Agonistic CD40 Antibody Research Articles

Abstract P3076: Immune Checkpoint Regulator Impairs Left Ventricular Remodeling In A Hypertension-Fibrosis Model Of Cardiac Injury.

Cancer treatments directly result in cardiovascular toxicity and may exacerbate typical age-related diseases such as hypertension and diabetes. Among cancer treatments contributing to survivorship, immunotherapies including immune checkpoint inhibitors (ICIs) are now widely utilized due to their groundbreaking efficacy. CD40 agonist (CD40ag) antibodies are immune checkpoint regulators that have emerged as promising candidates with remarkable efficacy across tumors including those thought to be resistant to established ICIs. To investigate CD40 signaling and its potential for adverse left ventricular remodeling in a hypertensive-fibrosis model of cardiac injury, C57BL/6J mice were pretreated with isotype or CD40ag antibodies 7 days prior to myocardial injury, then continued for a total of 35 days. Myocardial injury was induced by continuous infusion of angiotensin II and phenylephrine (AngII/PE) for 4 weeks. Masson’s trichrome stain revealed increased fibrosis and immunohistochemistry demonstrated increased immune cell abundance in heart sections. There was impaired cardiac function assessed by 2-D echocardiography in minimally sedated mice showing reduced ejection fraction and radial strain. Finally, CD40 agonism resulted in gene expression upregulation of multiple inflammatory cytokines and chemokines post-cardiac injury. These results reveal that pre-treatment with CD40 agonist prior to injury with AngII/PE infusion increased myocardial inflammation and impaired left ventricular remodeling.

Cell targeting and immunostimulatory properties of a novel Fcγ-receptor-independent agonistic anti-CD40 antibody in rhesus macaques

Targeting CD40 by agonistic antibodies used as vaccine adjuvants or for cancer immunotherapy is a strategy to stimulate immune responses. The majority of studied agonistic anti-human CD40 antibodies require crosslinking of their Fc region to inhibitory FcγRIIb to induce immune stimulation although this has been associated with toxicity in previous studies. Here we introduce an agonistic anti-human CD40 monoclonal IgG1 antibody (MAB273) unique in its specificity to the CD40L binding site of CD40 but devoid of Fcγ-receptor binding. We demonstrate rapid binding of MAB273 to B cells and dendritic cells resulting in activation in vitro on human cells and in vivo in rhesus macaques. Dissemination of fluorescently labeled MAB273 after subcutaneous administration was found predominantly at the site of injection and specific draining lymph nodes. Phenotypic cell differentiation and upregulation of genes associated with immune activation were found in the targeted tissues. Antigen-specific T cell responses were enhanced by MAB273 when given in a prime-boost regimen and for boosting low preexisting responses. MAB273 may therefore be a promising immunostimulatory adjuvant that warrants future testing for therapeutic and prophylactic vaccination strategies.

A phase 1b, open-label, two-part safety, tolerability, and efficacy study of a soluble beta-glucan (odetiglucan) in combination with a CD40 agonistic monoclonal antibody (CDX-1140) in patients with metastatic pancreatic adenocarcinoma whose disease did not progress during first-line (1L) chemotherapy.

TPS4201 Background: Metastatic pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal disease with a 5-year overall survival (OS) rate of 3%. Standard of care treatment is continuous cytotoxic chemotherapy which produces modest improvements in OS but is also associated with significant toxicity raising the need for novel treatment paradigms. Although immunotherapy has yet to produce clinical benefit for most patients with PDAC, preclinical modeling demonstrates its potential and show that combinations of myeloid agonists can trigger robust synergistic anti-tumor activity against PDAC tumors that are otherwise resistant to immunotherapy including immune checkpoint blockade (anti-CTLA4, anti-PD1). In this study, odetiglucan (OD), a novel beta glucan pathogen associated molecular pattern (PAMP), will be combined for the first time with CDX-1140, a fully human Ig2K agonistic monoclonal CD40 antibody in the maintenance setting after an induction phase of cytotoxic chemotherapy. OD and a CD40 agonist directly promote the activation and maturation of antigen presenting cells and shift the suppressive TME to enhance T-cell responses through non-redundant myeloid signaling pathways. We hypothesize that the combination of OD and CD40 agonist therapy has the potential to trigger anti-tumor immunity in PDAC and thus, extend and deepen responses to 1L cytotoxic chemotherapy. Methods: A two-part Phase 1b study of metastatic PDAC pts who have evidence of response or stable disease following 16-24 wks of chemotherapy is being conducted. Part A uses a 3+3 design; pts will receive OD 4 mg/kg QW + CDX-1140 1.5 mg/kg Q3W. Following a dose-limiting toxicity observation period, the dose will be held at 1.5 mg/kg or decreased to 0.72 mg/kg Q3W. Once preliminary safety is established, the cohort will be expanded to 15 pts. Part B pts will receive OD 4mg/kg + CDX-1140 at the dose identified in Part A Q3W. The study will enroll 30 pts. Main eligibility criteria: Metastatic PDAC having durable stable disease or response to 1L chemotherapy; serum ABA ≥20 µg/mL, and no prior anti-CD40 mAb or immunomodulatory treatment exposure. Primary endpoints are MTD, RP2D, and safety. Secondary endpoints include DOR, PFS, ORR, and OS and exploratory endpoints include evaluating immune pharmacodynamic responses in peripheral blood and tumor. Safety parameters will be listed and summarized using descriptive statistics. ORR will be tabulated by overall frequency; DOR, TTF, PFS and OS will be summarized and presented using Kaplan-Meier and waterfall plots. 5 US sites will participate. Clinical trial information: NCT04834778 .

