Nadine Ottoson

Cutting Edge: T Cell Migration Regulated by CXCR4 Chemokine Receptor Signaling to ZAP-70 Tyrosine Kinase

Chemokines regulate the homeostatic trafficking of lymphocytes and lymphocyte influx into sites of injury and inflammation. The signaling pathways by which chemokine receptors regulate lymphocyte migration remain incompletely characterized. We demonstrate that Jurkat T cells lacking the ZAP-70 tyrosine kinase exhibit reduced migration in response to the CXCR4 ligand CXCL12 when compared with wild-type Jurkat T cells. Expression of wild-type, but not kinase-inactive, ZAP-70 resulted in enhanced migration of ZAP-70-deficient Jurkat T cells. The tyrosine residue at position 292 in the interdomain B region of ZAP-70 exerts a negative regulatory effect on ZAP-70-dependent migration. Stimulation of Jurkat T cells with CXCL12 also resulted in ZAP-70-dependent tyrosine phosphorylation of the Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) adapter protein. Although CXCL12-dependent migration of SLP-76-deficient Jurkat T cells was impaired, re-expression of SLP-76 did not enhance migration. These results suggest a novel function for ZAP-70, but not SLP-76, in CXCR4 chemokine receptor signaling in human T cells.

Cutting Edge: A Novel Function for the SLAP-130/FYB Adapter Protein in β1 Integrin Signaling and T Lymphocyte Migration

The role of integrin-mediated signaling events in T cell function remains incompletely characterized. We report here that α4β1 integrin stimulation of H9 T cells and normal human T cell blasts results in rapid and transient tyrosine phosphorylation of the adapter protein, SH2 domain-containing 76-kDa protein (SLP-76)-associated phosphoprotein of 130 kDa (SLAP-130)/FYB at levels comparable to those observed following TCR stimulation. Stimulation of T cells via the α4β1 integrin enhances the association of tyrosine phosphorylated SLAP-130/FYB with the SH2 domain of the src tyrosine kinase p59fyn. Activation of normal T cells, but not H9 T cells, via α4β1 leads to tyrosine phosphorylation of SLP-76 as well as SLAP-130/FYB. Overexpression of SLAP-130/FYB in normal T cells enhances T cell migration through fibronectin-coated filters in response to the chemokine stromal cell-derived factor (SDF)-1α. These results identify SLAP-130/FYB as a new tyrosine phosphorylated substrate in β1 integrin signaling and suggest a novel function for SLAP-130/FYB in regulating T lymphocyte motility.

Regulation of β1 Integrin-Mediated Adhesion by T Cell Receptor Signaling Involves ZAP-70 but Differs from Signaling Events That Regulate Transcriptional Activity

Stimulation of the CD3/TCR results within minutes in an increase in T cell adhesion mediated by β1 integrins. The biochemical pathways that control CD3-mediated increases in β1 integrin-mediated adhesion remain poorly characterized. In this study, the role of the tyrosine kinase ZAP-70 in the regulation of β1 integrin activity by the CD3/TCR was investigated. CD3 stimulation did not increase β1 integrin-mediated adhesion of the ZAP-70-deficient Jurkat T cell line, P116, to the β1 integrin ligand fibronectin. Reintroduction of wild-type ZAP-70, but not a kinase-inactive variant, K369R, corrected the adhesive defect observed in P116 T cells. In addition, the kinase-inactive ZAP-70 mutant inhibited CD3-induced adhesion of primary human T cell blasts. Interestingly, a ZAP-70 mutant with a tyrosine to phenylalanine substitution at position 319 (Y319F) restored the adhesive defect in P116 T cells, even though Y319F ZAP-70 failed to fully reconstitute CD3-initiated NF-AT-dependent transcription and tyrosine phosphorylation of the LAT adapter protein. Finally, expression of mutants of LAT and the SLP-76 adapter protein that modulate CD3-mediated activation of an NF-AT reporter gene failed to block CD3-induced increases in β1 integrin-mediated adhesion. These observations support a model in which the tyrosine kinase activity of ZAP-70 kinase is critical for regulation of β1 integrin activity by CD3/TCR. However, the signaling events downstream of ZAP-70 that regulate CD3/TCR-mediated activation of β1 integrin function exhibit key differences when compared with the signaling pathways that regulate transcriptional events initiated by CD3/TCR stimulation.

