TJC4, a Differentiated Anti-CD47 Antibody with Novel Epitope and RBC Sparing Properties

Introduction Tumor cells overexpress CD47 which engages signal-regulatory protein (SIRPa) on macrophages to deliver a "do not eat" signal to avoid being phagocytosed. Blocking CD47 using SIRPa-Fc or anti-CD47 antibodies (Ab) has emerged as a promising strategy to neutralize CD47 and promote tumor eradication. However, targeting CD47 led to significant anemia and thrombocytopenia in both pre-clinical studies and phase I trials as CD47 is also expressed on normal red blood cells (RBCs) and platelets. I-Mab has developed a novel CD47 antibody, TJC4 also known as TJ011133, which was endowed with an RBC sparing property and unique binding epitope, may have better safety profile based on the pre-clinical data. Methods A naïve human single chain variable fragment (ScFv) library was subjected for the binders to human CD47-extracellular domain (ECD). All the binders with unique sequences were converted to full antibodies and screened against human RBCs and tumor cells, leading to the discovery of TJC4. A series of head to head experiments have been performed with other CD47 antibodies to compare the in vitro RBC binding and hemagglutination, ability to block the CD47-SIRPa interaction and enhance the macrophage mediated phagocytosis of tumor cells. Different in vivo tumor models were employed to evaluate the anti-tumor efficacy of TJC4 either by mono or combination treatment. In addition, a comprehensive analysis of the hematological parameters was assessed in cynomolgus monkeys receiving a single intravenous infusion or weekly repeated injections. To explore the underlined mechanism of the RBC sparing properties of TJC4, the binding pose and epitope were identified by X-ray crystallography and the influence of CD47 glycosylation in RBCs were further examined. Results TJC4 is a fully human anti-CD47 IgG4 antibody that shares a similar binding affinity to human and cynomolgus monkey CD47. Like other anti-CD47 antibodies, TJC4 blocks the interaction of CD47 and SIRPa, leading to the enhanced macrophage phagocytosis of various CD47+ tumor cell lines and primary AML cells. Mono-treatment of TJC4 completely eradicated tumor cells in a Raji cell xenograft model and significantly extended the overall survival of treated mice in an AML model. When combined with Rituximab, TJC4 showed a superior efficacy in a DLBCL model over the mono-treatment group. TJC4 has unique RBC sparing properties as evident by the negligible binding to healthy human RBCs and platelets respectively. Single dose or repeat dose treatment of TJC4 minimally and transiently impacts RBCs in cynomolgus monkeys and no other safety findings were observed up to the highest dose (100 mg/kg). No impact was observed in platelets. The unique functional properties of TJC4 can be explained in part by its structure when in complex with CD47, which reveals an almost straight head-to-head binding and a novel conformational epitope that is distinct from other CD47 antibodies. Upon the structural analysis of the binding epitope, we identified a potential N-linked glycosylation site located nearby the two critical epitopes on the CD47 protein. Due to the nature of the high glycosylation degree of membrane proteins by RBCs, the N-linked glycan is hypothesized to function as a "shield" to block the exposure of the epitopes and prevent the TJC4 binding to human RBCs. This hypothesis is validated by the restoration of TJC4 binding to the deglycosylated RBCs after the PNGase treatment. Conclusion In summary, TJC4 is a next generation therapeutic anti-CD47 antibody that is devoid of the hematological liabilities while maintaining anti-tumor efficacy. These attributes of TJC4 differentiate it from other CD47 targeting agents currently in clinical evaluation. Disclosures Meng: I-Mab Biopharma: Employment, Equity Ownership. Wang:I-Mab Biopharma: Employment, Equity Ownership. Guo:I-Mab Biopharma: Employment, Equity Ownership. Cao:I-Mab Biopharma: Employment, Equity Ownership. Shen:I-Mab Biopharma: Employment, Equity Ownership.

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A Novel CD20xCD3 Bispecific Fully Human Antibody Induces Potent Anti-Tumor Effects Against B Cell Lymphoma in Mice

Blood ◽

10.1182/blood.v124.21.4501.4501 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4501-4501 ◽

Cited By ~ 2

Author(s):

Bindu Varghese ◽

Jayanthi Menon ◽

Luis Rodriguez ◽

Lauric Haber ◽

Kara Olson ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Tumor Cells ◽

Bispecific Antibody ◽

Tumor Regression ◽

Employment Equity ◽

Cynomolgus Monkeys ◽

Nsg Mice ◽

Equity Ownership

Abstract Bispecific antibodies that redirect effector T cells to kill tumor cells have shown considerable promise in both pre-clinical and clinical studies. However, these bispecific formats can have short half-lives necessitating constant infusion of the molecules into patients. We report here on a novel full-length human IgG CD20xCD3 bispecific antibody (REGN1979) that targets CD20 expressed on normal and malignant B cells and CD3 expressed on T cells in humans and cynomolgus monkeys. Our results demonstrate CD20-target cell-dependent activation and cytokine release by T cells, and efficient redirected T cell lysis of target tumor cells. Raji B cell lymphomas grown as tumors in NOD SCID IL2R gamma deficient (NSG) mice and co-implanted with human peripheral blood mononuclear (PBMC) cells were completely inhibited when treated at the time of implantation with a low dose (0.004 mg/kg; 2x/week) of REGN1979. As expected, T cells were required for this tumor inhibition, since treatment in the absence of human T cells was not effective. REGN1979 bispecific antibody also demonstrated potent activity against other tumor cells expressing CD20, as it significantly delayed CD20-transduced B16F10.9 tumor growth in immune-competent mice. Most importantly, REGN1979 induced dramatic tumor regression in large advanced (500-900 mm3) Raji tumors, associated with long-lasting tumor control. The tumor-infiltrating lymphocytes (TILs) in B cell lymphomas in these untreated NSG mice were found to express the inhibitory receptors Tim-3 and PD-1 and were the predominant fraction of T cells in the tumors and in the circulation. T cells in mice treated with REGN1979 showed decreased Tim-3 and PD-1 expression in the circulation accompanied by complete tumor regression. In further studies, REGN1979 (dosed at 0.4 mg/kg; 2x/week) was superior to rituximab therapy (dosed at 8 mg/kg; 5x/week) and comparable to the CD19xCD3 BiTE (dosed at 0.5 mg/kg; 5x/week) in suppressing established Raji tumors (200-400mm3). Pre-clinical studies in cynomolgus monkeys to assess activity of the bispecific antibody for depleting B cells in circulation and various lymphoid organs showed that a single injection of REGN1979 (0.1 mg/kg) was more potent at depleting CD20+ B cells in the mesenteric lymph nodes than a high dose of rituximab (30 mg/kg). In separate studies, REGN1979 was also found to have a long half-life (>14 days) in the circulation of monkeys following depletion of B cells. These studies show potent activity of a new class of fully human bispecific antibodies for treating tumors, and support clinical testing of REGN1979 in patients with CD20+ cancers. Figure 1 Figure 1. Disclosures Varghese: Regeneron Pharmaceuticals: Employment, Equity Ownership. Menon:Regeneron Pharmaceuticals: Employment, Equity Ownership. Rodriguez:Regeneron Pharmaceuticals: Employment, Equity Ownership. Haber:Regeneron Pharmaceuticals: Employment, Equity Ownership. Olson:Regeneron Pharmaceuticals: Employment, Equity Ownership. Duramad:Regeneron Pharmaceuticals: Employment, Equity Ownership. Oyejide:Regeneron Pharmaceuticals: Employment, Equity Ownership. Smith:Regeneron Pharmaceuticals: Employment, Equity Ownership. Thurston:Regeneron Pharmaceuticals: Employment, Equity Ownership. Kirshner:Regeneron Pharmaceuticals: Employment, Equity Ownership.