A phase II trial with safety lead-in to evaluate the addition of sotigalimab, a CD40 agonistic monoclonal antibody, to standard-of-care doxorubicin for the treatment of advanced sarcoma.

11565 Background: Immune checkpoint inhibitors have limited efficacy in soft tissue sarcoma (STS) due to insufficient T-cell activation and infiltration by immunosuppressive macrophages. Sotigalimab (S), a high-affinity humanized monoclonal antibody for CD40, promotes antigen presentation, stimulates T-cell responses, and reprograms immunosuppressive macrophages. Preclinical studies with CD40 agonists revealed efficacy in immune “cold” tumor types and synergy with chemotherapy. Standard first-line doxorubicin (D) provides objective response rate (ORR) < 15% and median progression free survival (mPFS) of 4-6 mos in advanced STS. We therefore conducted this first-in-human study of D+S in STS. Methods: An open-label, single-arm, multi-center, phase 2 study evaluated D+S in advanced STS. Pts had ECOG PS ≤ 1 and any number of prior lines (but were anthracycline-naïve). The study initially enrolled all STS subtypes (except KS and GIST) but was amended in 12/2020 to limit enrollment to dedifferentiated liposarcoma (DDLPS), leiomyosarcoma (LMS), and undifferentiated pleomorphic sarcoma (UPS). Pts received D 75 mg/m2 IV D1 + S 0.3 mg/kg IV D1 for eight 21-day cycles, followed by S monotherapy. A safety lead-in was performed (first 6 patients). Primary endpoint was ORR. Secondary endpoints included PFS and safety. A Simon two-stage design was used. If ≥ 7/32 responded overall, the treatment would be considered promising (85% power, α = 0.05). A subset of patients underwent paired tumor biopsies. Results: 32 pts have enrolled (median age 62; 10 LMS, 10 UPS, 9 DDLPS, 3 other). 4 pts remain on treatment with median follow-up of 2.8 mos (1 UPS pt has not reached first imaging). D (75 mg/m2) + S (0.3 mg/kg) was safe and tolerable without dose limiting toxicity. Overall ORR was 16% (5/31). Objective responses occurred in UPS (2), LMS (1), other LPS (1), and epithelioid hemangioendothelioma (1). Overall mPFS was 7.5 mos (95% CI 5.6-14.9 mos) and PFS rate at 6 and 12-mos was 55% and 31%, respectively. mPFS by STS subtype was 11.9 mos (95% CI: 10.3 – NE) for DDLPS, 7.5 mos (95% CI: 1.4—NE) for UPS and 5.6 mos (95% CI: 1.3 – NE) for LMS. Overall, 17/31 (55%) pts experienced grade 3 or 4 adverse events (AEs): most commonly, neutropenia (32%), febrile neutropenia (19%), and anemia (16%). 16% of pts experienced cytokine release (all low-grade). Correlative analysis of biopsies with high-definition spatial proteomics is ongoing. Conclusions: D (75 mg/m2) + S (0.3 mg/kg) every 21 days is safe and tolerable in STS. Final analysis of the primary endpoint awaits further follow-up on recently enrolled patients. Subtype-specific analysis suggests improvement in median PFS for DDLPS over historical controls: 11.9 mos vs 4 mos (Stacchiotti S. Ann Oncol 2020; Livingston M. Sci Rep 2017). An expansion of the DDLPS cohort is planned. Clinical trial information: NCT03719430 .