Abstract A128: Imprime PGG, a novel, clinical-stage pathogen associated molecular pattern, modulates MDSC function, facilitating a coordinated antitumor immune response

The success of cancer immunotherapy is limited by multiple mechanisms of tumor-induced immunosuppression often a result of suppressive myeloid cells. M2-like tumor associated macrophages (TAM), myeloid-derived suppressor cells (MDSC), and tolerogenic dendritic cells (DC) constitute this immunosuppressive tumor microenvironment (TME) and have repeatedly been associated with poorer prognoses. Therapies designed to overturn this suppressive immune microenvironment could substantially enhance the efficacy of multiple immunotherapeutic approaches. Imprime PGG (Imprime) is a yeast β-1,3/1,6 glucan that has shown compelling efficacy in multiple Phase II trials with tumor-targeting and anti-angiogenic antibodies. As a pathogen associated molecular pattern (PAMP), Imprime activates myeloid cells (monocytes/macrophages, neutrophils, dendritic cells). Our previous ex vivo human and in vivo mouse studies have shown that Imprime promotes repolarization of M2 macrophages to an anti-tumor, M1-like orientation and enhances DC maturation, driving T cell expansion and the production of interferon gamma (IFNγ). We now show that Imprime can modulate the function of immature myeloid cells- the myeloid derived suppressor cells. MDSCs were generated from human cord blood by purifying CD34+ cells and culturing with GMCSF or GCSF ± tumor conditioned media. After 9 days, HLA-DR cells were depleted. The remaining cells were isolated and treated with Imprime or vehicle control. We found that incubating tumor-conditioned, vehicle-treated MDSCs with CD3/CD28-activated CD8 or CD4 T cells inhibited T cell proliferation. Imprime treatment significantly reduced this inhibition. Phenotypically, Imprime treatment elicited substantially increased expression of the co-stimulatory molecules CD80 and CD86 in both monocytic (Mo) and granulocytic (PMN) MDSCs, suggesting that these MDSCs now exhibited APC-like characteristics. In in vivo murine tumor models, Imprime was also able to modulate MDSC phenotype and function, which coincided with enhanced anti-tumor efficacy. Specifically, in the H441 human NSCLC xenograft model, Imprime when combined with 10mg/kg DC101 (murine anti-VEGFR2 Ab), significantly repressed tumor growth compared to the DC-101 alone or vehicle groups. Moreover, spleens from Imprime + DC101 treated-mice had reduced numbers of immunosuppressive, splenic Mo- or PMN-MDSC compared to either DC101 alone or vehicle control. The splenic Mo- or PMN-MDSCs from Imprime + DC101 treated mice that were present expressed significantly higher CD86. The Mo-MDSCs also expressed higher levels of iNOS, a key marker for activated macrophages. In another xenograft NSCLC model, H1299, the splenic MDSC after treatment with Imprime and α-VEGF-A Ab, bevacizumab (bev), showed increased iNOS expression and reduced Arg-1 expression- a shift typifying the M1 polarization state. Collectively, these data show that Imprime treatment can reshape the suppressive immune microenvironment of the tumor and elicit a robust anti-tumor immunity by targeting multiple myeloid lineage cells. Citation Format: Kathryn Fraser, Anissa Chan, Xiohong Qiu, Nadine Ottoson, Adria Bykowski Jonas, Jeremy Graff, Nandita Bose. Imprime PGG, a novel, clinical-stage pathogen associated molecular pattern, modulates MDSC function, facilitating a coordinated antitumor immune response [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A128.

