)1-based therapy. Sabestomig is intravenously infused over 1 hour following pretreatment with diphenhydramine and acetaminophen every 3 weeks, with planned doses ranging from 2 to 2000 mg across 8 cohorts (A1-A8); cohorts A1 to A4 (2, 7, 22.5 or 75 mg) follow an accelerated titration design with a single patient treated at each dose level, while cohorts A5 to A8 (225-2000 mg) follow a modified toxicity probability interval-2 algorithm. The primary endpoint of the dose escalation part of the study is safety, including dose-limiting toxicities (DLTs). Secondary endpoints include efficacy, pharmacokinetics, and immunogenicity. Data cutoffs were May 17, 2023 for safety and June 20, 2023 for efficacy. Results As of May 17, 2023, 14 patients were treated across the first 6 cohorts (A1-A6; 2-750 mg). They were predominantly male (64.3%) with Stage IV disease (85.7%) and a median age of 41.5 years (range, 25-77). Patients received a median of 6 prior lines of therapy (range, 3-13). Baseline disease characteristics are summarized in Table 1. Four patients received sabestomig 2-75 mg in cohorts A1-A4 (n=1 each), 5 patients received 225 mg in cohort A5, and 5 patients received 750 mg in cohort A6. Median duration of exposure to sabestomig was 8.9 weeks (range, 3.0-34.3) with a median of 3 cycles received (range 1-12). Sabestomig was well tolerated with no Grade ≥3 treatment-related adverse events (AEs) (Table 2). One Grade ≥3 AE occurred, which was fatal (sepsis secondary to gastric ulcer rupture, not related to sabestomig but deemed a DLT by the safety review committee). No immune-mediated AEs were reported, and no AEs led to treatment discontinuation. At the safety data cutoff, treatment was ongoing for 6 patients (42.9%). As of June 20, 2023, 11 patients had their first disease assessment and 4/11 had an objective response based on modified Lugano Criteria: 1 patient in cohort A3 (22.5 mg, a partial response [PR]) and all 3 patients who had their first disease assessment in cohort A6 (750 mg, 2 CRs and 1 PR). Two of these 4 responders (both in cohort A6) had experienced a best overall response of progressive disease during prior anti-PD-1 therapy; the other two initially had a response (1 CR [cohort A6] and 1 PR [cohort A3]) but subsequently progressed on anti-PD-1 therapy. Sustained PD-1 receptor occupancy (≥90%) was observed in peripheral blood at doses ≥225 mg. Conclusions Sabestomig was well tolerated with a manageable safety profile. Early efficacy data at 750 mg is encouraging with objective responses in 3/3 patients who had their first disease assessment, including those who were anti-PD-1 refractory or who relapsed on treatment. Updated data will be presented from ongoing and subsequent patients treated during dose escalation with doses of up to 2000 mg.

An alternatively spliced PD-L1 isoform PD-L1∆3, and PD-L2 expression in breast cancers: implications for eligibility scoring and immunotherapy response.

Targeting PD-1/PD-L1 has shown substantial therapeutic response and unprecedented long-term durable responses in the clinic. However, several challenges persist, encompassing the prediction of treatment effectiveness and patient responses, the emergence of treatment resistance, and the necessity for additional biomarkers. Consequently, we comprehensively explored the often-overlooked isoforms of crucial immunotherapy players, leveraging transcriptomic analysis, structural modeling, and immunohistochemistry (IHC) data. Our investigation has led to the identification of an alternatively spliced isoform of PD-L1 that lacks exon 3 (PD-L1∆3) and the IgV domain required to interact with PD-1. PD-L1∆3 is expressed more than the canonical isoform in a subset of breast cancers and other TCGA tumors. Using the deep learning-based protein modeling tool AlphaFold2, we show the lack of a possible interaction between PD-L1∆3 and PD-1. In addition, we present data on the expression of an additional ligand for PD-1, PD-L2. PD-L2 expression is widespread and positively correlates with PD-L1 levels in breast and other tumors. We report enriched epithelial-mesenchymal transition (EMT) signature in high PD-L2 transcript expressing (PD-L2 > PD-L1) tumors in all breast cancer subtypes, highlighting potential crosstalk between EMT and immune evasion. Notably, the estrogen gene signature is downregulated in ER + breast tumors with high PD-L2. The data on PD-L2 IHC positivity but PD-L1 negativity in breast tumors, together with our results on PD-L1∆3, highlight the need to utilize PD-L2 and PD-L1 isoform-specific antibodies for staining patient tissue sections to offer a more precise prediction of the outcomes of PD-1/PD-L1 immunotherapy.