CD40-dependent mechanism of virus control in a mouse model of HBV infection

Abstract An estimated 296 million people worldwide currently live with chronic hepatitis B virus (HBV) infection. Up to 90% of newborns and 30% of children with acute hepatitis B develop chronic infections, causing lifelong complications including liver cirrhosis and cancer. HBV persistence is a consequence of T and B cell dysfunction, so immunotherapy has been proposed as a means of HBV control. TNF receptor superfamily member CD40 has shown therapeutic value in overriding T cell exhaustion and inducing lymphocyte responses in solid tumors and viral infections. To determine the efficacy of CD40 targeting in generating HBV-resolving immune responses, mice were simultaneously transduced with adeno-associated virus encoding the HBV genome (AAV-HBV) and treated with the agonistic CD40 antibody FGK4.5 to activate CD40 signaling. AAV-HBV mice receiving FGK4.5 saw a transient reduction in HBV as determined by serum antigen analysis, while antigen levels in CD4-depleted mice treated with FGK4.5 became undetectable over seven weeks. HBV control was strongly associated with the generation of HBV-specific CD8 +T cells, increased serum ALT, and elevated expression of intrahepatic CD8 +T cell-related genes. Histological analysis of the liver revealed loss of hepatitis B core antigen-positive hepatocytes and increased inflammatory foci. Neither Treg cell depletion nor checkpoint inhibition in CD4-replete mice affected HBV control. Lastly, CD40 activation in CD4-depleted chronically infected mice also reduced both serum antigenemia and liver HBV RNA. Taken together, these results implicate a critical role of CD4 +T cells in maintaining HBV immune tolerance and further suggest CD40 as a potential therapeutic target for chronic HBV infection. Supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award numbers R01AI124006 and R01AI148354. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Abstract 4128: AJ17 potentiates agonist CD40 antibody efficacy and attenuates agonist CD40 induced PD-L1 expression and associated toxicity in murine tumor models

Abstract Agonist CD40 antibody (αCD40) has shown excellent anti-tumor efficacy in both pre and early clinical studies. However, in clinics, αCD40 has failed to prove its potential as αCD40 is associated with dose-limiting toxicity due to cytokine release syndrome (CRS), grade 3 to 4 hematological and liver toxicities. In addition, αCD40 induces immune checkpoint proteins (PD-L1, CTLA-4) which makes the tumor microenvironment (TME) immunosuppressive. In this context, this study investigates the therapeutic efficacy of a novel combination of αCD40 and AJ17, a small molecule inhibitor of receptor tyrosine kinase. Combination treatment showed a significant delay in the tumor growth and improved overall survival in mice bearing B16-F10 melanoma and 4T1 orthotopic tumor by increasing the population of CD8+ T cells and lowering the expression of PD-L1, Foxp3+, and arginase in both tumor and spleen. Most interestingly, AJ17 lowered the in vivo toxicity associated with αCD40 monotherapy by lowering the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and IL-6 in serum. In conclusion, this combination can be further explored in clinics to improve the in vivo anti-tumor efficacy of αCD40 while lowering the associated toxicity. Citation Format: Vidit Gaur, Anjali Barnwal, Bushra Ateeq, Jayanta Bhattacharyya. AJ17 potentiates agonist CD40 antibody efficacy and attenuates agonist CD40 induced PD-L1 expression and associated toxicity in murine tumor models. [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 4128.

Abstract 2929: Modular peptide cargo delivery by targeting CD40 enables ligandome driven, precision immunotherapy via the Adaptable Drug Affinity Conjugate (ADAC™) technology

