Patrick P. Ng

Therapeutic antitumor immunity by checkpoint blockade is enhanced by ibrutinib, an inhibitor of both BTK and ITK

Significance Antibodies that block the negative signals between PD1-Ligand on tumor cells and PD-1 on T cells are effective therapies against several types of cancer. Ibrutinib, a covalent inhibitor of BTK is an approved therapy for B-cell leukemia and lymphoma. But ibrutinib also inactivates ITK, an enzyme required for certain subsets of T lymphocytes (Th2 T cells). We found that the combination of anti–PD-L1 antibodies and ibrutinib led to impressive therapeutic effects not only in animal models of lymphoma but, surprisingly, also in models of breast cancer and colon cancer. Based on these preclinical results, we suggest that the combination of PD-1/PD-L1 blockade and ibrutinib be tested broadly in patients with lymphoma and also in other hematologic malignancies and solid tumors. Monoclonal antibodies can block cellular interactions that negatively regulate T-cell immune responses, such as CD80/CTLA-4 and PD-1/PD1-L, amplifying preexisting immunity and thereby evoking antitumor immune responses. Ibrutinib, an approved therapy for B-cell malignancies, is a covalent inhibitor of BTK, a member of the B-cell receptor (BCR) signaling pathway, which is critical to the survival of malignant B cells. Interestingly this drug also inhibits ITK, an essential enzyme in Th2 T cells and by doing so it can shift the balance between Th1 and Th2 T cells and potentially enhance antitumor immune responses. Here we report that the combination of anti–PD-L1 antibody and ibrutinib suppresses tumor growth in mouse models of lymphoma that are intrinsically insensitive to ibrutinib. The combined effect of these two agents was also documented for models of solid tumors, such as triple negative breast cancer and colon cancer. The enhanced therapeutic activity of PD-L1 blockade by ibrutinib was accompanied by enhanced antitumor T-cell immune responses. These preclinical results suggest that the combination of PD1/PD1-L blockade and ibrutinib should be tested in the clinic for the therapy not only of lymphoma but also in other hematologic malignancies and solid tumors that do not even express BTK.

An anti-transferrin receptor-avidin fusion protein exhibits both strong proapoptotic activity and the ability to deliver various molecules into cancer cells.

We have developed an antibody fusion protein (anti-rat TfR IgG3-Av) with the ability to deliver different molecules into cancer cells. It consists of avidin genetically fused to the CH3 region of a human IgG3 specific for the rat transferrin receptor. It forms strong, noncovalent interactions with biotinylated molecules such as glucose oxidase and β-galactosidase, and delivers them into the rat myeloma cell line Y3-Ag1.2.3 through receptor-mediated endocytosis. Importantly, the β-galactosidase retains activity after internalization. Furthermore, we have unexpectedly discovered that anti-rat TfR IgG3-Av, but not a recombinant anti-rat TfR IgG3 or a nonspecific IgG3-Av, possesses proapoptotic activities against Y3-Ag1.2.3 and the rat T cell lymphoma cell line C58 (NT) D.1.G.OVAR.1. These activities were not observed in two rat cell lines of nonhematopoietic lineage (bladder carcinoma BC47 and gliosarcoma 9L). Anti-human TfR IgG3-Av also demonstrated proapoptotic activity against the human erythroleukemia cell line K562. Studies showed that anti-rat TfR IgG3-Av exists as a dimer, suggesting that cross-linking of the surface transferrin receptor may be responsible for the cytotoxic activity. These findings demonstrate that it is possible to transform an antibody specific for a growth factor receptor that does not exhibit inhibitory activity into a drug with significant intrinsic cytotoxic activity against selected cells by fusing it with avidin. The antitumor activity may be enhanced by delivering biotinylated therapeutics into cancer cells. Further development of this technology may lead to effective therapeutics for in vivo eradication of hematological malignancies, and ex vivo purging of cancer cells in autologous transplantation.

