CD47-SIRPalpha Interaction Research Articles

HMBD004, a Novel Anti-CD47xCD33 Bispecific Antibody Displays Potent Anti-Tumor Effects in Pre-Clinical Models of AML

CD47 is the “don't-eat-me” signal and is ubiquitously expressed on normal cells where binding to SIRPalpha on macrophages inhibits phagocytosis. CD47 is commonly over-expressed in tumors where it correlates with immune evasion and poor prognosis. Blocking CD47-SIRPalpha interaction restores macrophage phagocytosis of tumor cells and anti-CD47 mAbs have shown anti-tumor efficacy in mouse models of solid tumors and hematological malignancies. Nevertheless, the widespread expression of CD47 decreases the bioavailability of anti-CD47 mAbs at the tumor due to antigen sink effects, and also risks off-target toxicities, most significantly from cross-linking of erythrocytes that show high expression of CD47. Therefore, increasing the specificity for binding CD47, and inhibiting the interaction with SIRPalpha, to tumor cells alone should increase the efficacy and safety of anti-CD47 therapy.CD33 is expressed on myeloid cells, but is highly over-expressed in 90% of AML patients, where it marks leukemic blast cells. The safety of CD33 as an antibody target for directing effector molecules to tumor cells has been convincingly demonstrated by the efficacy of gemtuzumab ozogamicin (Mylotarg), an antibody-drug conjugate (ADC) that binds CD33 with high affinity, in the treatment of relapsed AML. However, ADCs are notable for their significant risk of toxicities, and gemtuzumab ozogmicin was withdrawn for this reason in 2010. Still, other companies continue development on next generation anti-CD33 ADCs, e.g. vadastuximab talirine.Hummingbird Bioscience has used its proprietary Rational Antibody Development Platform for the design and development of novel therapeutic antibodies against CD47. Computational sequence and structural analyses predicted a highly specific surface epitope, with strong antigenicity and good safety profile, where antibody binding would inhibit binding to SIRPalpha. Subsequently, an in-house mouse immunization and screening strategy efficiently yielded a monoclonal antibody (mAb, 11a1) that bound with low nM (KD 5 nM) affinity and high specificity to the desired epitope on human CD47. In vitro, 11a1 demonstrated 100% inhibition of SIRPalpha binding at 1 nM, and potently induced phagocytosis of Raji cells by isolated macrophages.11a1 was subsequently humanized by CDR grafting and back-mutation, and the resulting humanized mAb maintained high affinity binding to CD47. Hummingbird Bioscience then constructed a bispecific anti-CD47xCD33 antibody (bsAb), based on a standard 1+1 IgG format, with a humanized anti-CD47 variable domain as an effector arm and the gemtuzumab variable domain as an anti-CD33 specificity arm. This bsAb (HMBD004) maintained the potent inhibition of SIRPalpha binding and induction of phagocytosis but, unlike many CD47 mAbs, caused negligible hemagglutination (i.e. cross-linking) of erythrocytes in vitro . Furthermore, HMBD004 showed preferential sorting of CD47+ CD33+ cells during FACS of mixed populations of CD47+ cells. Finally, in vivo CDX mouse models of AML were established by engrafting NCr nude mice with the CD47+ CD33+ AML cell line, HL-60, via subcutaneous implantation. Treatment of these mice with HMBD004 showed a significant decrease in tumor burden and increased PFS.Together, these data highlight the benefit of using CD33 binding to enhance the specificity, efficacy and safety of anti-CD47 antibody therapy in AML. Hummingbird Bioscience is currently advancing HMBD004 towards the clinic in 2018. DisclosuresNo relevant conflicts of interest to declare.

Alteration of CD47 Expression in Human and Murine Tumor Models Changes M1/M2 Phagocytosis Effect in Vitro and In Vivo