Abstract LB-080: Imprime PGG, a β-glucan PAMP (pathogen-associated molecular pattern) activates the direct killing functions of innate immune cells in concert with tumor targeting antibodies

Imprime PGG (Imprime) is a soluble yeast 1,3/1,6 β-glucan PAMP (pathogen-associated molecular pattern). As a PAMP, Imprime triggers innate immune function, activating the direct killing functions of innate immune cells, facilitating MDSC differentiation and macrophage repolarization as well as enabling dendritic cell maturation and antigen presentation, driving T cell expansion and activation. In the clinic, Imprime is administered intravenously and is well-tolerated. In multiple clinical trials (> 400 subjects), including randomized phase 2 studies in NSCLC, Imprime has consistently shown promising increases in both objective tumor response and patient survival. To date, the clinical experience with Imprime has centered on combinations with tumor targeting monoclonal antibodies (Mabs). For instance, Imprime combined with rituximab and alemtuzumab in CLL patients yielded a 65% complete response rate (vs 37% historical CR rate for alemtuzumab plus rituximab). We sought to better characterize the effect of Imprime in concert with tumor-targeting mAbs. We show that Imprime enhances the effector functions of multiple innate immune cell lineages. We first evaluated the generation of Reactive Oxygen Species (ROS) in neutrophils isolated from human healthy volunteer whole blood. These neutrophils, but not those from vehicle treated whole blood, specifically recognized B cell lymphomas (Raji) only after opsonization with anti-CD20 Mabs (rituximab, ofatumumab, obinatuzumab), generating a substantial ROS burst that coincided with enhanced tumor cell cytotoxicity. Similarly, increased antibody dependent cellular phagocytosis (ADCP) mediated by monocyte-derived macrophages was evident against antibody-opsonized lymphomas (Z138 B cell lymphomas with obinutuzumab) and solid tumor cells (SKBr3 breast cancer cells with trastuzumab) from Imprime-treated whole blood. Likewise, increased Natural Killer (NK) cell-mediated antibody dependent cellular cytotoxicity (ADCC) was evident only after Imprime treatment against antibody-opsonized cancer cells (SKBR3 with trastuzumab, K562 erythroleukemia cells with anti-glycophorin-A). In vivo, we now show that Imprime administered intravenously significantly enhances the anti-tumor efficacy of trastuzumab in a patient derived xenograft model of breast cancer, reducing mean tumor volume to ∼ 50% of that achieved by trastuzumab alone. In the B16 lung experimental metastasis model, the addition of Imprime to the anti-TRP1 tumor targeting antibody TA-99 significantly reduces both the number and size of B16 lung metastases. Together, these data show that Imprime stimulates the innate immune system, augmenting the anti-tumor efficacy of a diverse array of tumor targeting antibodies in multiple tumor types. Citation Format: Steven M. Leonardo, Ross B. Fulton, Keith B. Gorden, Katy Fraser, Ben Harrison, Takashi Kangas, Adria Jonas, Yumi Yokoyama, Nadine Ottoson, Nandita Bose, Jeremy R. Graff. Imprime PGG, a β-glucan PAMP (pathogen-associated molecular pattern) activates the direct killing functions of innate immune cells in concert with tumor targeting antibodies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-080.

Abstract 3280: Imprime PGG synergizes with anti-angiogenic antibodies to repolarize the immune microenvironment, suppressing xenograft tumor growth in vivo