Camel nanobody-based B7-H3 CAR-T cells show high efficacy against large solid tumours

Rational design of chimeric antigen receptor T (CAR-T) cells based on the recognition of antigenic epitopes capable of evoking the most potent CAR activation is an important objective in optimizing immune therapy. In solid tumors, the B7-H3 transmembrane protein is an emerging target that harbours two distinct epitope motifs, IgC and IgV, in its ectodomain. Here, we generate dromedary camel nanobodies targeting B7-H3 and demonstrate that CAR-T cells, based on the nanobodies recognizing the IgC but not IgV domain, had potent antitumour activity against large tumors in female mice. These CAR-T cells are characterized by highly activated T cell signaling and significant tumor infiltration. Single-cell transcriptome RNA sequencing coupled with functional T-cell proteomics analysis uncovers the top-upregulated genes that might be critical for the persistence of polyfunctional CAR-T cells in mice. Our results highlight the importance of the specific target antigen epitope in governing optimal CAR-T activity and provide a nanobody-based B7-H3 CAR-T product for use in solid tumor therapy.

Interaction between hTIM-1 and Envelope Protein Is Important for JEV Infection.

Japanese encephalitis virus (JEV), a mosquito-borne zoonotic virus, is one of the most important causes of human viral encephalitis. JEV relies on various attachment or entry co-factors to enter host cells. Among these co-factors, hTIM-1 has been identified as an attachment factor to promote JEV infection through interacting with phosphatidylserine (PS) on the viral envelope. However, the reasons why JEV prefers to use hTIM-1 over other PS binding receptors are unknown. Here, we demonstrated that hTIM-1 can directly interact with JEV E protein. The interaction between hTIM-1 and JEV relies on specific binding sites, respectively, ND114115 in the hTIM-1 IgV domain and K38 of the E protein. Furthermore, during the early stage of infection, hTIM-1 and JEV are co-internalized into cells and transported into early and late endosomes. Additionally, we found that the hTIM-1 soluble ectodomain protein effectively inhibits JEV infection in vitro. Moreover, hTIM-1-specific antibodies have been shown to downregulate JEV infectivity in cells. Taken together, these findings suggested that hTIM-1 protein directly interacts with JEV E protein and mediates JEV infection, in addition to the PS-TIM-1 interaction.

Discovery of TIGIT inhibitors by phage display

Abstract TIGIT is an emerging immune checkpoint receptor expressed higher than PD-1 in some tumors especially anti-PD1 resistant tumors. By competing with the co-stimulatory receptor CD226/DNAM-1 for binding to CD155/PVR, TIGIT transduces inhibitory signals and suppresses the immune response. Therefore, we sought to discover TIGIT inhibitors for improving tumor immunotherapy. Through directional cloning, bacterial transformation and phage amplification techniques, we generated a novel genetically encoded, phage-displayed bicyclic peptide library comprised of more than a million peptide sequences. Depending on the two combinations of different disulfide bonds, each peptide backbone is predicted to be folded into either endo-bicyclic or exo-bicyclic structure. We conducted three rounds of phage display panning against recombinant TIGIT IgV domain that enriched high-affinity peptides and identified promising TIGIT binding peptides. Phage ELISA showed binding of phage-presented peptides to recombinant TIGIT IgV and competitive ELISA evaluated the ability of the peptides to disrupt interactions between TIGIT and PVR. This work validated the efficiency of the phage-displayed bicyclic peptide library for discovery of ligands targeting protein receptors and led to identification of novel TIGIT inhibitors. AAI Careers in Immunology Fellowship