IntroductionCD47 expression in human tumor cell represents a resistance mechanism escaping the macrophage surveillance system. To reinstate macrophage function in recognizing and engulfing tumor cells, unblocking CD47-SIRPalpha interaction and thereby re-activating macrophage activity has been an important strategy in immune-oncology therapy. To validate the strategy, we profiled a large panel of tumor models including tumor cell lines, PDX models in Crown tissue bank and clinical samples for CD47 expression. Despite of its expression in the majority of tumor cell models, the expression level of CD47 vary in flow cytometry examination. Interestingly a portion of tumor cells do not express CD47 at all.To facilitate in vitro screening for effective macrophage therapy, we established an in vitro assay platform using CD14+ monocytes as starting material. We also engineered human and murine cell models to overexpress human CD47 at one end and delete endogenous CD47 at the other end, and observed robust phagocytosis effect in these models. Finally, the findings in the in vitro assays were validated in MC38-hCD47 homograft in vivo.Material and MethodsTumor cell lines were CFSE-labeled and incubated with human monocyte-derived M1 or M2 macrophages (T:M 1:1) in the presence of 20μg/ml IgG1 isotype control, or anti-CD47 blocking antibodies B6H12 and BRIC126 and non-blocking antibody 2D3, for 2hrs. Cells were then harvested and stained with a macrophage marker (CD14+). Phagocytosed tumor cells were identified by flow cytometry as CD14+CFSE+. The phagocytosis index was calculated by dividing the number of macrophages engulfing tumor cells by the total number of macrophages. CD47 overexpression and deletion clones (KARPAS299, SK-OV3, and MC38 background) were developed in house. The CD14+ cell isolation kit was from Miltenyi. M1/M2 differentiation reagents were purchased from R&D and Stem Cell. Anti-CD47 antibodies B6H12 and 2D3 and mouse IgG1 were purchased from Thermo Fisher, and BRIC126 from GeneTex. B6H12 for animal study was sourced from Bioxcell.ResultsTo facilitate in vitro screening for effective macrophage therapy, we sought to establish a robust yet reproducible assay platform using CD14+ monocytes as starting material. Through macrophage differentiation we were able to achieve > 80% of M1 and M2 populations, respectively, though variation exists. Differentiated M1/M2 were then co-cultured with CFSE-labeled target tumor cells for subsequent flow cytometry analysis. Strong phagocytosis effect was observed with CD47 expression tumor cells when anti-CD47 antibody BRICK126, which blocks the CD47-SIRPalpha interaction, was introduced. In contrast, aCD274 (PD-L1) antibody didn't yield more than background phagocytosis effect in the same assay suggesting the specific activation effect of BRICK126 triggering M1/M2 functionality.To prove the CD47-specific effect, we engineered CARPAS299 and SK-OV3 tumor cell line models and MC38 murine cell model to overexpress human CD47 at one end and delete endogenous CD47 at the other end. The recombinant and isogenic models were then subject to the CD47 biology examination and more profound changes in phagocytosis responses were observed.To validate our findings in vivo, we introduced engineered MC38-hCD47 clone in to C57/BL6 mice to measure homograft tumor growth kinetics and pharmacological responses to B6H12, another CD47 blocking agent. Details of results will be presented.ConclusionDisrupting the CD47-SIRPa anti-phagocytic axis is a novel immunotherapy approach against cancer. This study reveals that CD47 expression level varies in different tumor cell models. Our in vitro screen demonstrated that M1 and M2 macrophages resulted in comparable anti-CD47 phagocytic activities, and suggests that Karpas299 with higher CD47 expression level may be a better model than SK-OV3 in cell engulfing study. We also developed Karpas299-hCD47, SK-OV-3-hCD47 and MC38-hCD47 stable clones to facilitate in vitro and in vivo screening towards macrophage therapy, and discovered strong positive correlation between CD47 expression level and phagocytic activity in in vitro and in vivo systems. DisclosuresNo relevant conflicts of interest to declare.

Abstract 4556: Phagocytosis response of macrophage to CD47 expression of tumor cells in anti-CD47 therapy

Abstract Macrophage as an important surveillance system is escaped by human cancer cells expressing CD47. To reinstate macrophage functionality in clearing tumor cells targeting CD47 and thereby blocking phagocytosis inhibitory CD47-SIRPalpha interaction has been an important strategy in immune-oncology therapy. We profiled a large panel of tumor cell lines, Crown's proprietary PDX models and clinical lung cancer tumors for CD47 expression. Although it has been reported as a ubiquitously expressed immune-escape marker in cancer cells, the expression levels of CD47 vary among different tumor cell lines and models when the cells were examined by flow cytometry. It is of interest that a portion of tumor cells in fact do not present CD47. To tackle the cause for the loss of CD47 expression, whole-exome sequencing was performed to examine the intactness of the gene in the CD47 loss models. The results will be presented. We next established a robust and reproducible in vitro assay platform to support anti-CD47 drug research. We applied a macrophage differentiation system using isolated CD14+ monocytes as starting material, and were able to achieve >80% of M1 and M2 populations, respectively, through the 6-day differentiation. Differentiated macrophage was co-cultured with target tumor cells labeled for flow cytometry analysis. In the presence of blocking anti-CD47 antibody BRICK126, steady and specific phagocytosis effect was observed when target tumor cells express high level of CD47 markers. BRIC126 antibody blocks the CD47-SIRPalpha interaction. In contrast, aCD274 antibody did not yield more than background phagocytosis effect in the same assay. To prove the CD47-specific effect, we constructed isogenic cell models from a couple of parental cell models in that CD47 gene is knocked out in one sub-cell line and overexpressed in another sub-cell line. The isogenic systems were then subject to the CD47 biology assay and more profound changes in phagocytosis responses were observed. Furthermore, the in vitro findings were validated in in vivo setting. Citation Format: Huajun Yang, Zhongliang Li, Phillip Wang, Beibei Tang, Qinyun Ma, Frank Xing, Qian Shi. Phagocytosis response of macrophage to CD47 expression of tumor cells in anti-CD47 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4556.