Abstract Agonistic anti-CD40 therapy has shown impressive efficacy in preclinical murine models, models inert to toxicity of high systemic drug exposure. Clinical data consist of pharmacodynamic responses reported as non-specific cytokine release, transient drop of CD40-expressing cells in blood and induction of co-stimulatory ligands/receptors on antigen-presenting cells, but so far clinically meaningful efficacy data on monotherapy use is lacking. Clinical data also indicate a bell-shaped dose-response curve, with excessive immune activation leading to immune exhaustion when combined with check-point inhibitors[1]. Intratumoral route of administration, bispecific antibodies for tumor drug localization or Antibody-Drug Conjugates (ADCs) carrying antigenic cargo are or have been assessed, but have associated clinical utility enigmas. Herein we present a novel solution ensuring modular peptide delivery along with an efficient T cell priming strategy; a bivalent anti-CD40 agonistic antibody equipped with a peptide-binding single chain variable fragment (scFv) that binds a short peptide tag (pTag) in the low nM range. The resulting drug strategy makes use of synthetic long peptide production of any tumor-associated antigen (TAA) of choice where the pTag itself ensures simple conjugate production to the protein, through affinity linkage, leading to a final rapid in-hospital mixing step. Data suggest that the novel candidate drug (STRIKE2001), now developed based on the ADAC concept[2], retains similar agonistic activity in its bispecific, IgG2 format and does not inhibit CD40L binding to the CD40 protein. Data further show impressive expansion of endogenous tumor-specific T cells (as measured by tetramer staining), along with effective anti-tumor responses in the TC1 model. In addition, by using the subcutaneous delivery strategy CD40 agonist exposure to liver and spleen is vastly reduced, limiting systemic immune toxicity/exhaustion risks[3]. [1] Padrón, L.J., et al. Sotigalimab and/or nivolumab with chemotherapy in first-line metastatic pancreatic cancer: clinical and immunologic analyses from the randomized phase 2 PRINCE trial. Nat Med 28, 1167-1177 (2022). https://doi.org/10.1038/s41591-022-01829-9 [2] Eltahir, M., et al. (2022), An Adaptable Antibody-Based Platform for Flexible Synthetic Peptide Delivery Built on Agonistic CD40 Antibodies. Adv. Therap., 5: 2200008. https://doi.org/10.1002/adtp.202200008 [3] Sandin, L., et al. Locally Delivered CD40 Agonist Antibody Accumulates in Secondary Lymphoid Organs and Eradicates Experimental Disseminated Bladder Cancer. Cancer Immunol Res 1 January 2014; 2 (1): 80-90. https://doi.org/10.1158/2326-6066.CIR-13-0067 Citation Format: Aman Mebrahtu, Ida Lauren, Rosanne Veerman, Alexandros Kostakis, Gözde Gucluler Akpinar, Oskar Andersson, Lindvi Gudmundsdotter, Tina Furebring, Helena Persson, Pierre Donnes, Johan Rockberg, Sara Mangsbo. Modular peptide cargo delivery by targeting CD40 enables ligandome driven, precision immunotherapy via the Adaptable Drug Affinity Conjugate (ADAC™) technology [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 2929.

Abstract 3287: A bedside to bench study of anti-PD-1, anti-CD40, and anti-CSF1R indicates that more is not necessarily better

Abstract Stimulating innate immunity can potentially enable us to overcome resistance to PD-(L)1 blockade. We previously conducted a phase 1 trial of cabiralizumab (anti-CSF1R) with sotigalimab (CD40 agonistic antibody) and nivolumab. Our purpose was to determine safety and the effects of this regimen on circulating and tumor-infiltrating immune cells and to determine the activity of this regimen in a phase 1b trial for melanoma patients whose disease had progressed on anti-PD-(L)1. CyTOF analysis on circulating immune cells taken before and during treatment revealed a reduction in non-classical monocytes and an increase in dendritic cells. Patients with prolonged stable disease had less T-regulatory cells and more circulating antigen presenting cells after treatment compared to patients that were treated for a shorter time. In the phase 1b component of the trial in 13 melanoma patients, objective response rates were: 1 confirmed partial response (7.7%), 1 unconfirmed partial response (7.7%), 5 stable disease (38.5%) and 6 disease progression (42.6%). Despite therapy-induced changes in circulating immune cells and previous preclinical studies supporting rationale for this combination, responses in humans were insufficient to proceed to the second stage of the phase 1b trial. Given the challenges with translating doses from mice to humans, we proceeded to study various doses of anti-CSF1R in combination with CD40 agonist and anti-PD-1 in a murine model. Higher dose anti-CSF1R in mice was associated with increased tumor growth, worse survival and by single-cell RNA-sequencing analyses, we identified a more suppressive monocyte/macrophage profile in murine tumors. Our study suggests that more anti-CSF1R might not be better. Further optimization of cabiralizumab dosing is necessary to evaluate the clinical potential in combination with anti-PD-1 and anti-CD40 in a difficult-to treat patient population whose therapeutic options are limited. Citation Format: Dijana Djureinovic, Sarah A. Weiss, Irina Krykbaeva, Rihao Qu, Ioannis Vathiotis, Myrto Moutafi, Lin Zhang, Ana L. Perdigoto, Wei Wei, Gail Anderson, William Damsky, Michael Hurwitz, Barbara Johnson, Amit Mahajan, Frank Hsu, Kathryn Miller-Jensen, Yuval Kluger, Mario Sznol, Susan M. Kaech, Marcus Bosenberg, Lucia Jilaveanu, Harriet M. Kluger. A bedside to bench study of anti-PD-1, anti-CD40, and anti-CSF1R indicates that more is not necessarily better [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 3287.

