Catalog No.	PA007380.m2cLA
Product Name	Anti-Mouse CD152/CTLA4 Antibody (9D9) | PA007380.m2cLA
Supplier Name	Syd Labs, Inc.
Brand Name	Syd Labs
Synonyms	cytotoxic T-lymphocyte associated antigen 4, cytotoxic T-lymphocyte antigen 4, CD152, CTLA4
Summary	The in vivo grade recombinant mouse monoclonal antibody (clone: 9D9) specifically binds to the mouse CTLA-4 protein.
Clone	9D9
Isotype	Mouse IgG2c kappa
Specificity/Sensitivity	The in vivo grade recombinant mouse monoclonal antibody (clone: 9D9) specifically binds to the mouse CTLA-4 protein.
Applications	ELISA, flow cytometry, neutralization, functional assays such as bioanalytical PK and ADA assays, and those assays for studying biological pathways affected by the mouse CTLA-4 protein.
Form Of Antibody	0.2 uM filtered solution, pH 7.4, no stabilizers or preservatives.
Endotoxin	< 1 EU per 1 mg of the protein by the LAL method.
Purity	>95% by SDS-PAGE under reducing conditions.
Shipping	The antibody is shipped with ice pack. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage	Use a manual defrost freezer and avoid repeated freeze-thaw cycles. 12 months from date of receipt, -20 to -70°C as supplied. 1 month from date of receipt, 2 to 8°C as supplied.
Note	Recombinant mouse IgG2b isotype control, Recombinant mouse IgG2c LALAPG isotype control, and Recombinant mouse IgG2a LALAPG isotype control are available. Condition of sample preparation and optimal sample dilution should be determined experimentally by the investigator.
Order Offline	Phone: 1-617-401-8149 Fax: 1-617-606-5022 Email: message@sydlabs.com Or leave a message with a formal purchase order (PO) Or credit card.

Description

PA007380.m2cLA: Recombinant Anti-mouse CD152/CTLA4 Antibody(Clone: 9D9), Mouse IgG2c kappa, In vivo Grade

Background

The 9D9 antibody blocks the mouse cytotoxic T lymphocyte antigen-4 (CTLA-4). Blocking CTLA-4 removes an inhibitory signal from reducing the activity of T lymphocytes.

Cytotoxic T-lymphocyte antigen-4 (CTLA-4) is an inhibitory molecule that competes with the stimulatory CD28 for binding to B7 on antigen presenting cells. CTLA-4 and CD28 are both presented on the surface of T-cells.

1、Hijacking antibody-induced CTLA-4 lysosomal degradation for safer and more effective cancer immunotherapy
Yan Zhang,et al.Cell Res. 2019.PMCID: PMC6796842
“It remains unclear why the clinically used anti-CTLA-4 antibodies, popularly called checkpoint inhibitors, have severe immunotherapy-related adverse effects (irAEs) and yet suboptimal cancer immunotherapeutic effects (CITE). Here we report that while irAE-prone Ipilimumab and TremeIgG1 rapidly direct cell surface CTLA-4 for lysosomal degradation, the non-irAE-prone antibodies we generated, HL12 or HL32, dissociate from anti-ctla-4 in vivo antibody after endocytosis and allow CTLA-4 recycling to cell surface by the LRBA-dependent mechanism. Disrupting CTLA-4 recycling results in robust CTLA-4 downregulation by all anti-CTLA-4 antibodies and confers toxicity to a non-irAE-prone anti-CTLA-4 mAb. Conversely, increasing the pH sensitivity of TremeIgG1 by introducing designed tyrosine-to-histidine mutations prevents antibody-triggered lysosomal CTLA-4 downregulation and dramatically attenuates irAE. Surprisingly, by avoiding CTLA-4 downregulation and due to their increased bioavailability, pH-sensitive anti-CTLA-4 antibodies are more effective in intratumor regulatory T-cell depletion and rejection of large established tumors. Our data establish a new paradigm for cancer research that allows for abrogating irAE while increasing CITE of anti-CTLA-4 antibodies.”