Abstract B002: CD47 expression predicts efficacy of macrophage-mediated phagocytosis of tumor cells

Abstract Most human cancers express CD47 to escape the macrophage surveillance. To reinstate macrophage functionality in clearing tumor cells, targeting CD47 has been an important strategy in immune-oncology therapy. We profiled a large panel of tumor cell lines of both solid and blood tumors that are frequently used in cancer drug research and in Mixeno® animal study in Crown Bioscience for CD47. Despite its being reported as a ubiquitously expressed immune-escape marker in cancer cells, the expression levels of CD47 vary among different tumor cell lines when the cells were examined by flow cytometry. It is of interest that a portion of tumor cells in fact do not present CD47 despite gene expression data, such as RNA-seq data, suggesting that they do. To establish a robust and reproducible in vitro assay platform to support anti-CD47 drug research, we applied a macrophage differentiation system using isolated CD14+ monocytes as starting material, and were able to achieve > 80% of M1 and M2 populations, respectively, through the 6-day differentiation. Differentiated macrophage was cocultured with target tumor cells labeled for flow cytometry analysis. In the presence of anti-CD47 antibody, steady phagocytosis effect was observed with aCD47 BRIC126 antibody and target tumor cells expressing high level of CD47 markers. BRIC126 antibody blocks the CD47-SIRPalpha interaction. To prove the finding, we constructed isogenic cell models from the same parental cells in that CD47 gene is knocked out in one sub-cell line and overexpressed in another sub-cell line. The isogenic systems are also subject to the CD47 biology studies and can be served as positive and negative controls in phagocytosis assays. Detailed data will be discussed. Citation Format: Huajun Yang, Zhongliang Li, Beibei Tang, Frank Xing, Qian Shi. CD47 expression predicts efficacy of macrophage-mediated phagocytosis of tumor cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B002.

Abstract B54: Antagonizing CD47-SIRP alpha interaction with a bispecific antibody: A novel cancer immunotherapy approach