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Highly Differentiated Anti-CD47 Antibody, AO-176, Potently Inhibits Hematologic Malignancies Alone and in Combination

Blood ◽

10.1182/blood-2019-126298 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1844-1844

Author(s):

John Richards ◽

Myriam N Bouchlaka ◽

Robyn J Puro ◽

Ben J Capoccia ◽

Ronald R Hiebsch ◽

...

Keyword(s):

Multiple Myeloma ◽

Combination Therapy ◽

Tumor Growth ◽

Growth Inhibition ◽

Tumor Cells ◽

Tumor Growth Inhibition ◽

Employment Equity ◽

Equity Ownership

AO-176 is a highly differentiated, humanized anti-CD47 IgG2 antibody that is unique among agents in this class of checkpoint inhibitors. AO-176 works by blocking the "don't eat me" signal, the standard mechanism of anti-CD47 antibodies, but also by directly killing tumor cells. Importantly, AO-176 binds preferentially to tumor cells, compared to normal cells, and binds even more potently to tumors in their acidic microenvironment (low pH). Hematological neoplasms are the fourth most frequently diagnosed cancers in both men and women and account for approximately 10% of all cancers. Here we describe AO-176, a highly differentiated anti-CD47 antibody that potently targets hematologic cancers in vitro and in vivo. As a single agent, AO-176 not only promotes phagocytosis (15-45%, EC50 = 0.33-4.1 µg/ml) of hematologic tumor cell lines (acute myeloid leukemia, non-Hodgkin's lymphoma, multiple myeloma, and T cell leukemia) but also directly targets and kills tumor cells (18-46% Annexin V positivity, EC50 = 0.63-10 µg/ml) in a non-ADCC manner. In combination with agents targeting CD20 (rituximab) or CD38 (daratumumab), AO-176 mediates enhanced phagocytosis of lymphoma and multiple myeloma cell lines, respectively. In vivo, AO-176 mediates potent monotherapy tumor growth inhibition of hematologic tumors including Raji B cell lymphoma and RPMI-8226 multiple myeloma xenograft models in a dose-dependent manner. Concomitant with tumor growth inhibition, immune cell infiltrates were observed with elevated numbers of macrophage and dendritic cells, along with increased pro-inflammatory cytokine levels in AO-176 treated animals. When combined with bortezomib, AO-176 was able to elicit complete tumor regression (100% CR in 10/10 animals treated with either 10 or 25 mg/kg AO-176 + 1 mg/kg bortezomib) with no detectable tumor out to 100 days at study termination. Overall survival was also greatly improved following combination therapy compared to animals treated with bortezomib or AO-176 alone. These data show that AO-176 exhibits promising monotherapy and combination therapy activity, both in vitro and in vivo, against hematologic cancers. These findings also add to the previously reported anti-tumor efficacy exhibited by AO-176 in solid tumor xenografts representing ovarian, gastric and breast cancer. With AO-176's highly differentiated MOA and binding characteristics, it may have the potential to improve upon the safety and efficacy profiles relative to other agents in this class. AO-176 is currently being evaluated in a Phase 1 clinical trial (NCT03834948) for the treatment of patients with select solid tumors. Disclosures Richards: Arch Oncology Inc.: Employment, Equity Ownership, Other: Salary. Bouchlaka:Arch Oncology Inc.: Consultancy, Equity Ownership. Puro:Arch Oncology Inc.: Employment, Equity Ownership. Capoccia:Arch Oncology Inc.: Employment, Equity Ownership. Hiebsch:Arch Oncology Inc.: Employment, Equity Ownership. Donio:Arch Oncology Inc.: Employment, Equity Ownership. Wilson:Arch Oncology Inc.: Employment, Equity Ownership. Chakraborty:Arch Oncology Inc.: Employment, Equity Ownership. Sung:Arch Oncology Inc.: Employment, Equity Ownership. Pereira:Arch Oncology Inc.: Employment, Equity Ownership.

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Effective Targeting of PRAME-Positive Tumors with Bispecific T Cell-Engaging Receptor (TCER®) Molecules

Blood ◽

10.1182/blood-2019-129958 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3368-3368

Author(s):

Sebastian Bunk ◽

Martin Hofmann ◽

Felix Unverdorben ◽

Meike Hutt ◽

Gabriele Pszolla ◽

...