Anti-angiogenic antibodies (Ab) such as bevacizumab (αVEGF) and ramucirumab (αVEGFR2) suppress tumor growth by disrupting the leaky, tortuous vasculature characteristic of growing tumors. Recent work now indicates that these Ab may also promote a shift from an immunosuppressive tumor microenvironment to one more permissive for immune recognition and tumor eradication. These data suggest that combining anti-angiogenic Abs with immunotherapies, particularly those that may also drive repolarization of the immunosuppressive tumor microenvironment, may enhance therapeutic efficacy. Imprime PGG (Imprime) is a β glucan PAMP (Pathogen Associated Molecular Pattern) that has demonstrated promising efficacy in phase 2 randomized clinical trials with the bevacizumab (bev)-based therapy. Preclinical mechanistic work has shown that Imprime can promote repolarization of the suppressive M2 macrophages and MDSCs that typically reside within the tumor microenvironment. We now show that, when combined with DC101 (murine anti-VEGFR2 Ab), Imprime significantly enhances the inhibition of H441 human NSCLC xenograft tumor growth in athymic nude mice. Moreover, we also show that the combination of Imprime plus DC101 promotes a more pronounced and significant shift in myeloid function than either agent alone. Specifically, mice treated with Imprime plus DC101 had reduced numbers of immunosuppressive, splenic MDSCs and an increase in the number of CD68+F4/80+ cells expressing the critical co-stimulatory marker CD86, indicating an increase in activated splenic macrophages. Tumor associated macrophages from these mice also showed significantly increased expression of CD86. qRT-PCR analyses of these tumor tissues likewise revealed that the combination specifically elicited a profound shift in the polarization state of the microenvironment, increasing M1 markers (TNFα, iNOS, IL-6) and decreasing M2 markers (CD206, IL-10, TGFβ and CCL22). Similarly, in H1299 NSCLC xenograft-bearing mice, the addition of Imprime to bev also elicited a profound shift in the polarization state of myeloid cells. Macrophages and neutrophils from spleen and tumor tissue of mice treated with the combination showed significant upregulation of CD86. Moreover, when compared to mice treated only with bev, splenic MDSCs from Imprime plus bev treated mice showed increased iNOS expression and reduced Arg-1 expression- a shift typifying the M1 polarization state. These data reveal that the addition of Imprime to anti-angiogenic Ab therapy prompts a substantial shift in the tumor immune microenvironment in situ and enhances the efficacy of anti-angiogenic therapy. Citation Format: Kathryn Fraser, Nadine Ottoson, Xiahong Qiu, Anissa Chan, Adria Jonas, Takashi Kangas, Jeremy Graff, Nandita Bose. Imprime PGG synergizes with anti-angiogenic antibodies to repolarize the immune microenvironment, suppressing xenograft tumor growth in vivo . [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3280.

Abstract A3: Imprime PGG triggers a coordinated anti-cancer immune response in concert with anti-angiogenic antibodies, re-polarizing the immune microenvironment to suppress tumor growth

Imprime PGG (Imprime) is a soluble, yeast-derived 1,3-1,6 β-glucan in clinical development for the treatment of cancer in combination with other anti-cancer therapies. Imprime acts as a Pathogen Associated Molecular Pattern (PAMP) and can be recognized by cells of the innate immune system. Preclinical data using human whole blood from healthy volunteers show that Imprime binding to innate immune cells triggers a coordinated immune response that includes repolarization of M2 macrophages, activation of neutrophils and maturation of dendritic cells. This response ultimately leads to cross-talk with the adaptive immune system driving T cell expansion and the production of interferon gamma (IFNγ). In a randomized phase 2 clinical study in stage IV NSCLC patients, treatment with Imprime plus bevacizumab (bev; anti-VEGF antibody), carboplatin and paclitaxel showed a median overall survival of 16.1 months versus 11.6 months in patients not receiving Imprime. We sought to explore a mechanistic understanding for this promising clinical activity. Angiogenic factors, such as VEGF, not only drive the formation of new leaky vessels but also facilitate the establishment of a suppressive immune microenvironment enabling tumor survival and growth. Recent work has shown that anti-angiogenics not only block neovascularization but may also promote a shift in the immune microenvironment, enabling immune recognition and destruction of the tumor. We therefore sought to evaluate whether Imprime, may complement the effect of anti-angiogenics on the immune microenvironment. We tested Imprime in combination with either bev or DC101 (anti-VEGFR2) in distinct NSCLC xenograft models in athymic nude mice. Once tumors reached a mean size of 100mm3, mice were treated with Imprime, bev or DC101. H1299 and H441 tumor-bearing mice were used in the bev and DC101 studies, respectively. In the bev study, Imprime plus bev induced >75% tumor growth inhibition in ∼50% of mice vs 20% in the bev alone groups. Both macrophages and neutrophils from spleen and tumor tissue of combination-treated mice showed significant upregulation of the activation marker CD86 compared to tissues from bev alone treated mice. Moreover, splenic MDSCs in combination-treated mice showed significantly increased iNOS2 expression with reduced Arg-1 expression compared to bev alone treated mice. Tumors from the Imprime plus bev groups showed significantly reduced expression of the potent immunosuppressor, TGFβ, when compared to tumors from mice treated only with bev-with the greatest reduction evident in the tumors with the greatest growth inhibition. In the H441 tumor-bearing mice treated with Imprime and DC101, a significant suppression of tumor growth compared with DC101 alone was also observed and additional mechanistic studies in this model are ongoing. These data show for the first time that Imprime-based treatment prompts a shift in the immune microenvironment of a tumor in situ, eliciting enhanced tumor growth inhibition in concert with anti-angiogenic therapy. Citation Format: Kathryn Fraser, Nadine Ottoson, Xiahong Qiu, Anissa SH Chan, Adria Jonas, Takashi Kangas, Jeremy Graff, Nandita Bose. Imprime PGG triggers a coordinated anti-cancer immune response in concert with anti-angiogenic antibodies, re-polarizing the immune microenvironment to suppress tumor growth. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A3.