Abstract CD47 is a ubiquitous cell surface glycoprotein that serves as a negative regulator of numerous innate immune functions such as phagocytosis, neutrophil inflammatory responses or dendritic cell maturation and activation. In particular, the interaction of CD47 with Signal Regulatory Protein Alpha (SIRP alpha) on myeloid cells is a mechanism of self-recognition, a ‘don't eat me’ signal used by healthy cells to impede their elimination by phagocytes. Cancer cells often up-regulate CD47 expression, which helps them to evade immune surveillance and killing. What is more, increased CD47 levels are generally predictive of poor clinical outcome. Inhibiting CD47-SIRP alpha interaction represents therefore an attractive, generally applicable therapeutic strategy. Yet, achieving that goal with an anti-CD47 monoclonal antibody (Mab) may be challenging in practice, given the ubiquitous expression of the target. The “drug sink” represented by erythrocytes, platelets and other CD47-expressing cells may lead to a rapid elimination of an anti-CD47 Mab through target-mediated drug disposition; another likely consequence of ubiquitous CD47 expression are on-target toxicities like, e.g., anemia. To overcome these limitations, we have developed dual-targeting bispecific antibodies to CD47 and different Tumor-Associated Antigens (TAAs). Dual targeting CD47/TAA bispecific antibodies bind preferentially to TAA-expressing cancer cells and enable selective CD47 neutralization at the same time sparing healthy TAA-negative cells. Various TAAs are currently being pursued at Novimmune (e.g., CD19, mesothelin, or glypican 3). Such dual targeting CD47/TAA bispecific antibodies have the kappa/lambda body format i.e., they are fully human bispecific IgGs composed of a high-affinity anti-TAA arm, a CD47-neutralizing arm, and an unmodified IgG1 Fc. So far, only CD47/CD19 kappa/lambda bodies have been thoroughly characterized in vitro and in vivo. Various human B cell leukemia and lymphoma lines were used to demonstrate selective, CD19-dependent binding of these CD47/CD19 kappa/lambda bodies. As anticipated, the neutralization of the CD47-SIRP alpha interaction was also CD19-dependent, and led to the enhancement of Fc-mediated cancer cell killing through ADCP (antibody mediated cellular phagocytosis) or ADCC (antibody mediated cellular cytotoxicity). Efficient killing of CD19-positive cancer cells was also observed in vivo using a localized tumor xenograft model with NOD/SCID mice implanted with Raji Burkitt lymphoma cells. A dose dependent inhibition of tumor growth or tumor regression was observed, depending on the potency of the CD47-neutralizing arm used to generate the kappa/lambda body. In parallel, a single dose PK study in cynomolgus demonstrated favorable elimination kinetics, comparable to human IgG Mab. Moreover, no effects on hematological parameters were apparent at either of the doses tested (0.5 and 10 mg/kg). The example of CD47/CD19 kappa/lambda bodies illustrates the power of the dual-targeting approach: A favorable safety profile and pharmacokinetics could be achieved in parallel to potent inhibition of the CD47-SIRP alpha interaction and effective cancer cell killing. Antagonizing CD47-SIRP alpha interaction with bispecific antibodies is therefore an attractive immune-potentiating strategy. Because of its distinct mechanism of action and the ability to that harnesses the innate immunity to fight cancer, CD47/TAA bispecific antibodies would be good candidates for combination therapies with immune checkpoint inhibitors or other immunotherapeutics. Citation Format: Krzysztof Masternak, Zoe Johnson, Vanessa Buatois, Francois Rousseau, Giovanni Magistrelli, Valery Moine, Ulla Ravn, Franck Gueneau, Lucile Broyer, Susana Salgado Pires, Maureen Deehan, Nicolas Fischer, Walter Ferlin, Marie Kosco-Vilbois. Antagonizing CD47-SIRP alpha interaction with a bispecific antibody: A novel cancer immunotherapy approach. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B54.

Abstract 2482: Neutralizing CD47 in cancer cells with dual targeting kappa/lambda bodies

Abstract Neutralizing CD47, the ‘don't eat me signal’ hijacked by different tumor types, is a novel generally applicable therapeutic strategy. Because of a distinct mechanism of action and the ability to stimulate the innate anti-tumor immunity, CD47-neutralizing agents are poised as attractive candidates for combination therapies in association with other immunotherapies. However, the development of general CD47 antagonists could be hindered by the ubiquitous and abundant expression of CD47 on virtually all healthy cells. To overcome potential pharmacological and clinical liabilities of a general CD47 antagonist, we have developed bispecific kappa/lambda bodies, which selectively target CD47 in cancer cells. These kappa/lambda bodies: (i) are full-length bispecific IgGs, (ii) bind with high affinity and neutralize the CD47-SIRP alpha interaction in cancer cells expressing a tumor-associated antigen (TAA), and (iii) mediate efficient cell killing of TAA-positive cancer cells in vitro through Fc-dependent mechanisms such as ADCP (antibody mediated cellular phagocytosis) and ADCC (antibody mediated cellular cytotoxicity). We are currently developing two molecules of this type, one targeting CD47 and CD19 (for B cell malignancies), the other targeting CD47 and mesothelin (for various mesothelin-positive solid tumors). The efficacy of the CD47/CD19 kappa/lambda body was demonstrated in vivo, using two B-cell lymphoma xenograft models in NOD/SCID mice. We also performed a pharmacokinetics study in non-human primates with the CD47/CD19 lead candidate, with the objective of assessing the potential “antigen sink” effect related to ubiquitous CD47 expression on erythrocytes, platelets and other cells. Encouragingly, the CD47/CD19 kappa/lambda body administered in a single dose to cynomolgus monkeys, at 0.5 and 10 mg/kg, showed an acceptable pharmacokinetic profile and the absence of hematological toxicities. The example of the CD47/CD19 kappa/lambda body illustrates the power of the dual-targeting approach for addressing a ubiquitous cell surface receptor such as CD47. Citation Format: Krzysztof Masternak, Lucile Broyer, Elie Dheilly, Stefano Majocchi, Valéry Moine, Giovanni Magistrelli, François Rousseau, Ulla Ravn, Franck Gueneau, Pauline Malinge, Sébastien Calloud, Maud Charreton-Galby, Mireille Guerrier, Nessie Costes, Nicolas Bosson, Gérard Didelot, Lucie Bernard, Vanessa Buatois, Laura Cons, Laurence Chatel, Anne Papaioannou, Zoë Johnson, Walter Ferlin, Marie Kosco-Vilbois, Nicolas Fischer. Neutralizing CD47 in cancer cells with dual targeting kappa/lambda bodies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2482. doi:10.1158/1538-7445.AM2015-2482