Keyword(s):

T Cell ◽

Tumor Cells ◽

Half Life ◽

Normal Tissue ◽

Malignant Diseases ◽

Proof Of Concept ◽

Employment Equity ◽

Target Peptide ◽

Equity Ownership ◽

Nog Mice

T cell receptors (TCRs) naturally recognize human leukocyte antigen (HLA)-bound peptides derived from foreign and endogenous proteins regardless of their extracellular or intracellular location. Preferentially expressed antigen in melanoma (PRAME) has been shown to be expressed at high levels in a variety of cancer cells while being absent or present only at very low levels in normal adult tissues except testis. In contrast to most other cancer/testis antigens, PRAME is expressed not only in solid tumors but also in leukemia and myeloma cells. Immunotherapy with bispecific T cell engagers has emerged as a novel and promising treatment modality for malignant diseases, however, antibody-based approaches (ie. blinatumomab) are restricted to few surface antigens such as CD19 or BCMA. Immatics has developed bispecific T cell-engaging receptors (TCER®) that are fusion proteins consisting of an affinity-maturated TCR and a humanized T cell-recruiting antibody with an effector function-silenced IgG1 Fc part. TCER® molecules confer extended half-life together with antibody-like stability and manufacturability characteristics. The molecular design allows for effective redirection of T cells towards target peptide-HLA selectively expressed in tumor tissues. Here we present proof-of-concept data from a TCER® program targeting a PRAME-derived peptide bound to HLA-A*02:01. We confirmed the abundant presence of the target peptide-HLA in several cancer indications and its absence in relevant human normal tissues by using the XPRESIDENT® target discovery engine, which combines quantitative mass spectrometry, transcriptomics and bioinformatics. Yeast surface display technology was used to maturate the stability and affinity of a parental human TCR recognizing PRAME with high functional avidity and specificity. During maturation we applied XPRESIDENT®-guided off-target toxicity screening, incorporating the world's largest normal tissue immunopeptidome database, to deselect cross-reactive candidate TCRs. The maturated TCRs were engineered into the TCER® scaffold and production in Chinese hamster ovary (CHO) cells generated highly stable molecules with low tendency for aggregation as confirmed during stress studies. Following TCR maturation, the TCER® molecules exhibited an up to 10,000-fold increased binding affinity towards PRAME when compared to the parental TCR. The high affinity correlated with potent in vitro anti-tumor activity requiring only low picomolar concentrations of TCER® molecules to induce half-maximal lysis of tumor cells expressing the target at physiological levels. Furthermore, using a tumor xenograft model in immunodeficient NOG mice, we could demonstrate significant growth inhibition of established tumors upon intravenous injection of TCER® molecules. Pharmacokinetic profiling in NOG mice determined a terminal half-life of more than 4 days, compatible with a once weekly dosing regimen in patients. For the safety assessment, we measured killing of more than 20 different human normal tissue cell types derived from high risk organs. Notably, we could confirm a favorable safety window for selected TCER® molecules, which induced killing of most normal tissue cells only at significantly higher concentrations than required for killing of tumor cells. To further support safety of TCER® molecules, we also performed a comprehensive characterization of potential off-target peptides selected from the XPRESIDENT® normal tissue database based on its high similarity to the sequence of the target peptide or based on data from alternative screening approaches. In summary, the efficacy, safety and manufacturability data to be presented provide preclinical proof-of-concept for a novel bispecific T cell-engaging receptor (TCER®) molecule targeting PRAME for treatment of various malignant diseases. Disclosures Bunk: Immatics: Employment. Hofmann:Immatics: Employment. Unverdorben:Immatics: Employment. Hutt:Immatics: Employment. Pszolla:Immatics: Employment. Schwöbel:Immatics: Employment. Wagner:Immatics: Employment. Yousef:Immatics: Employment. Schuster:Immatics: Employment. Missel:Immatics: Employment. Schoor:Immatics: Employment. Weinschenk:Immatics: Employment, Equity Ownership. Singh-Jasuja:Immatics: Employment, Equity Ownership. Maurer:Immatics: Employment. Reinhardt:Immatics: Employment, Equity Ownership.

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Blockade of XBP1 Splicing by Inhibition of IRE1α Is a Promising Therapeutic Option in Multiple Myeloma

Blood ◽

10.1182/blood.v118.21.133.133 ◽

2011 ◽

Vol 118 (21) ◽

pp. 133-133 ◽

Cited By ~ 2

Author(s):

Naoya Mimura ◽

Mariateresa Fulciniti ◽

Gullu Gorgun ◽

Yu-Tzu Tai ◽

Diana D. Cirstea ◽

...

Keyword(s):