Abstract 6362: Overcoming resistance to immunotherapy due to loss of antigen presentation

Abstract Resistance to immune checkpoint inhibitors represents a major therapeutic challenge, as only 40% of patients with melanoma (and less with other tumor types) have a long-term response to anti-PD-1 therapy. Resistance can arise because of somatic mutations in cancer cells that allow evasion of T cell-mediated killing. One commonly acquired resistance mutation in melanoma, loss of beta-2 microglobulin (B2m), prevents T cell killing by hiding cancer cells from CD8+ T cell recognition. To understand the failed immune response against resistant tumors, we used single-cell RNA-seq to characterize tumor-infiltrating immune cells in antigen presentation-deficient human melanoma biopsies and CRISPR-modified mouse melanoma tumors. Our data demonstrate an increase in immunosuppressive M2-like macrophages and absence of CD8+ T cells in B2m-null tumors. To overcome this resistance, we treated tumor-bearing mice with CD40 agonist antibody, which promotes differentiation of macrophages towards a pro-inflammatory phenotype and increases dendritic cell priming of CD8+ T cells. Treatment with CD40 agonist reduced tumor growth and improved tumor clearance in B2m-null melanoma and colorectal cancer models. To determine how CD40 agonist treatment works, we depleted different immune populations from the tumor microenvironment. We hypothesized that by depleting M2 macrophages, CD40 agonist treatment would remove an immunosuppressive brake to allow natural killer (NK) cells to kill tumor cells lacking MHC expression. To our surprise, NK cells were not required for the efficacy of CD40 agonist. Instead CD8+ T cells were required, even though the CD8+ T cells cannot directly recognize the tumor cells. scRNA-seq identified a transcriptionally unique state of CD8+ T cells that is recruited to the tumor microenvironment after CD40 agonist treatment. These CD8+ T cells produce IFNγ, which is required for the efficacy of CD40 agonist treatment. These data demonstrate that CD8+ T cells, a key mediator of anti-tumor immunity, can still be recruited to control tumors deficient in antigen presentation. More broadly, they suggest that strategies to activate CD8+ T cells may be effective even in the context of acquired resistance to checkpoint inhibitor therapy. Citation Format: Brian C. Miller, Yacine Choutri, Rose Al Abosy, Amy Huang, Emily K. Cox, Matthew P. Zimmerman, Wan Lin Chong, Katherine J. Vietor, Jenna Collier, Sarah A. Weiss, Debattama Sen, W. Nicholas Haining, Arlene H. Sharpe. Overcoming resistance to immunotherapy due to loss of antigen presentation [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 6362.

Abstract 2740: Safe and effective delivery of antibody fragments to the solid tumor microenvironment

Abstract Promising immunotherapeutics, particularly agonists, have shown limited clinical benefit, in part because of dose-limiting toxicities, caused by potent immune activation in systemic compartments, with limited evidence of activity in the tumor microenvironment (TME). The Tumor Specific Immunogene (T-SIGn) platform is designed to generate vectors that can express combinations of transgenes producing immunotherapeutics selectively within the tumor microenvironment. T-SIGn is clinically validated for intravenous delivery, enabling the vectors to reach primary and metastatic sites to replicate and produce their therapeutic transgene payloads specifically in malignant epithelial cells. Transgene (TG) cassettes of T-SIGn vectors can be up to approximately 3.5Kb, allowing the expression of multiple distinct TGs. NG-350A is a clinical stage T-SIGn vector, with an established safety/tolerability profile, that expresses a full-length agonist CD40 antibody, and shows evidence of target engagement and adaptive immune activation in patients with different epithelial solid tumors via prolonged, dose-dependent increases in IL-12p70, IFNγ and IL-17A detected in the circulation of patients about 2 weeks after the initiation of the day 1, 3, 5 dosing regimen. NG-350A is currently being evaluated in phase I clinical studies in combination with Pembrolizumab or Ipilimumab. We have been characterizing numerous smaller fragment antibody formats that enable us to combine their expression with other antibody fragments or immunomodulatory molecules as a T-SIGn vector. An ScFv anti-CD40 fragment antibody design was identified as a potent immunotherapeutic to induce the secretion of IL-12p70 cytokine following stimulation of human monocyte-derived dendritic cells, as well as an enhanced antigen-specific T-cell activation and macrophage conversion to a more pro-inflammatory phenotype. When encoded together with CXCL9 and other immunostimulatory cytokines, the anti-CD40 ScFv has been selected as a potential follow-up to NG-350A that synergistically drives enhanced in vitro T-cell priming, activation and migration. Other checkpoint inhibitor and coactivator antibodies, as both ScFv and camelid VHH formats, have also been encoded individually, in combination with the anti-CD40 antibody fragment or other fragment antibodies to create a set of viral vectors that are being evaluated preclinically as future T-SIGn vector candidates. Data from these studies with initial vectors encoding combinations of functionally active antibody fragments will be presented. Collectively, these clinical and preclinical datasets demonstrate the broad utility of the T-SIGn platform for expressing rationally-designed combinations of antibodies and other natural and synthetic agents within tumors and thus enabling the development of new and potent immunotherapies for patients with solid epithelial cancers. Citation Format: Rachel Bergin, Amandine Pelletier, Manuela Zonca, Alice Muntzer, Meg Snowden, Samuel Hardy, Carla Cerqueira, Tae Hyun-Jang, Rochelle Lear, Masumi Fujinobu, Maria Stella Sasso, Samantha Bucktrout, Brian R. Champion. Safe and effective delivery of antibody fragments to the solid tumor microenvironment [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 2740.