Abstract 3233: A novel triple humanized B-hPD-1/hPD-L1/hCCR8 mouse model to evaluate in vivo efficacy of combination therapies

Abstract Regulatory T cells (Tregs) play an immunosuppressive role in the tumor microenvironment (TME), such that altering or depleting Treg function has emerged as an important anti-tumor strategy. Targeting CCR8, which is highly expressed in tumor-infiltrating Tregs, has shown its potency in reducing tumor burden and increasing responsiveness to immune-checkpoint blockade therapy. Collectively, this suggests a need for preclinical models that support Treg-targeted therapies. To address this, Biocytogen developed a triple humanized (B-hPD-1/hPD-L1/hCCR8) mouse model to evaluate in vivo efficacy of combination therapies. In this model, the full coding sequence of murine Ccr8 was replaced by the full coding sequence of human CCR8, while murine Pd-1 and Pd-l1 IgV domains were replaced by their human counterpart gene sequences. Human PD-1 and PD-L1 protein expression was confirmed in B-hPD-1/hPD-L1/hCCR8 mice by flow cytometry, and human CCR8 protein expression was observed in MC38 tumors harvested from B-hPD-1/hPD-L1/hCCR8 mice. Finally, combination therapy using anti-human PD-1 and anti-human CCR8 antibodies were effective in controlling B-hPD-L1 MC38 tumor growth in B-hPD-1/hPD-L1/hCCR8 mice. Altogether, this line provides a powerful preclinical model for in vivo evaluation of therapeutic antibodies. Citation Format: Linlin Wang, Xiao Liu, James Jin, Yuxiang Yu. A novel triple humanized B-hPD-1/hPD-L1/hCCR8 mouse model to evaluate in vivo efficacy of combination therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3233.

Phosphoantigen sensing combines TCR-dependent recognition of the BTN3A IgV domain and germline interaction with BTN2A1

Vγ9Vδ2 Tcells play critical roles in microbial immunity by detecting target cells exposed to pathogen-derived phosphoantigens (P-Ags). Target cell expression of BTN3A1, the "P-Ag sensor," and BTN2A1, a direct ligand for Tcell receptor (TCR) Vγ9, is essential for this process; however, the molecular mechanisms involved are unclear. Here, we characterize BTN2A1 interactions with Vγ9Vδ2 TCR and BTN3A1. Nuclear magnetic resonance (NMR), modeling, and mutagenesis establish a BTN2A1-immunoglobulin V (IgV)/BTN3A1-IgV structural model compatible with their cell-surface association in cis. However, TCR and BTN3A1-IgV binding to BTN2A1-IgV is mutually exclusive, owing to binding site proximity and overlap. Moreover, mutagenesis indicates that the BTN2A1-IgV/BTN3A1-IgV interaction is non-essential for recognition but instead identifies a molecular surface on BTN3A1-IgV essential to P-Ag sensing. These results establish a critical role for BTN3A-IgV in P-Ag sensing, in mediating direct or indirect interactions with the γδ-TCR. They support a composite-ligand model whereby intracellular P-Ag detection coordinates weak extracellular germline TCR/BTN2A1 and clonotypically influenced TCR/BTN3A-mediated interactions to initiate Vγ9Vδ2 TCR triggering.

The IgV domain of the poliovirus receptor alone is immunosuppressive and binds to its receptors with comparable affinity

PVR (poliovirus receptor) functions as a ligand that signals through TIGIT and CD96 to induce suppression of T-cell and NK-cell responses. Alternatively, PVR binds to CD226, resulting in a co-stimulatory signal. To date, TIGIT antibody antagonists have been developed to restore immune functions and allow PVR to signal though CD226 in the context of cancer immunotherapy. Due to PVR receptor heterogeneity, agonizing either of these pathways with a recombinant form of the PVR extracellular domain represents a therapeutic strategy for either immunosuppression or activation. Here, we developed a minimal murine PVR-Fc fusion construct, consisting of only the IgV domain of PVR (vdPVR-Fc), and assessed its ability to dampen inflammatory responses in a murine model of psoriasis. vdPVR-Fc and PVR-Fc containing the full-length extracellular domain bound to TIGIT, CD96 and CD226 with similar low nanomolar affinities as defined by surface plasmon resonance. vdPVR-Fc was also able to suppress the in-vitro proliferation of murine CD4+ and CD8+ T-cells in mixed splenocyte cultures. Importantly, vdPVR-Fc delayed the onset, and reduced inflammatory responses (scaling and thickness) in a murine model of psoriasis. Collectively, our results suggest that the minimal IgV domain of PVR is sufficient to dampen immune responses in-vitro and attenuate symptoms of psoriasis in-vivo.