Growth Inhibition ◽

Er Stress ◽

Board Of Directors ◽

Tumor Cells ◽

Therapeutic Option ◽

Rt Pcr ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership

Abstract Abstract 133 Multiple myeloma (MM) cells are characterized by high protein synthesis resulting in chronic endoplasmic reticulum (ER) stress, which is adaptively managed by the unfolded protein response (UPR). Therefore blockade of UPR could provide a novel therapeutic option in MM. Upon UPR, inositol-requiring enzyme 1α (IRE1α) is activated by auto-phosphorylation, resulting in activation of its endoribonuclease domain to cleave XBP1 mRNA from XBP1 unspliced form (XBP1u: inactive) to generate the XBP1 spliced form (XBP1s: active). XBP1s protein in turn regulates genes responsible for protein folding and degradation, playing a pro-survival signaling role in the UPR. In this study, we specifically examined whether IRE1α-XBP1 pathway is a potential therapeutic target in MM. We first examined the biologic significance of IRE1α by knockdown using lentiviral shRNA and observed significant growth inhibition in IRE1α knockdown cells. We next examined the impact of inhibition of XBP1 splicing using a novel small molecule IRE1α endoribonuclease domain inhibitor MKC-3946 (MannKind, Valencia CA). MKC-3946 blocked not only the basal level, but also inducible (by tunicamycin) XBP1s, evidenced by RT-PCR analysis in RPMI8226 cells, without affecting phosphorylation of IRE1α. Importantly, MKC-3946 also inhibited XBP1s in primary tumor cells from MM patients. We also confirmed functional inhibition of XBP1s, with target genes including SEC61A1, p58IPK, and ERdj4 downregulated by MKC-3946 treatment. Importantly, MKC-3946 triggered growth inhibition in MM cell lines, without toxicity in normal mononuclear cells. Furthermore, it significantly enhanced cytotoxicity induced by bortezomib or 17-AAG in RPMI8226 and INA6 cells, as well as primary tumor cells from MM patients. Both bortezomib and 17-AAG induced ER stress with XBP1s, which was markedly blocked by MKC-3946. Moreover, apoptosis induced by bortezomib or 17-AAG was enhanced by MKC-3946, associated with increased CHOP mRNA and protein, a proapoptotic factor triggered by ER stress. We next demonstrated that XBP1s was induced by bortezomib in INA6 cells co-cultured with bone marrow (BM) stromal cells, which was inhibited by MKC-3946, associated with enhanced cytotoxicity induced by the combination. Finally, MKC-3946 inhibited XBP1s in a model of in vivo ER stress induced by tunicamycin. To evaluate the anti-MM effect of MKC-3946, we used the subcutaneous RPMI8226 xenograft model in mice. MKC-3946 significantly reduced MM tumor growth in the treatment versus control group, associated with prolonged overall survival. We also confirmed that MKC-3946 treatment significantly inhibited XBP1s in excised tumors, assessed by RT-PCR. In order to examine the activity of MKC-3946 on MM cell growth in the context of the human BM microenvironment in vivo, we used the SCID-hu model, in which INA6 cells are directly injected into a human bone chip implanted subcutaneously in SCID-mice. MKC-3946 treatment significantly inhibited tumor growth compared with vehicle control. Moreover, XBP1s in excised tumor cells was inhibited, evidenced by RT-PCR. In conclusion, these data demonstrate that blockade of XBP1s by MKC-3946 triggers MM cell growth inhibition in vivo and prolongs host survival. Taken together, our results demonstrate that blockade of XBP1 splicing by inhibition of IRE1α endoribonuclease domain is a potential novel therapeutic option in MM. Disclosures: Tam: MannKind Corporation: Employment, Equity Ownership. Zeng:MannKind Corporation: Employment, Equity Ownership. Patterson:MannKind Corporation: Employment, Equity Ownership. Richardson:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Munshi:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; MannKind: Membership on an entity's Board of Directors or advisory committees.

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LY2928057, An Antibody Targeting Ferroportin, Is a Potent Inhibitor Of Hepcidin Activity and Increases Iron Mobilization In Normal Cynomolgus Monkeys

Blood ◽

10.1182/blood.v122.21.3433.3433 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3433-3433 ◽

Cited By ~ 11

Author(s):

Derrick R Witcher ◽

Donmienne Leung ◽

Karen A Hill ◽

David C De Rosa ◽

Jianghuai Xu ◽

...

Keyword(s):

Iron Homeostasis ◽

Potent Inhibitor ◽

Peptide Hormone ◽

Beta Thalassemia ◽

Iron Release ◽

Employment Equity ◽

Hek 293 ◽

Cynomolgus Monkeys ◽

Equity Ownership

Abstract Hepcidin, a 25-amino acid peptide hormone which is primarily synthesized and secreted from the liver, is a key regulator of iron homeostasis. It regulates dietary iron absorption, plasma iron concentrations, and tissue iron distribution through interactions with ferroportin, the only known mammalian cellular iron exporter. Hepcidin induces the internalization and subsequent degradation of ferroportin. The reduction in iron release caused by the loss of ferroportin, combined with the continuing demand for iron by erythropoietic precursors, results in a decrease in circulating iron levels. Dysregulation of the hepcidin-ferroportin axis contributes to the pathogenesis of different anemias. Decreased synthesis of hepcidin may cause systemic iron overload in iron-loading anemias such as beta-thalassemia; whereas overproduction of hepcidin may contribute to the development of anemia in inflammatory disorders, malignancies, and chronic kidney disease. LY2928057 is a novel humanized IgG4 monoclonal antibody that binds to human ferroportin with a high affinity, blocks the binding of human hepcidin to ferroportin, and is a potent inhibitor of hepcidin activity in a recombinant ferroportin expressing HEK 293 cell-based assay. In addition, this antibody was able to significantly inhibit hepcidin-induced increase in ferritin levels using Caco-2 cells, a human enterocyte cell line that naturally expresses ferroportin. LY2928057 does not block the efflux of iron from ferroportin, nor does this antibody cause the internalization of this transporter in vitro. Administration of LY2928057 results in a dose dependent increase in serum iron and hepcidin in normal cynomolgus monkeys. LY2928057 may provide therapeutic benefit for patients with hepcidin-related anemia by stabilizing ferroportin located on the cell surface, thus restoring iron export and erythropoiesis. LY2928057 is currently in clinical evaluation. Disclosures: Witcher: Eli Lilly and Company: Employment, Equity Ownership. Leung:Eli Lilly and Company: Employment, Equity Ownership. Hill:Eli Lilly and Company: Employment, Equity Ownership. De Rosa:Eli Lilly and Company: Employment, Equity Ownership. Xu:Eli Lilly and Company: Employment, Equity Ownership. Manetta:Eli Lilly and Company: Employment, Equity Ownership. Wroblewski:Eli Lilly and Company: Employment, Equity Ownership. Benschop:Eli Lilly and Company: Employment, Equity Ownership.

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A Novel and Highly Potent CAR T Cell Drug Product for Treatment of BCMA-Expressing Hematological Malignances

Blood ◽

10.1182/blood.v126.23.3094.3094 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3094-3094 ◽

Cited By ~ 5

Author(s):

Alena A. Chekmasova ◽

Holly M. Horton ◽

Tracy E. Garrett ◽

John W. Evans ◽

Johanna Griecci ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Cell Lines ◽