2、Activity of murine surrogate antibodies for durvalumab and tremelimumab lacking effector function and the ability to deplete regulatory T cells in mouse models of cancer
Darren J Schofield,et al.MAbs. 2021.PMCID: PMC7831362
“Preclinical studies of PD-L1 and anti-ctla-4 in vivo antibody blockade have relied heavily on mouse syngeneic tumor models with intact immune systems, which facilitate dissection of immunosuppressive mechanisms in the tumor microenvironment. Commercially developed monoclonal antibodies (mAbs) targeting human PD-L1, PD-1, and CTLA-4 may not demonstrate cross-reactive binding to their mouse orthologs, and surrogate anti-mouse antibodies are often used in their place to inhibit these immune checkpoints. In each case, multiple choices exist for surrogate antibodies, which differ with respect to species of origin, affinity, and effector function. To develop relevant murine surrogate antibodies for the anti-human PD-L1 mAb durvalumab and the anti-human CTLA-4 mAb tremelimumab, rat/mouse chimeric or fully murine mAbs engineered for reduced effector function were developed and compared with durvalumab and tremelimumab. Characterization included determination of target affinity, in vivo effector function, pharmacokinetic profile, and anti-tumor efficacy in mouse syngeneic tumor models. Results showed that anti–PD-L1 and anti–CTLA-4 murine surrogates with pharmacologic properties similar to those of durvalumab and tremelimumab demonstrated anti-tumor activity in a subset of commonly used mouse syngeneic tumor models. This activity was not entirely dependent on antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis effector function, or regulatory T-cell depletion, as antibodies engineered to lack these features showed activity in models historically sensitive to checkpoint inhibition, albeit at a significantly lower level than antibodies with intact effector function.”

3、CTLA-4 antibody-drug conjugate reveals autologous destruction of B-lymphocytes associated with regulatory T cell impairment
Musleh M Muthana,et al.eLife. 2023.PMCID: PMC10735222
“Germline anti-CTLA-4 in vivo antibodydeficiency causes severe autoimmune diseases characterized by dysregulation of Foxp3+ Tregs, hyper-activation of effector memory T cells, and variable forms autoimmune cytopenia including gradual loss of B cells. Cancer patients with severe immune-related adverse events (irAE) after receiving anti-CTLA-4/PD-1 combination immunotherapy also have markedly reduced peripheral B cells. The immunological basis for B cell loss remains unexplained. Here, we probe the decline of B cells in human CTLA-4 knock-in mice by using anti-human CTLA-4 antibody Ipilimumab conjugated to a drug payload emtansine (Anti-CTLA-4 ADC). The anti-CTLA-4 ADC-treated mice have T cell hyper-proliferation and their differentiation into effector cells which results in B cell depletion. B cell depletion is mediated by both CD4 and CD8 T cells and at least partially rescued by anti-TNF-alpha antibody. These data revealed an unexpected antagonism between T and B cells and the importance of regulatory T cells in preserving B cells.”

4、Toxicological and pharmacological assessment of AGEN1884, a novel human IgG1 anti-CTLA-4 antibody
Randi B Gombos,et al.PLoS One. 2018.PMCID: PMC5884502
“CTLA-4 and CD28 exemplify a co-inhibitory and co-stimulatory signaling axis that dynamically sculpts the interaction of antigen-specific T cells with antigen-presenting cells. Anti-CTLA-4 antibodies enhance tumor-specific immunity through a variety of mechanisms including: blockade of CD80 or CD86 binding to CTLA-4, repressing regulatory T cell function and selective elimination of intratumoral regulatory T cells via an Fcγ receptor-dependent mechanism. AGEN1884 is a novel IgG1 antibody targeting CTLA-4. It potently enhanced antigen-specific T cell responsiveness that could be potentiated in combination with other immunomodulatory antibodies. AGEN1884 was well-tolerated in non-human primates and enhanced vaccine-mediated antigen-specific immunity. AGEN1884 combined effectively with PD-1 blockade to elicit a T cell proliferative response in the periphery. Interestingly, an IgG2 variant of AGEN1884 revealed distinct functional differences that may have implications for optimal dosing regimens in patients. Taken together, the pharmacological properties of AGEN1884 support its clinical investigation as a single therapeutic and combination agent.”