Abstract 2: Phosphorylation of BNIP3 is a switch between life and death in cancer cells

Abstract Antibodies vs CD47 (CD47mAbs) that block the CD47-SIRPalpha interaction promote the phagocytosis of cancer cells and have efficacy in several tumor models. A select few CD47mAbs also directly kill cancer cells by lowering cellular cAMP levels leading to mitochondrial damage and cell death. These CD47mAbs are thus referred to as “dual-function” mAbs. Activation of protein kinase A prevents CD47mAb-mediated death suggesting that phosphorylation of one or more target proteins in the cancer cell can block the death mechanism. BNIP3, a member of the BH3-only family, is induced by hypoxia and oncogenes and is necessary for induction of cell death by dual-function CD47mAbs. BNIP3 can activate autophagy, a pro-survival function, and can also induce cell death by damaging mitochondria. The role of BNIP3 in cancer is controversial and context-dependent with some cancers over-expressing BNIP3 compared to low levels of expression in normal tissue, while other cancers cannot tolerate BNIP3 expression and silence the gene, often by methylation of the BNIP3 promoter. This suggests that cancers that tolerate BNIP3 expression employ an as yet unknown mechanism to protect themselves from its toxic effects. The C-terminal transmembrane (TM) domain of BNIP3 (residues 164-184) penetrates the outer mitochondrial membrane allowing the extreme C-terminal ten residue tail of BNIP3 (RRLTTSTSTF, residues 185-194) to extend across the intermembrane space to bind OPA1 on the inner mitochondrial membrane, a key step in the death mechanism. We used a phosphosite-specific antibody to detect phosphorylation of BNIP3 immunoprecipitated from Jurkat leukemia cells at residue T188 which resides in a canonical protein kinase A site (RRLT, amino acids 185-188). Using mass spectrometry of 6His-tagged BNIP3 isolated from HEK293 cells treated with 8BrcAMP, we identified as many as 4 additional phosphorylated sites in the C-terminal tail sequence (residues 189-194, TSTSTF). We generated phosphomimetic (S/T to D) and unphosphorylated (S/T to A or N) mutations at these residues and expressed the mutant BNIP3 proteins in 293 cells. All of the BNIP3 mutants associated with mitochondria but only the phosphomimetic mutants prevented BNIP3-induced mitochondrial damage and cell death. In contrast, mutation of the phosphorylated S/T residues to unphosphorylated residues resulted in rapid and extensive cell death. Importantly, phosphomimetic C-terminal BNIP3 residues blocked cell death without preventing autophagy, providing evidence that the two roles of BNIP3 can be regulated independently. We replicated these results in one lung cancer and three breast cancer cell lines. These findings suggest that phosphorylation at the C-terminus of BNIP3 is a switch that determines the pro-survival vs pro-death effects of BNIP3. Dual-function CD47mAbs may act by dephosphorylating BNIP3 expressed in tumor cells thus unleashing its killing potential. Citation Format: William Frazier, Katherine Liu, Julie Dimitry, Benjamin Capoccia, Pamela Manning, Robert Karr. Phosphorylation of BNIP3 is a switch between life and death in cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2. doi:10.1158/1538-7445.AM2015-2

Alteration of integrin-associated protein (CD47) on experimental porcine eperythrozoonosis

To evaluate the alteration of CD47 on RBCs of pigs infected with M. suis, we induced the experimental porcine eperythrozoonosis and collected the blood samples at the different time points. The result of analysis by flow cytometry after reaction with mouse-anti-human CD47 and caprine-anti-mouse IgG-FITC reagents indicated that the CD47 quantity on RBCs changed correlatively with the course of PE. The lowest value of M1 (percentage of the positive cells in fluorescence intensity) occurred on day 7 post inoculation at the peak of parasitemia and decreased 82% compared with the control sample. And then, the values of M1 on day 14 and 21 rised slowly but were still significantly different with the controls (p < 0.01). These suggested that the quantity of CD47 on RBCs altered progressively with the phases of the PE disease. The decrease of CD47 on RBCs maybe weaken the inhibitory CD47-SIRPalpha interaction and provide positive signals for phagocytosis of macrophages resulting in the removal of the RBCs from the circulation. In conclusion, CD47, a marker on RBCs, maybe play an important role on the mechanism of hemolysis caused by infection of M. suis.