Drug Product ◽

Single Chain ◽

Employment Equity ◽

Car T Cells ◽

Car T ◽

Equity Ownership

Abstract Recently, B cell maturation antigen (BCMA) expression has been proposed as a marker for identification of malignant plasma cells in patients with multiple myeloma (MM). Nearly all MM and some lymphoma tumor cells express BCMA, while normal tissue expression is restricted to plasma cells and a subset of mature B cells. Targeting BCMA maybe a therapeutic option for treatment of patients with MM and some lymphomas. We are developing a chimeric antigen receptor (CAR)-based therapy for the treatment of BCMA-expressing MM. Our anti-BCMA CAR consists of an extracellular single chain variable fragment (scFv) antigen recognition domain derived from an antibody specific to BCMA, fused to CD137 (4-1BB) co-stimulatory and CD3zeta chain signaling domains. Selection of our development candidate was based on the screening of four distinct anti-BCMA CARs (BCMA01-04) each comprised of unique single chain variable fragments. One candidate, BCMA02 (drug product name bb2121) was selected for further studies based on the robust frequency of CAR-positive cells, increased surface expression of the CAR molecule, and superior in vitro cytokine release and cytolytic activity against the MM cell lines. In addition to displaying specific activity against MM (U226-B1, RPMI-8226 and H929) and plasmacytoma (H929) cell lines, bb2121 was demonstrated to react to lymphoma cell lines, including Burkitt's (Raji, Daudi, Ramos), chronic lymphocytic leukemia (Mec-1), diffuse large B cell (Toledo), and a Mantle cell lymphoma (JeKo-1). Based on receptor density quantification, bb2121 can recognize tumor cells expressing less than 1000 BCMA molecules per cell. The in vivo pharmacology of bb2121 was studied in NSG mouse models of human MM and Burkitt's lymphoma. NSG mice were injected subcutaneously (SC) with 107 RPMI-8226 MM cells. After 18 days, mice received a single intravenous (IV) administration of vehicle or anti-CD19Δ (negative control, anti-CD19 CAR lacking signaling domain) or anti-BCMA CAR T cells, or repeated IV administration of bortezomib (Velcade®; 1 mg/kg twice weekly for 4 weeks). Bortezomib, which is a standard of care for MM, induced only transient reductions in tumor size and was associated with toxicity, as indicated by substantial weight loss during dosing. The vehicle and anti-CD19Δ CAR T cells failed to inhibit tumor growth. In contrast, treatment with bb2121 resulted in rapid and sustained elimination of the tumors, increased body weights, and 100% survival. Flow cytometry and immunohistochemical analysis of bb2121 T cells demonstrated trafficking of CAR+ T cells to the tumors (by Day 5) followed by significant expansion of anti-BCMA CAR+ T cells within the tumor and peripheral blood (Days 8-10), accompanied by tumor clearance and subsequent reductions in circulating CAR+ T cell numbers (Days 22-29). To further test the potency of bb2121, we used the CD19+ Daudi cell line, which has a low level of BCMA expression detectable by flow cytometry and receptor quantification analysis, but is negative by immunohistochemistry. NSG mice were injected IV with Daudi cells and allowed to accumulate a large systemic tumor burden before being treated with CAR+ T cells. Treatment with vehicle or anti-CD19Δ CAR T cells failed to prevent tumor growth. In contrast, anti-CD19 CAR T cells and anti-BCMA bb2121 demonstrated tumor clearance. Adoptive T cell immunotherapy approaches designed to modify a patient's own lymphocytes to target the BCMA antigen have clear indications as a possible therapy for MM and could be an alternative method for treatment of other chemotherapy-refractory B-cell malignancies. Based on these results, we will be initiating a phase I clinical trial of bb2121 for the treatment of patients with MM. Disclosures Chekmasova: bluebird bio, Inc: Employment, Equity Ownership. Horton:bluebird bio: Employment, Equity Ownership. Garrett:bluebird bio: Employment, Equity Ownership. Evans:bluebird bio, Inc: Employment, Equity Ownership. Griecci:bluebird bio, Inc: Employment, Equity Ownership. Hamel:bluebird bio: Employment, Equity Ownership. Latimer:bluebird bio: Employment, Equity Ownership. Seidel:bluebird bio, Inc: Employment, Equity Ownership. Ryu:bluebird bio, Inc: Employment, Equity Ownership. Kuczewski:bluebird bio: Employment, Equity Ownership. Horvath:bluebird bio: Employment, Equity Ownership. Friedman:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership.

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Dual-Function Anti-CD47mAbs Induce Tumor Cell Death and Promote Phagocytosis Resulting in Enhanced in Vivo Efficacy

Blood ◽

10.1182/blood.v124.21.991.991 ◽

2014 ◽

Vol 124 (21) ◽

pp. 991-991

Author(s):

Pamela T. Manning ◽

Benjamin J. Capoccia ◽

Michael P. Rettig ◽

Ronald R. Hiebsch ◽

Robert W. Karr ◽

...

Keyword(s):