5、CTLA-4 Immunohistochemistry and Quantitative Image Analysis for Profiling of Human Cancers
Charles Brown,et al.J Histochem Cytochem. 2019.PMCID: PMC6882065
“There is an important need in immuno-oncology to develop reliable immunohistochemistry (IHC) to assess the expression of anti-mouse CTLA-4 (CD152)+ tumor-infiltrating lymphocytes in human cancers and quantify them with image analysis (IA). We used commercial polyclonal and monoclonal antibodies and characterized three chromogenic cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) assays with suitable specificity and sensitivity for use in formalin-fixed, paraffin-embedded (FFPE) tissues. We found variable numbers of anti-mouse CTLA-4 (CD152)+ lymphocytes in multiple types of cancer and secondary lymphoid organs (SLOs) and other normal human tissues. Combining CTLA-4 with CD3, CD4, or CD8 by immunofluorescence showed that CTLA-4+ lymphocytes in SLOs and tumors were typically CD3+ and CD4+, but not CD8+. Individual lymphocytes expressed CTLA-4 either as primarily granular cytoplasmic staining or as excentric globular deposits. The CTLA-4/FoxP3 (forkhead box P3 protein) duplex IHC demonstrated that CTLA-4+/FoxP3− lymphocytes predominated in the germinal centers of SLOs and tumor tertiary lymphoid structures (TLSs), whereas CTLA-4+/FoxP3+ lymphocytes populated the T-cell zone of SLOs and TLSs, plus tumor stroma. IA scoring was highly comparable with pathologist scoring for CTLA-4 and CTLA-4/FoxP3 assays and a FoxP3 single IHC. Our findings show that CTLA-4 IHC can be used to reliably label lymphocytes in FFPE human tissues, making it possible to investigate the role of CTLA-4 in the tumor microenvironment.”

6、Leukemic B Cell CTLA-4 Suppresses Co-stimulation of T cells
Priscilla Do,et al.J Immunol. 2020.PMCID: PMC6478536
“Clinical benefit of anti-mouse CTLA-4 (CD152) blockade on T cells is known, yet the impact of its expression on cancer cells remains unaddressed. We define an immunosuppressive role for tumor expressed anti-mouse CTLA-4 (CD152) using chronic lymphocytic leukemia (CLL) as a disease model. CLL, among other cancer cells, are CTLA-4+. Co-culture with activated human T cells induced surface CTLA-4 on primary human CLL B cells. CTLA-4 on CLL-derived human cell lines decreased CD80 expression on co-cultured CD80+ cells, with restoration upon CTLA-4 blockade. Co-culture of anti-mouse CTLA-4 (CD152)+ CLL cells with CD80-GFP+ cell lines revealed transfer of CD80-GFP into CLL tumor cells, similar to CTLA-4+ T cells able to trans-endocytose CD80. Co-culture of T cells with CTLA-4+ CLL cells decreased IL2 production. Using a human CTLA-4 knock-in mouse lacking FcγR function, anti-tumor efficacy was observed by blocking murine CTLA-4 on tumor cells in isolation of the T cell effect and Fc-mediated depletion. These data implicate tumor CTLA-4 in cancer cell-mediated immunosuppression in vitro and to have a functional role on tumor cells in vivo.”