Abstract LB-199: Imprime PGG modulates immunosuppressive myeloid components of the tumor microenvironment and drives enhanced antitumor efficacy in combination with checkpoint inhibitor therapies

Checkpoint inhibitor therapies (CPI) have shown great promise, however in a limited percentage of patients. One of the key mechanisms behind the limited efficacy of CPI therapy is immune resistance mediated by immunosuppressive myeloid cells at the tumor microenvironment (TME), namely M2 macrophages and myeloid-derived suppressor cells (MDSC). Multiple therapeutic interventions are being developed to target these cell types with the intention of reshaping the TME and enhancing the effector functions of the infiltrating cytotoxic T cells.Molecules containing pathogen associated molecular patterns (PAMPS) are one of the unique combination partners that can sensitize tumors to respond to CPI. Imprime PGG (Imprime), an intravenously administered soluble yeast β-1,3/1,6 glucan PAMP, is being clinically developed in combination with tumor-targeting antibodies, anti-angiogenics, and CPI. Imprime has shown promising results in two randomized phase 2 studies in non-small cell lung cancer. Mechanistic studies have shown Imprime to repolarize M2 macrophages and MDSC in in vitro human systems as well as multiple xenograft models. The objective of this study was to evaluate Imprime’s ability to counteract immunosuppression and thereby influence the effector functions of T cells in a syngeneic tumor model. To this end, we first evaluated the anti-tumor efficacy of Imprime in combination with anti-PD-1 or anti-PD-L1 antibody in the MC-38 colon cancer model and found that both combinations repressed tumor growth more effectively than either single agent. Flow cytometric evaluation of single cell suspensions of spleen and tumor tissue after one week of Imprime dosing revealed that the tumor associated macrophages showed a shift to an M1-like phenotype with increased expression of PD-L1, CD86, inducible nitric oxide synthase, MHC class II and downmodulation of Arginase-1. qRT-PCR analyses also demonstrated an increase in transcripts for M1 markers (Il12b p35, Ifng, Tnfa) with a coincident decrease in transcripts for M2 markers (VEGF, Fizz1, CCL17). Adaptive immune resistance, increased PD-L1 expression on the tumor cells as a result of immune activation was observed. In the spleen, the monocytic MDSC also showed increased expression of M1 markers. Furthermore, increased number of CD8 cells in the spleen were of effector memory phenotype with enhanced expression of PD-1, granzyme B, and Ki-67. At the tumor site, the CD8 cells from Imprime treated mice demonstrated increased proliferative and cytokine producing capabilities (IL-2, IFNg, and TNFa) in response to CD3/CD28 stimulation. Collectively, these data show that Imprime’s ability to remold the TME such that the myeloid cells are less suppressive and the cytotoxic T cells are more functionally active can have a tremendous impact on overcoming resistance to CPI therapy. Citation Format: Adria B. Jonas, Anissa SH Chan, Xiaohong Qiu, Kathryn Fraser, Nadine Ottoson, Takashi Kangas, Richard Walsh, Steven M. Leonardo, Ross Fulton, Keith Gorden, Mark Uhlik, Jeremy Graff, Nandita Bose. Imprime PGG modulates immunosuppressive myeloid components of the tumor microenvironment and drives enhanced antitumor efficacy in combination with checkpoint inhibitor therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-199. doi:10.1158/1538-7445.AM2017-LB-199