Abstract 2656: Reversible chemical modification of antibodies: A complementary approach to tuning FcγR binding that maintains anti-tumor activity while mitigating peripheral immune activation

Abstract Modification of effector function has proven to be an effective modality for optimizing activity and tolerability of therapeutic antibodies. Currently available methods to modulate effector function include the introduction of point mutations in the Fc region and glycan engineering of the antibody. Here we present an alternative and complementary method of tuning effector function utilizing a conjugation-based approach. This methodology uses conjugation of polyethylene glycol (PEG) to native cysteines of an antibody to impair FcγR binding of antibodies to innate immune effector cells. Utilizing maleimide or disulfide conjugation techniques, attenuated effector function can be either permanent or restored over time through a de-conjugation process. Impacts of PEGylation on FcγR binding, signaling, and restoration of function were assessed in vitro and in vivo. As a proof-of-concept, the lead technology was applied to an agonist CD40 antibody, which resulted in significant reductions in systemic cytokine production in hCD40 mice and non-human primates, while demonstrating retained efficacy and improved pharmacokinetics. Additionally, we combined the conjugation technology with glycan engineering and FcγR enhancing point mutations to impart unique effector function profiles to clinical antibodies. This simple, modular approach can be rapidly applied to existing antibodies to reduce immune-driven toxicities, such as infusion reactions, and optimize effector function activity. Citation Format: Philip N. Moquist, Chris I. Leiske, Noah A. Bindman, Xinqun Zhang, Nicole Duncan, Weiping Zeng, Serena W. Wo, Abbie Wong, Clark M. Henderson, Karalyne Crowder, Haley D. Neff-LaFord, Django Sussman, Shyra J. Gardai, Matthew R. Levengood. Reversible chemical modification of antibodies: A complementary approach to tuning FcγR binding that maintains anti-tumor activity while mitigating peripheral immune activation [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 2656.

Abstract 6332: A humanized anti-cd40 antibody with anti-tumor pharmacological activity and cynomolgus monkey safety

Abstract LNF1901 is a humanized CD40 agonistic antibody. LNF1901 can bind to CD40 protein of human(EC50=23.6ng/mL) and monkey(EC50=23.5ng/mL), but cannot bind to CD40 protein of rat and mouse. LNF1901 had strong activation effect on DC cells(EC50=0.04ug/mL) and had strong ADCC and CDC effects. AT a dose of 20mg/kg, the TGITW of LNF1901 was 59.9% in Female NPG mice were inoculated with Raji/CIK/DC subcutaneous transplanted tumor model. Following intravenous infusion of LNF1901 at doses of 6, 30, or 150 mg/kg to cynomolgus monkeys weekly for 4 weeks, the highest non-severely toxic dose (HNSTD) was considered to be 150 mg/kg.LNF1901 had demonstrated manageable safety and promising antitumor activity in vivo and in vitro tests. Citation Format: Zhenyu Li, Lili Zhao, Guimin Zhang, Bin Li, Zhong Liu, Guangyan Li, Zhongsong Zhu. A humanized anti-cd40 antibody with anti-tumor pharmacological activity and cynomolgus monkey safety [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 6332.

Abstract 1531: Human anti-CD40 agonistic antibodies with enhanced FcgR engagement activate immune cell and promote anti-tumor efficacy