Cell Death ◽

Cancer Cells ◽

Tumor Cells ◽

Tumor Burden ◽

Dual Function ◽

Employment Equity ◽

Hematologic Cancer ◽

Equity Ownership

Abstract Recent success in immunomodulation of cancer has targeted immune checkpoints such as CTLA-4, PD-1 and PDL-1 to enhance adaptive immunity by stimulating production of tumor-selective, cytotoxic T cells. Anti-CD47mAbs enhance innate immunity by increasing the phagocytosis of tumor cells by macrophages leading to processing and presentation of tumor antigens to prime the adaptive T cell response. Many cancers, including hematologic cancers, up-regulate the expression of CD47 presumably to avoid immune destruction. Increased CD47 expression protects cancer cells from phagocytosis by sending a “don't eat me” signal to macrophages via SIRPalpha, an inhibitory receptor that prevents phagocytosis of CD47-bearing cells. CD47mAbs that block the CD47/SIRPalpha interaction (“blocking-only” mAbs) enhance phagocytosis of cancer cells in vitro. We have identified two CD47mAbs, Vx-1000 and Vx-1004, both of which block the CD47/SIRPalpha interaction and promote phagocytosis of tumor cells by macrophages equally well. However, Vx-1004 also has the unique property of killing cancer cells, but not normal blood cells, via a direct, cell-autonomous, cytotoxic mechanism. Therefore, Vx-1004 is a dual-function antibody. Vx-1004 selectively kills a variety of hematologic cancer cells in vitro, while Vx-1000, the blocking-only mAb, does not as assessed by annexin V staining and flow cytometry (Figure 1). In dose-response studies, cell death in leukemia cells was induced in 2 hrs by <1 ug="" vx-1004="" whereas="" normal="" peripheral="" blood="" mononuclear="" cells="" are="" resistant="" to="" the="" induction="" of="" cell="" death="" by="" following="" incubation="" with="" 10="" for="" 24="" hrs="" both="" these="" cd47mabs="" bind="" many="" species="" cd47="" including="" mouse="" and="" human="" p=""> To determine if the tumor-toxic activity of Vx-1004 confers enhanced efficacy in vivo compared to Vx-1000, we compared them in two mouse hematologic cancer models: murine acute promyelocytic leukemia (APL) and B cell lymphoma (BCL). Briefly, 1x106 GFP-labeled C57BL/6 APL cells were injected IV into wild-type C57BL/6 mice that were then treated IP with 0.4 mg/kg of either Vx-1000 or Vx-1004 on the day of tumor injection and on days 3 and 6 following tumor injection, a very low dose and limited dosing regimen. On day 25, the blood of these mice was analyzed for the number of circulating APL cells. As shown in Figure 2, Vx-1000 did not significantly reduce tumor burden compared to the control group. In contrast, Vx-1004 significantly reduced tumor burden compared to controls, demonstrating greater efficacy of the dual-function CD47mAb. In addition, enhanced efficacy of Vx-1004 compared to Vx-1000 was demonstrated in BCL (Figure 3). In this model, NSG mice were injected with 1x106 murine A20 lymphoma cells subcutaneously and then treated with 0.4mg/kg/day of the CD47mAbs IP for the first five days following tumor injection. In this model, Vx-1000 also failed to inhibit tumor growth compared to controls while Vx-1004 significantly reduced tumor burden at 35 days compared to both the control and Vx-1000 groups, nearly four weeks after treatment was stopped. These data demonstrate increased anti-cancer efficacy with a dual-function CD47mAb that not only blocks the CD47/SIRPalpha interaction to increase phagocytosis of cancer cells, but also selectively kills cancer cells. These studies indicate that dual-function CD47mAbs may have better anti-tumor activity in vivo and support their use in human clinical trials. Figure 1 Figure 1. Disclosures Manning: Corvus Pharmaceutical: Employment, Equity Ownership. Capoccia:Corvus Pharmaceutical: Employment, Equity Ownership. Hiebsch:Corvus Pharmaceutical: Employment, Equity Ownership. Karr:Corvus Pharmaceutical: Employment, Equity Ownership. Frazier:Corvus Pharmaceutical: Consultancy, Equity Ownership.

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TNB-486, a Novel Fully Human Bispecific CD19 x CD3 Antibody That Kills CD19-Positive Tumor Cells with Minimal Cytokine Secretion

Blood ◽

10.1182/blood-2019-123226 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4070-4070

Author(s):

Harbani Malik ◽

Ben Buelow ◽

Udaya Rangaswamy ◽

Aarti Balasubramani ◽

Andrew Boudreau ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

B Cell ◽

Tumor Cells ◽

Cytokine Secretion ◽

Employment Equity ◽

Equity Ownership

Introduction The restricted expression of CD19 in the B-cell lineage makes it an attractive target for the therapeutic treatment of B-cell malignancies. Many monoclonal antibodies and antibody drug conjugates targeting CD19 have been developed, including bispecific T-cell redirecting antibodies (T-BsAbs). In addition, anti-CD19 chimeric antigen receptor T-cells (CAR-T) have been approved to treat leukemia and lymphoma. However, despite the impressive depth of responses achieved by T-cell redirecting approaches such as T-BsAbs and CAR-T cells, toxicity from over-activation of T-cells remains a substantial limitation for this type of therapy, in particular neurotoxicity. In designing TNB-486, a novel CD19 x CD3 T-BsAb, we endeavored to retain activity against CD19-positive tumor cells while limiting the cytokine secretion thought to underlie toxicity from T-cell redirecting therapies. Utilizing TeneoSeek, a next generation sequencing (NGS)-based discovery pipeline that leverages in silico analysis of heavy chain only/fixed light chain antibody (HCA/Flic, respectively) sequences to enrich for antigen specific antibodies, we made a high affinity αCD19 HCA and a library of αCD3 Flic antibodies that showed a >2 log range of EC50s for T cell activation in vitro. Of note, the library contained a low-activating αCD3 that induced minimal cytokine secretion even at concentrations that mediated saturating T-cell dependent lysis of lymphoma cells (when paired with an αCD19 HCA). We characterized the relative efficacy and potential therapeutic window of this unique molecule, TNB-486, in vitro and in vivo and compared it to two strongly activating bispecific CD19 x CD3 antibodies similar to those currently available and in clinical development. Methods Affinity measurements of the αCD19 moiety were made via Biacore (protein) and flow cytometry (cell surface). Stability measurements were made by subjecting the molecule to thermal stress and the %aggregation was measured by Size Exclusion Chromatography. T-cell activation was measured via flow cytometry (CD69 and CD25 expression) and cytokine was measured by ELISA (IL-2, IL-6, IL-10, INF-ɣ, and TNFα) in vitro. Lysis of B-cell tumor cell lines (Raji, RI-1, and Nalm6) was measured via flow cytometry in vitro. In vivo, NOG mice were engrafted subcutaneously with NALM-6 or SUDHL-10 cells and intravenously with human peripheral blood mononuclear cells (huPBMC), and the mice treated with multiple doses of TNB-486 or negative or positive control antibody. Tumor burden was evaluated via caliper measurement. Pharmacodynamic/Pharmacokinetic (PK/PD) studies were performed in NOG mice. A pharmacokinetic (PK) study was performed in BALB/c mice, and a tolerability and PK study are ongoing in cynomolgus monkeys. Results TNB-486 bound to cell surface CD19 with single digit nanomolar affinity (~3nM). EC50s for cytotoxicity were in the single-digit nanomolar range for TNB-486, and sub-nanomolar for the strongly activating controls; TNB-486 maximum achievable lysis was identical to the positive controls. TNB-486 induced significantly less cytokine release for all cytokines tested compared to the positive controls even at doses saturating for tumor lysis. No off-target activation was observed in the absence of CD19 expressing target cells. In vivo, TNB-486 eradicated all CD19-positive tumors tested (NALM-6 and SUDHL10) at doses as little as 1µg administered every four days after tumors had reached ~200mm3. TNB-486 showed a PK profile consistent with other IgG molecules in mice (T1/2 ~6 days in mice). Conclusions TNB-486 induced comparable lysis of CD19-positive tumor cells as the strongly activating control bispecific antibodies while inducing significantly reduced cytokine secretion, even at doses saturating for tumor lysis in vitro. In vivo TNB-486 eradicated all tested CD19 positive tumor cell lines in established tumor models. No off-target binding was observed. In summary, TNB-486 shows promise as a lymphoma therapeutic differentiated from T-cell targeted therapies currently in the clinic and in clinical trials. Disclosures Malik: Teneobio, Inc.: Employment, Equity Ownership. Buelow:Teneobio, Inc.: Employment, Equity Ownership. Rangaswamy:Teneobio, Inc.: Employment, Equity Ownership. Balasubramani:Teneobio, Inc.: Employment, Equity Ownership. Boudreau:Teneobio, Inc.: Employment, Equity Ownership. Dang:Teneobio, Inc.: Employment, Equity Ownership. Davison:Teneobio, Inc.: Employment, Equity Ownership. Force Aldred:Teneobio, Inc.: Equity Ownership. Iyer:Teneobio, Inc.: Employment, Equity Ownership. Jorgensen:Teneobio, Inc.: Employment, Equity Ownership. Pham:Teneobio, Inc.: Employment, Equity Ownership. Prabhakar:Teneobio, Inc.: Employment, Equity Ownership. Schellenberger:Teneobio, Inc.: Employment, Equity Ownership. Ugamraj:Teneobio, Inc.: Employment, Equity Ownership. Trinklein:Teneobio, Inc.: Employment, Equity Ownership. Van Schooten:Teneobio, Inc.: Employment, Equity Ownership.