Abstract B008: Imprime PGG, an intravenously administered beta glucan PAMP activates the innate immune system: A phase I clinical study to evaluate immunopharmacodynamic responses

Imprime PGG (Imprime), in combination with both tumor-targeting and anti-angiogenic antibodies, has shown promising efficacy in multiple phase 2 clinical trials. In numerous pre-clinical in vivo tumor models, Imprime also enhances the efficacy of immune checkpoint inhibitor antibodies in addition to tumor-targeting and anti-angiogenic antibodies. Imprime is a yeast-derived, soluble β-1,3/1,6 glucan that acts as a Pathogen Associated Molecular Pattern (PAMP) to trigger activation of innate immune effector cells (macrophages, monocytes, neutrophils, dendritic cells (DC)), which orchestrate a coordinated anti-cancer immune response with cells of the adaptive immune system. Ex vivo studies with human whole blood have shown that Imprime forms an immune complex with endogenous anti-beta glucan antibodies (ABA) to trigger a constellation of innate immune functions. These include complement activation via the classical complement pathway, select chemokine production, phenotypic activation and enhanced tumor cell killing by neutrophils and macrophages. Imprime also activates antigen-presenting cells (e.g. macrophages, DC), enabling T cell expansion and activation. In vivo, intravenous (IV) injection of Imprime in C57BL/6 mice increases select chemokine expression, triggers neutrophil and monocyte mobilization into circulation and secondary lymphoid organs, and also enhances DC maturation and antigen-specific T-cell priming. In this study, we show that the immunopharmacodynamic (IPD) responses elicited by IV administration of Imprime in healthy human subjects are consistent with the innate immune responses observed in ex vivo human and in vivo mouse studies. Healthy human volunteers (18-65 yr) were administered single (Cohort 1) or multiple (once weekly for 3 wks-Cohort 2) doses of Imprime PGG (4 mg/kg) by IV infusion over 2-3 hrs. Physical examination with vital signs, adverse event solicitation and timed blood sampling for IPD changes were performed. IPD endpoints included complement protein levels, circulating blood cell lineage counts, ABA concentrations, circulating immune complex (CIC) levels, cytokine and chemokine concentrations, as well as binding and activation of blood leukocytes. Cohort 1 and 2 results show that the complement activation proteins C5a and SC5b-9 were significantly increased in the plasma at the end of infusion (EOI) of Imprime. The formation of Imprime:ABA complexes was evident in a substantial drop of free ABA and a concomitant increase in CIC in the serum also at the EOI. IL-8 and MCP-1 were consistently detected between EOI and 1 hr post infusion. Additional chemokines, including MIP-1α, MIP-1β, and IP-10 were also detected in some of the subjects. A significant increase in the neutrophil and monocyte counts was seen in the blood after infusion. Cellular analyses showed Imprime binding to neutrophils, monocytes, and subsets of DC (classical and inflammatory) 15-30 mins after the start of infusion. Additionally, 24 hrs after completion of Imprime administration, a population of non-classical monocytes (CD14/CD16 positive), which are known to have higher antigen presentation capability and thus express higher levels of the activation markers CD86, PD-L1, and HLA-DR, was observed. Importantly, these IPD responses were evident only in subjects with higher ABA levels. Collectively, these data provide the first evidence that, when dosed IV in healthy human subjects, Imprime elicits a constellation of innate immune activating events that are consistent with efficacy in preclinical tumor models. Importantly, these human data also provide the first evidence linking pre-treatment ABA levels and Imprime induced IPD changes, suggesting the plausibility of using pre-treatment ABA levels in the selection of patients most likely to benefit from Imprime-based therapy. Citation Format: Nadine C. Ottoson, Richard D. Huhn, Jamie Lowe, Ben Harrison, Jose Iglesias, Blaine Rathmann, Takashi Kangas, Lindsay R. Wurst, Xiaohong Qiu, Anissa Chan, Adria Bykowski Jonas, Kathryn Fraser, Richard M. Walsh, Katie Ertelt, Steven M. Leonardo, Ross Fulton, Keith Gorden, Mark A. Matson, Mark Uhlik, Jeremy Graff, Nandita Bose. Imprime PGG, an intravenously administered beta glucan PAMP activates the innate immune system: A phase I clinical study to evaluate immunopharmacodynamic responses [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B008.