Abstract Checkpoint inhibitors represent a major advance in cancer immunotherapy, and combinatorial immunotherapies with secondary drivers of anti-tumor immunity provide beneficial effects for patients that do not show a strong endogenous immune response. CD40 is a member of the TNF family of receptors that has been shown to play a crucial role in enhancing B cell and dendritic cell activity and fostering anti-tumor immune responses. Engagement of the inhibitory Fcg-receptor (FcgR) IIb is an absolute requirement for in vivo antitumor activity of agonistic anti-CD40 monoclonal antibodies, and Fc region mutant designs of anti-CD40 antibodies had been discovered to enhance FcgRIIb engagement. With Twist’s precision DNA writing technologies, we have created phage display VHH and scFv libraries with diversity greater than 1 × 1010 for optimal discovery. In this study, we performed high affinity binding of the antibodies by SPR and cell surface binding. The leads are reformatted on human IgG1, IgG4, and IgG1 mutant. The in vitro properties of the CD40 agonistic antibodies demonstrate enhanced FcgRIIb engagement by NFkB activation. B cell activation is also detected by upregulation of CD86 and IL6 secretion. In humanized hCD40 mice model, we observed the engineered anti-CD40 agonists enhance anti-tumor immune function in vivo. These studies suggest that our antibodies can be potential drug for cancer immunotherapy. Citation Format: Linya Wang, Tom Z. Yuan, Mouna Villalta, Zhen Han, Hansika Wadhwa, Kara Y. Chan, Marisa Yang, Tammy Htoy, Paul VanDyke, Carson Holliday, Hector Franco, Hoa Giang, Fumiko Axelrod, Aaron Sato. Human anti-CD40 agonistic antibodies with enhanced FcgR engagement activate immune cell and promote anti-tumor efficacy [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 1531.

Sustained Intratumoral Administration of Agonist CD40 Antibody Overcomes Immunosuppressive Tumor Microenvironment in Pancreatic Cancer (Adv. Sci. 9/2023)

Intratumoral Immunotherapy for Pancreatic Cancer In article number 2206873, Hsuan-Chen Liu, Corrine Ying Xuan Chua, Alessandro Grattoni, and co-workers leveraged a nanofluidic implant to demonstrate the efficacy of sustained intratumoral CD40Ab immunotherapy against pancreatic adenocarcinoma. The intratumoral strategy transformed the tumor immune microenvironment to a phenotype conducive to tumor elimination without systemic toxicity and achieved abscopal response against distant untreated tumors.

Neutrophil-activating therapy for the treatment of cancer.

Despite their cytotoxic capacity, neutrophils are often co-opted by cancers to promote immunosuppression, tumor growth, and metastasis. Consequently, these cells have received little attention as potential cancer immunotherapeutic agents. Here, we demonstrate in mouse models that neutrophils can be harnessed to induce eradication of tumors and reduce metastatic seeding through the combined actions of tumor necrosis factor, CD40 agonist, and tumor-binding antibody. The same combination activates human neutrophils invitro, enabling their lysis of human tumor cells. Mechanistically, this therapy induces rapid mobilization and tumor infiltration of neutrophils along with complement activation in tumors. Complement component C5a activates neutrophils to produce leukotriene B4, which stimulates reactive oxygen species production via xanthine oxidase, resulting in oxidative damage and Tcell-independent clearance of multiple tumor types. These data establish neutrophils as potent anti-tumor immune mediators and define an inflammatory pathway that can be harnessed to drive neutrophil-mediated eradication of cancer.

Sustained Intratumoral Administration of Agonist CD40 Antibody Overcomes Immunosuppressive Tumor Microenvironment in Pancreatic Cancer.

Agonist CD40 monoclonal antibodies (mAb) is a promising immunotherapeutic agent for cold-to-hot tumor immune microenvironment (TIME) conversion. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal cancer known as an immune desert, and therefore urgently needs more effective treatment. Conventional systemic treatment fails to effectively penetrate the characteristic dense tumor stroma. Here, it is shown that sustained low-dose intratumoral delivery of CD40 mAb via the nanofluidic drug-eluting seed (NDES) can modulate the TIME to reduce tumor burden in murine models. NDES achieves tumor reduction at a fourfold lower dosage than systemic treatment while avoiding treatment-related adverse events. Further, abscopal responses are shown where intratumoral treatment yields growth inhibition in distant untreated tumors. Overall, the NDES is presented as a viable approach to penetrate the PDAC immune barrier in a minimally invasive and effective manner, for the overarching goal of transforming treatment.

Treatment of pancreatic cancer with irreversible electroporation and intratumoral CD40 antibody stimulates systemic immune responses that inhibit liver metastasis in an orthotopic model

BackgroundPancreatic cancer (PC) has a poor prognosis, and most patients present with either locally advanced or distant metastatic disease. Irreversible electroporation (IRE) is a non-thermal method of ablation used clinically...

GPC2 antibody–drug conjugate reprograms the neuroblastoma immune milieu to enhance macrophage-driven therapies

BackgroundAntibody–drug conjugates (ADCs) that deliver cytotoxic drugs to tumor cells have emerged as an effective and safe anticancer therapy. ADCs may induce immunogenic cell death (ICD) to promote additional endogenous...