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Ofatumumab, a Human Mab Targeting a Membrane-Proximal Small-Loop Epitope On CD20, Induces Potent NK Cell-Mediated ADCC.

Blood ◽

10.1182/blood.v114.22.1725.1725 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1725-1725 ◽

Cited By ~ 7

Author(s):

Jenny L Craigen ◽

Wendy J.M. Mackus ◽

Patrick Engleberts ◽

Sam R Miller ◽

Sue Speller ◽

...

Keyword(s):

B Cell ◽

Nk Cells ◽

Tumor Cells ◽

Fc Receptor ◽

Target Cells ◽

Employment Equity ◽

Mixed Effect ◽

Blinded Study ◽

Small Loop ◽

Equity Ownership

Abstract Abstract 1725 Poster Board I-751 Introduction CD20 represents a well established target for immunotherapy of B-cell malignancies such as follicular lymphoma (FL). The standard therapy for FL is the monoclonal antibody (mAb) rituximab given as a single agent or combined with chemotherapy. Antibody-dependent cytotoxicity (ADCC) has been suggested to be an important in vivo mechanism of action of CD20 mAb as differences in response rates to rituximab exist between individuals expressing different allotypes of the polymorphic Fc receptor FcgRIIIa. Individuals homozygous for the FcgRIIIa 158V allotype respond significantly better than individuals expressing the FcgRIIIa 158F allotype. Ofatumumab, a unique human mAb targeting a novel membrane-proximal small-loop epitope on CD20, has been shown to be associated with highly efficient killing of primary tumor cells through complement-dependent cytotoxicity. Here we studied the ability of ofatumumab to induce ADCC by NK cells from FcgRIIIa 158V and 158F homozygous healthy donors and compared it to that of rituximab. Methods Blood was drawn (with informed consent) from ten healthy volunteers homozygous for FcgRIIIa 158V, and ten homozygous for FcgRIIIa 158F, selected from a panel of 479 donors and matched for age, sex and race. NK cells were purified, after which we assessed Fc-mediated antibody binding to NK cells, and ADCC, in a blinded study. Glycan profiling was performed for ofatumumab and rituximab by HPAEC-PAD analysis. MAb binding was measured by flow cytometry in a competition binding assay in which binding of FITC-labelled CD16 mAb 3G8 was blocked by the binding of ofatumumab or rituximab. ADCC was assessed by measuring Europium release from the B cell line ARH77 target cells upon incubation with a concentration curve of ofatumumab or rituximab in the presence of NK effector cells (effector:target ratio 5:1). Statistical analysis was performed using a non-linear mixed effect model fitting sigmoidal concentration-responses curves to data from each donor for each antibody. The model provided estimates of mean EC50 for each antibody and donor group, and allowed differences between these means to be tested for statistical significance. Results Biochemical analysis of clinical grade ofatumumab and rituximab batches indicated their quality to be comparable with similar amounts of aggregates present. The level of core-fucosylation was determined to be similar and ranged from 4 – 6 % for both mAbs. Monomeric ofatumumab bound more strongly to NK cells expressing FcgRIIIa 158 V/V (EC50: 900 mg/ml [95% CI 680-1200 mg/ml]) than FcgRIIIa 158 F/F (EC50: 3970 mg/ml [95% CI 2940-5370 mg/ml]). The approximately 4.4 fold difference in affinity was statistically significant (p<0.0001). A similar 4.2 fold difference (p<0.0001) was found for rituximab (FcgRIIIa 158 V/V EC50: 1370 mg/ml [95% CI 1020-1850 mg/ml] and FcgRIIIa 158 F/F EC50: 5720 mg/ml [95% CI 4170-7850 mg/ml]). Rituximab bound 1.4 fold less tightly to both FcgRIIIa allotypes than ofatumumab (p<0.0001). Ofatumumab induced a potent NK-mediated ADCC with both FcgRIIIa 158V/V expressing (EC50: 6.4 ng/ml [95% CI 4.8-8.5 ng/ml]) as well as FcgRIIIa 158 F/F expressing NK cells (EC50: 17.6 ng/ml [95% CI 13.4-23.1 ng/ml]). The observed ∼2.7 fold difference in potency was significant (p< 0.0001). Notably, higher concentrations of rituximab compared to ofatumumab were required to induce ADCC by NK cells obtained from both types of donors. Thus, rituximab induced ADCC by FcgRIIIa 158V/V and 158F/F expressing NK cells at EC50s of 12 ng/ml [95% CI 11-13 ng/ml] and 31 ng/ml [95% CI 28-34 ng/ml], respectively. This 1.8 fold difference in ADCC potency between ofatumumab and rituximab was statistically significant (p<0.0001). Conclusions We assessed FcgRIIIa affinity and potency to induce ADCC by purified NK cells for ofatumumab and rituximab in a blinded study. Expected differences in affinity for the 158V and 158F allotypes of FcgRIIIa were observed for both ofatumumab and rituximab. These differences correlated with a stronger ADCC by FcgRIIIa 158V/V compared to 158F/F expressing NK cells. Significantly, ofatumumab was able to induce ADCC more potently than rituximab for both Fc receptor allotypes. Ofatumumab binds CD20 stably and at a distinct membrane-proximal epitope compared to rituximab. Our data suggest that these binding characteristics may positively impact ofatumumab's ability to direct killing of tumor cells via ADCC. Disclosures Craigen: GSK: Employment, Equity Ownership. Mackus:Genmab: Employment, Equity Ownership. Engleberts:Genmab: Employment, Equity Ownership. Miller:GSK: Employment, Equity Ownership. Speller:GSK: Employment, Equity Ownership. Chamberlain:GSK: Employment, Equity Ownership. Davis:GSK: Employment, Equity Ownership. McHugh:GSK: Employment, Equity Ownership. Bullmore:GSK: Employment, Equity Ownership. Cox:GSK: Employment, Equity Ownership. Wetten:GSK: Employment, Equity Ownership. Perdock:Genmab: Employment, Equity Ownership. Bakker:Genmab: Employment. van de Winkel:Genmab: Employment, Equity Ownership. Parren:Genmab: Employment.