Abstract A014: Endogenous anti-β-glucan antibodies and FcgRIIA (CD32a) single nucleotide polymorphisms (SNP) as potential predictive biomarkers for the efficacy of Imprime PGG immunotherapy in cancer patients

Imprime PGG (Imprime), a soluble yeast 1,3/1,6 β-glucan, is being developed as a novel cancer immunotherapy in conjunction with anti-tumor antibodies in several cancers. Randomized Phase 2 clinical trials of Imprime in the 1st-line treatment of stage IV non-small cell lung cancer have shown promising efficacy in terms of both objective tumor response and survival. Mechanistic studies have demonstrated that Imprime forms an immune complex with endogenous IgG and IgM anti-β-glucan antibodies (ABA). The immune complex then binds to and primes innate immune cells, including macrophages, monocytes and neutrophils, to kill antibody-targeted cancer cells via a complement receptor 3-dependent mechanism. In this study, we sought to investigate how the formation of this Imprime-ABA immune complex might influence patient selection strategies. We have shown that there is a threshold level of ABA required for binding of Imprime-ABA immune complex to the innate immune effectors. In human healthy volunteers (N=143) IgG ABA concentrations vary from 8 to 1448 μg/ml. Imprime treatment of whole blood from individuals with ABA concentrations above a threshold yields immune complex formation that allows both classical and alternative complement pathways leading to complement opsonization of the complex. This complex then binds innate immune cells, triggering the modulation of cell surface receptors and production of selective chemokines such as IL-8. We now show that the predominant subclass of endogenous IgG ABA specific to Imprime is IgG2. Additionally, we show that binding of Imprime to immune cells critically involves FcgRIIA, the only Fc receptor capable of binding IgG2 immune complexes. Blocking FcgRIIA significantly inhibits binding to both neutrophils and monocytes in whole blood. This observation was further confirmed by binding of Imprime to HEK cells overexpressing FcgRIIA. In order to evaluate the effect that ABA IgG subclass has on Imprime, chimeric ABAs with identical affinity but differing in the human IgG subclass (IgG1 vs. IgG2) were generated using the heavy and light chain variable domains from a mouse ABA hybridoma. IgG1 or IgG2 ABA added to whole blood of healthy donors with low ABA levels was able to increase Imprime binding, consistent with previous data showing that Imprime binding is dependent on ABA levels. However, the relative increase in binding due to the addition of IgG1 or IgG2 ABA varied among donors. While addition of either IgG1 or IgG2 ABA was equivalent in increasing binding in some donors, IgG1 was more efficacious than IgG2 in other donors. These results led us to look at allelic variants of the FCGR2A gene. In humans, there is a SNP in FCGR2A, R131H, that defines two alleles whose protein products differ in affinity for IgG2, with H variant designating the higher affinity allele and the R variant indicating lower affinity. The variable binding of Imprime in donors with addition of chimeric ABA was found to be related to the FCGR2A genotype. Individuals homozygous for the R genotype showed lower binding with IgG2 chimeric ABA than with IgG1 chimeric ABA. The findings were similar with exogenously added naturally occurring ABA purified from serum. The effect of the R131H SNP on Imprime binding in relation to endogenous ABA levels was further confirmed in a survey of 140 healthy volunteers. The prevalence and functional relevance of this SNP on Imprime binding to immune cells in cancer patients is being evaluated. Together, these data implicate both the concentration of ABA in serum and FCGR2A genotype as critical determinants of the ability of an individual to bind to Imprime and suggest that either or both may enable the selection of patients most likely to derive therapeutic benefit from Imprime treatment. Citation Format: Nandita Bose, Anissa Chan, Adria Jonas, Nadine Ottoson, Xiaohong Qiu, Lindsay Wurst, Steven Leonardo, Peter Maimonis, Katie Ertelt, Ben Harrison, Keith Gorden. Endogenous anti-β-glucan antibodies and FcgRIIA (CD32a) single nucleotide polymorphisms (SNP) as potential predictive biomarkers for the efficacy of Imprime PGG immunotherapy in cancer patients. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A014.