Abstract C013: Response to combination immunotherapy in a mouse model of PDAC is a function of baseline T cell content

Abstract Findings from the recently reported phase 2 PRINCE trial for patients with metastatic pancreatic cancer suggest potential clinical benefit with certain combinations of chemotherapy and immunotherapy. Overall survival following gemcitabine/nab-paclitaxel and either PD-1 blocking antibody or CD40 agonistic antibody (aCD40) correlated with the prevalence of distinct T cell subsets in circulation at baseline and with the extent of certain tumor infiltrating T cells (Padron et al., Nature Medicine, 2022). Here, we modeled the therapeutic effect of aCD40/PD-1/CTLA-4 therapy in the absence of chemotherapy in mice bearing tumors derived from the autochthonous pancreatic ductal adenocarcinoma (PDAC) KPC model and studied the dependency of tumor regression on the degree of tumor infiltrating T cells. We used a panel of KPC tumor cell clones that exhibit reproducible and transplantable levels of T cell infiltration ranging between high, intermediate, and low amounts. Mice were subcutaneously injected with clonal PDAC lines and treated 14-21 days later with either aCD40 alone or aCD40/PD-1/CTLA-4 (all i.p., n=5-8 mice per cohort of T cell high, intermediate and low clones). Systemic immune dynamics were surveyed by bleeding mice on day 0, 7, and 14 of treatment and analyzing for immune cell activation and exhaustion markers. In mice bearing T cell high and intermediate tumors, aCD40/PD-1/CTLA-4 induced higher rates of complete tumor regressions (71% for T cell high; 25% for T cell intermediate) and prolonged survival (P < 0.001 for T cell high; P = 0.013 for T cell intermediate) compared to untreated controls. Treatment with aCD40 alone was effective in mice with T cell intermediate tumors, leading to regressions in 29% of mice. In mice bearing T cell low tumors, however, tumor regressions after aCD40/PD-1/CTLA-4 were less frequent (12.5%), although survival was improved significantly following the treatment (P <0.001). aCD40 alone improved overall survival of T cell low bearing mice (P = 0.026) without inducing tumor regressions. Regardless of T cell high, intermediate, or low tumors, aCD40/PD-1/CTLA-4 increased the proportions of circulating activated and exhausted CD4+ and CD8+ T cells. aCD40/PD-1/CTLA-4 also increased T cell infiltration into T cell low tumors, but this enhanced infiltration did not correlate with complete regressions. Thus, using tumor clones derived from the same autochthonous genetic model, we show that response to combination immunotherapy is a function of baseline T cell content, with ‘hot’ tumors responding the best and ‘cold’ tumors responding the worst. Additional tumor cell-intrinsic factors such as those revealed in other mouse experiments and in the human samples from the PRINCE study likely restrict a robust anti-tumor immune response even when T cells have been recruited. Citation Format: Noah C. Cheng, Nune Markosyan, Robert H. Vonderheide. Response to combination immunotherapy in a mouse model of PDAC is a function of baseline T cell content [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C013.

Injectable Nanoparticle-Based Hydrogels Enable the Safe and Effective Deployment of Immunostimulatory CD40 Agonist Antibodies.

When properly deployed, the immune system can eliminate deadly pathogens, eradicate metastatic cancers, and provide long‐lasting protection from diverse diseases. Unfortunately, realizing these remarkable capabilities is inherently risky as disruption to immune homeostasis can elicit dangerous complications or autoimmune disorders. While current research is continuously expanding the arsenal of potent immunotherapeutics, there is a technological gap when it comes to controlling when, where, and how long these drugs act on the body. Here, this study explored the ability of a slow‐releasing injectable hydrogel depot to reduce dose‐limiting toxicities of immunostimulatory CD40 agonist (CD40a) while maintaining its potent anticancer efficacy. A previously described polymer‐nanoparticle (PNP) hydrogel system is leveraged that exhibits shear‐thinning and yield‐stress properties that are hypothesized to improve locoregional delivery of CD40a immunotherapy. Using positron emission tomography, it is demonstrated that prolonged hydrogel‐based delivery redistributes CD40a exposure to the tumor and the tumor draining lymph node (TdLN), thereby reducing weight loss, hepatotoxicity, and cytokine storm associated with standard treatment. Moreover, CD40a‐loaded hydrogels mediate improved local cytokine induction in the TdLN and improve treatment efficacy in the B16F10 melanoma model. PNP hydrogels, therefore, represent a facile, drug‐agnostic method to ameliorate immune‐related adverse effects and explore locoregional delivery of immunostimulatory drugs.