7、The CTLA-4 x OX40 bispecific antibody ATOR-1015 induces anti-tumor effects through tumor-directed immune activation
Anne Månsson Kvarnhammar,et al.J Immunother Cancer. 2019.PMCID: PMC6458634
“Background
The CTLA-4 blocking antibody ipilimumab has demonstrated substantial and durable effects in patients with melanoma. While anti-mouse CTLA-4 (CD152) therapy, both as monotherapy and in combination with PD-1 targeting therapies, has great potential in many indications, the toxicities of the current treatment regimens may limit their use. Thus, there is a medical need for new CTLA-4 targeting therapies with improved benefit-risk profile.
Methods
ATOR-1015 is a human anti-mouse CTLA-4 (CD152) x OX40 targeting IgG1 bispecific antibody generated by linking an optimized version of the Ig-like V-type domain of human CD86, a natural CTLA-4 ligand, to an agonistic OX40 antibody. In vitro evaluation of T-cell activation and T regulatory cell (Treg) depletion was performed using purified cells from healthy human donors or cell lines. In vivo anti-tumor responses were studied using human OX40 transgenic (knock-in) mice with established syngeneic tumors. Tumors and spleens from treated mice were analyzed for CD8+ T cell and Treg frequencies, T-cell activation markers and tumor localization using flow cytometry.
Results
ATOR-1015 induces T-cell activation and Treg depletion in vitro. Treatment with ATOR-1015 reduces tumor growth and improves survival in several syngeneic tumor models, including bladder, colon and pancreas cancer models. It is further demonstrated that ATOR-1015 induces tumor-specific and long-term immunological memory and enhances the response to PD-1 inhibition. Moreover, ATOR-1015 localizes to the tumor area where it reduces the frequency of Tregs and increases the number and activation of CD8+ T cells.
Conclusions
By targeting CTLA-4 and OX40 simultaneously, ATOR-1015 is directed to the tumor area where it induces enhanced immune activation, and thus has the potential to be a next generation CTLA-4 targeting therapy with improved clinical efficacy and reduced toxicity. ATOR-1015 is also expected to act synergistically with anti-PD-1/PD-L1 therapy. The pre-clinical data support clinical development of ATOR-1015, and a first-in-human trial has started (NCT03782467).”

8、PD-L1:CD80 Cis-Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways
Yunlong Zhao,et al.Immunity. 2019.PMCID: PMC6935268
“Combined immunotherapy targeting the immune checkpoint receptors cytotoxic T-lymphocyte-associated protein 4 (anti-mouse CTLA-4 (CD152)) and programmed cell death 1 (PD-1), or CTLA-4 and the PD-1 ligand (PD-L1) exhibits superior anti-tumor responses compared with single-agent therapy. Here, we examined the molecular basis for this synergy. Using reconstitution assays with fluorescence readouts, we found that PD-L1 and the CTLA-4 ligand CD80 heterodimerize in cis but not trans. Quantitative biochemistry and cell biology assays revealed that PD-L1:CD80 cis-heterodimerization inhibited both PD-L1:PD-1 and CD80:CTLA-4 interactions through distinct mechanisms but preserved the ability of CD80 to activate the T cell co-stimulatory receptor CD28. Furthermore, PD-L1 expression on antigen-presenting cells (APCs) prevented CTLA-4-mediated trans-endocytosis of CD80. Atezolizumab (anti-PD-L1), but not anti-PD-1, reduced cell surface expression of CD80 on APCs, and this effect was negated by co-blockade of CTLA-4 with ipilimumab (anti-CTLA-4). Thus, PD-L1 exerts an immunostimulatory effect by repressing the CTLA-4 axis; this has implications to the synergy of anti-PD-L1 and anti-CTLA-4 combination therapy.”