The CIS association of CD47 with integrin Mac-1 regulates macrophage responses by stabilizing the extended integrin conformation

CD47 is a ubiquitously expressed cell surface integrin-associated protein. Recently, we have demonstrated that integrin Mac-1 (αMβ2, CD11b/CD18, CR3), the major adhesion receptor on the surface of myeloid cells, can be coprecipitated with CD47. However, the molecular basis for the CD47-Mac-1 interaction and its functional consequences remain unclear. Here, we demonstrated that CD47 regulates macrophage functions directly interacting with Mac-1. In particular, adhesion, spreading, migration, phagocytosis, and fusion of CD47-deficient macrophages were significantly decreased. We validated the functional link between CD47 and Mac-1 by co-immunoprecipitation analysis using various Mac-1-expressing cells. In HEK293 cells expressing individual αM and β2 integrin subunits, CD47 was found to bind both subunits. Interestingly, a higher amount of CD47 was recovered with the free β2 subunit than in the complex with the whole integrin. Furthermore, activating Mac-1-expressing HEK293 cells with PMA, Mn2+, and activating antibody MEM48 increased the amount of CD47 in complex with Mac-1, suggesting CD47 has a greater affinity for the extended integrin conformation. Notably, on the surface of cells lacking CD47, fewer Mac-1 molecules could convert into an extended conformation in response to activation. Additionally, we identified the binding site in CD47 for Mac-1 in its constituent IgV domain. The complementary binding sites for CD47 in Mac-1 were localized in integrin epidermal growth factor-like domains 3 and 4 of the β2 and calf-1 and calf-2 domains of the α subunits. These results indicate that Mac-1 forms a lateral complex with CD47, which regulates essential macrophage functions by stabilizing the extended integrin conformation.

Investigating ps surface sensitivity and calcium dependency of hTIM-1 and hTIM-4.

Transmembrane immunoglobulin and mucin domain (TIM) protein plays an important role in the immune system. TIM protein utilizes the phosphatidylserine (PS) binding pocket of the IgV domain to recognize PS lipids on the surface of target cells, and the binding is calcium-mediated. Although all members of the TIM protein family possess PS-binding pockets, they are involved in different immune response and are expressed on the surface of different immune cells. To understand the variation of intercellular recognition mechanisms, it is important to investigate their surface sensitivities to PS and their dependency on calcium for binding. Previous work in our group showed that murine TIM1 (mTIM1) and TIM4 (mTIM4) have different PS surface sensitivities. Moreover, mTIM1 and mTIM4 can bind, to different extent, to the membrane without calcium ions, but calcium ions enhance binding. Since literature shows that genes of mTIM1 and mTIM4 are orthologs of human TIM1 (hTIM1) and TIM4 (hTIM4), respectively, we aim to understand the PS surface sensitivities and calcium dependency of hTIM1 and hTIM4, and elucidate if the difference of their PS surface sensitivities and calcium dependency preserve between murine and human TIM. hTIM1 and hTIM4 have three tryptophan amino acids, but only one of them buries into the membrane when they bind to the membrane. Using a tryptophan fluorescence assay, we quantify PS surface sensitivities and calcium dependency of hTIM1 and hTIM4. We have observed that hTIM1 binds to 7:3 POPC:POPS vesicles without calcium and the fluorescence spectrum blue shifts. By investigating factors of membrane-binding mechanism for hTIM1 and hTIM4, we help elucidate difference in TIM binding between human and murine, and may provide further insights on intercellular recognition mechanisms.