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Glutaminase Inhibitor CB-839 Synergizes with Pomalidomide in Preclinical Multiple Myeloma Models

Blood ◽

10.1182/blood.v124.21.4720.4720 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4720-4720 ◽

Cited By ~ 3

Author(s):

Francesco Parlati ◽

Mathew Gross ◽

Julie Janes ◽

Evan Lewis ◽

Andy MacKinnon ◽

...

Keyword(s):

Multiple Myeloma ◽

Clinical Trials ◽

Growth Inhibition ◽

Tumor Cells ◽

Single Agent ◽

Aspartate Transcarbamylase ◽

Employment Equity ◽

Two Agents ◽

Equity Ownership ◽

Hematological Tumor

Abstract Many hematological tumor cells are dependent on glutamine for growth and survival. Glutamine is the most abundant amino acid in plasma and can be utilized by tumor cells for production of energy and generation of building blocks for the synthesis of macromolecules. Small molecule CB-839 inhibits glutaminase (GLS) activity thereby blocking cellular glutamine utilization resulting in an anti-tumor effect in several hematological tumor types including multiple myeloma (MM), acute lymphocytic leukemia, and several types of non-Hodgkin’s lymphoma [Parlati et al. Blood 2013 122:4226]. Phase 1 clinical trials have been initiated to test the safety, pharmacokinetics, pharmacodynamics, and clinical activity of single agent CB-839 in several hematological malignancies. In anticipation of potential combinations of CB-839 with standard of care agents in future MM clinical trials, we tested the effects of CB-839 in combination with the IMiD, pomalidomide (POM). POM caused complete growth inhibition in MM.1S cells with an EC50 of 16 nM as opposed to partial growth inhibition in RPMI8226 cells, with an EC50 of 130 nM. CB-839 caused complete growth inhibition in MM.1S cells with an EC50 value of 26 nM and produced a cytotoxic effect in RPMI8226 cells with an EC50 of 160 nM. When combined, CB-839 enhanced the anti-proliferative activity of POM in both POM-sensitive MM.1S and POM-resistant RPMI8226 cells resulting in a synergistic anti-tumor effect as demonstrated by combination index values between 0.18-0.62 (mean= 0.36) for the MM.1S and 0.25-0.72 (mean= 0.38) for the RPMI8226 cells. To investigate the mechanism that underlies the observed synergy, RPMI8226 cells were treated for 24 hours and changes in proteins and metabolites were measured by reverse-phase-protein array and LC/MS, respectively. When treated with CB-839 alone, RPMI8226 cells respond by decreasing mTOR pathway signaling proteins (e.g. phospho-mTOR, phospho-p70S6K, phospho-PRAS40, phospho-S6), decreasing the amount of oncogenic proteins (c-Myc and c-Kit), and increasing programmed cell death pathway proteins (e.g. cleaved caspase 7, cleaved PARP), consistent with the cytotoxic activity observed for CB-839. Several of these changes were further enhanced in the presence of POM (e.g. phospho-p70S6K, phospho-S6, phospho-PRAS40, c-kit, c-Myc), however only the enhanced decrease in c-Myc reached statistical significance. Metabolite analysis showed changes with CB-839 consistent with GLS inhibition (e.g. decreases in glutamate, aspartate, succinate and malate and increases in glutamine). On the other hand, single agent POM caused very modest changes in the metabolite profile. When the two agents were combined, metabolite levels were consistent with those observed with single agent CB-839, with the notable exception of carbamoyl-aspartate where lower levels were measured in the combination group in comparison to cells treated with either agent alone. Carbamoyl-aspartate is an intermediate in the pyrimidine biosynthesis pathway and is synthesized by the multi-catalytic enzyme CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, dihydroorotase), an enzyme that is regulated by mTOR [Ben-Sahra et al. (2013) Science339: 1323-8]. These observations suggest that CB-839 dampens mTOR signaling and POM may further attenuate this response, possibly contributing to the synergistic anti-tumor effect. These data motivated testing the anti-tumor effect of the combination of CB-839 and POM in mice bearing RPMI8226 xenografts. Oral dosing with single agent CB-839 and POM resulted in tumor growth inhibition (TGI) of 64% and 46%, respectively, whereas the combination of the two agents resulted in a TGI of 97%. Efficacious doses of CB-839 and POM alone or in combination were well tolerated with no effect on animal body weight. These promising results indicate that GLS inhibition with CB-839 in combination with POM may provide therapeutic benefit in MM and provide motivation for future clinical studies. Disclosures Parlati: Calithera Biosciences: Employment, Equity Ownership. Gross:Calithera Biosciences: Employment, Equity Ownership. Janes:Calithera Biosciences: Employment, Equity Ownership. Lewis:Calithera Biosciences: Employment, Equity Ownership. MacKinnon:Calithera Biosciences: Employment, Equity Ownership. Rodriguez:Calithera Biosciences: Employment, Equity Ownership. Shwonek:Calithera Biosciences: Employment, Equity Ownership. Bennett:Calithera Biosciences: Employment, Equity Ownership.

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