9、Differential control of human Treg and effector T cells in tumor immunity by Fc-engineered anti–CTLA-4 antibody
Danbee Ha,et al.Proc Natl Acad Sci U S A. 2018.PMCID: PMC6329979
“Anti–CTLA-4 mAb is efficacious in enhancing tumor immunity in humans. CTLA-4 is expressed by conventional T cells upon activation and by naturally occurring FOXP3+CD4+ Treg cells constitutively, raising a question of how anti–CTLA-4 mAb can differentially control these functionally opposing T cell populations in tumor immunity. Here we show that FOXP3high potently suppressive effector Treg cells were abundant in melanoma tissues, expressing CTLA-4 at higher levels than tumor-infiltrating CD8+ T cells. Upon in vitro tumor-antigen stimulation of peripheral blood mononuclear cells from healthy individuals or melanoma patients, Fc-region–modified anti–CTLA-4 mAb with high antibody-dependent cell-mediated cytotoxicity (ADCC) and cellular phagocytosis (ADCP) activity selectively depleted CTLA-4+FOXP3+ Treg cells and consequently expanded tumor-antigen–specific CD8+T cells. Importantly, the expansion occurred only when antigen stimulation was delayed several days from the antibody treatment to spare CTLA-4+ activated effector CD8+T cells from mAb-mediated killing. Similarly, in tumor-bearing mice, high-ADCC/ADCP anti–CTLA-4 mAb treatment with delayed tumor-antigen vaccination significantly prolonged their survival and markedly elevated cytokine production by tumor-infiltrating CD8+ T cells, whereas antibody treatment concurrent with vaccination did not. Anti–CTLA-4 mAb modified to exhibit a lesser or no Fc-binding activity failed to show such timing-dependent in vitro and in vivo immune enhancement. Thus, high ADCC anti–CTLA-4 mAb is able to selectively deplete effector Treg cells and evoke tumor immunity depending on the CTLA-4–expressing status of effector CD8+ T cells. These findings are instrumental in designing cancer immunotherapy with mAbs targeting the molecules commonly expressed by FOXP3+ Treg cells and tumor-reactive effector T cells.”

10、Blockade of CTLA-4 and Tim-3 pathways induces fetal loss with altered cytokine profiles by decidual CD4+T cells
Songcun Wang,et al.Cell Death Dis. 2019.PMCID: PMC6325160
“The single and/or combination use of immune checkpoint blockade therapies in human infectious diseases and cancer are rapidly expanding. Despite early efforts, substantial uncertainty remains about the safety and efficacy of immune checkpoint blockade in some populations. Cytotoxic T-lymphocyte-associated protein 4 anti-mouse CTLA-4 (CD152) and T-cell immunoglobulin mucin-3 (Tim-3) are the major targetable co-inhibitory receptors on T cells. Here we showed that in animal studies, treatment with either anti-mouse CTLA-4 (CD152) or Tim-3-blocking antibody caused greater susceptibility to fetal loss with altered cytokine profiles by decidual CD4+T (dCD4+T) cells. CTLA-4 and Tim-3 pathways appeared to play key roles in maintaining maternal-fetal tolerance by regulating the function of dCD4+T cells. In addition, the abnormality in number and functionality of dCTLA-4+Tim-3+CD4+T cells was associated with miscarriage. These findings underscored the important roles of the CTLA-4 and Tim-3 pathways in regulating dCD4+T cells function and maintaining normal pregnancy. Our study also emphasized the importance of careful consideration of reproductive safety when choosing immune checkpoint blockade therapies in real world clinical care.”

Related Recombinant IgG Reference Antibodies:
Recombinant mouse lgG2b isotype control antibody
Recombinant mouse IgG2c LALAPG isotype control antibody
Recombinant mouse IgG2a LALAPG isotype control antibody

Syd Labs provides the following research grade anti-CTLA-4 antibody biosimilars:
Ipilimumab Biosimilar, research grade, anti-human CTLA-4 monoclonal antibody
Tremelimumab Biosimilar, research grade, anti-human CTLA-4 monoclonal antibody

Recombinant CTLA-4 Proteins:
Biotinylated Human CTLA-4 Protein
Cynomolgus CTLA-4 Protein
Human CTLA-4 Protein

Anti-Mouse CD152/CTLA4 Antibody (9D9) from: Recombinant Anti-mouse CTLA-4 Monoclonal Antibody(Clone: 9D9), Mouse IgG2c-L234A L235A P329G (LALAPG) Kappa, In vivo Grade: PA007380.m2cLA Syd Labs

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