A vaccine directed to B cells and produced by cell-free protein synthesis generates potent antilymphoma immunity

Clinical studies of idiotype (Id) vaccination in patients with lymphoma have established a correlation between the induced anti-Id antibody responses and favorable clinical outcomes. To streamline the production of an Id vaccine, we engineered a small diabody (Db) molecule containing both a B-cell–targeting moiety (anti-CD19) and a lymphoma Id. This molecule (αCD19-Id) was designed to penetrate lymph nodes and bind to noncognate B cells to form an antigen presentation array. Indeed, the αCD19-Id molecule accumulated on B cells in vivo after s.c. administration. These noncognate B cells, decorated with the diabody, could then stimulate the more rare Id-specific B cells. Peptide epitopes present in the diabody linker augmented the response by activating CD4+ helper T cells. Consequently, the αCD19-Id molecule induced a robust Id-specific antibody response and protected animals from tumor challenge. Such diabodies are produced in a cell-free protein expression system within hours of amplification of the specific Ig genes from the B-cell tumor. This customized product can now be available to vaccinate patients before they receive other, potentially immunosuppressive, therapies.

Conjugation of an anti–transferrin receptor IgG3-avidin fusion protein with biotinylated saporin results in significant enhancement of its cytotoxicity against malignant hematopoietic cells

We have previously developed an antibody fusion protein composed of a mouse/human chimeric IgG3 specific for the human transferrin receptor genetically fused to avidin (anti-hTfR IgG3-Av) as a universal delivery system for cancer therapy. This fusion protein efficiently delivers biotinylated FITC into cancer cells via TfR-mediated endocytosis. In addition, anti-hTfR IgG3-Av alone exhibits intrinsic cytotoxic activity and interferes with hTfR recycling, leading to the rapid degradation of the TfR and lethal iron deprivation in certain malignant B-cell lines. We now report on the cytotoxic effects of a conjugate composed of anti-hTfR IgG3-Av and biotinylated saporin 6 (b-SO6), a toxin derived from the plant Saponaria officinalis that inhibits protein synthesis. Conjugation of anti-hTfR IgG3-Av with b-SO6 enhances the cytotoxic effect of the fusion protein in sensitive cells and also overcomes the resistance of malignant cells that show low sensitivity to the fusion protein alone. Our results show for the first time that loading anti-hTfR IgG3-Av with a biotinylated toxin enhances the cytotoxicity of the fusion protein alone. These results suggest that anti-hTfR IgG3-Av has great potential as a therapeutic agent for a wide range of applications due to its intrinsic cytotoxic activity plus its ability to deliver biotinylated molecules into cancer cells. [Mol Cancer Ther 2007;6(11):2995–3008]

B-cell receptors expressed by lymphomas of hepatitis C virus (HCV)-infected patients rarely react with the viral proteins

Chronic hepatitis C virus (HCV) infection has been implicated in the induction and maintenance of B-cell lymphomas. The strongest evidence for this derives from clinical observations of tumor regressions upon antiviral treatments. Here we used multiple methods to test the hypothesis that the expansion of HCV-specific B cells gives rise to lymphomas. We obtained lymphoma tissues from HCV-infected lymphoma patients, including some that later regressed upon antiviral treatments. We expressed the lymphoma B-cell receptors as soluble immunoglobulin Gs and membrane IgMs, and analyzed their reactivity with HCV proteins and with HCV virions. We confirmed previous reports that HCV-associated lymphomas use a restricted immunoglobulin variable region gene repertoire. However, we found no evidence for their binding to the HCV antigens. We conclude that most lymphomas of HCV-infected patients do not arise from B cells aimed at eliminating the virus.

Cell-free production of Gaussia princeps luciferase - antibody fragment bioconjugates for ex vivo detection of tumor cells

Antibody fragments (scFvs) fused to luciferase reporter proteins have been used as highly sensitive optical imaging probes. Gaussia princeps luciferase (GLuc) is an attractive choice for a reporter protein because it is small and bright and does not require ATP to stimulate bioluminescence-producing reactions. Both GLuc and scFv proteins contain multiple disulfide bonds, and consequently the production of active and properly folded GLuc-scFv fusions is challenging. We therefore produced both proteins individually in active form, followed by covalent coupling to produce the intended conjugate. We used an Escherichia coli-based cell-free protein synthesis (CFPS) platform to produce GLuc and scFv proteins containing non-natural amino acids (nnAAs) for subsequent conjugation by azide-alkyne click chemistry. GLuc mutants with exposed alkyne reactive groups were produced by global replacement of methionine residues in CFPS. Antibody fragment scFvs contained a single exposed azide group using a scheme for site-specific incorporation of tyrosine analogs. Incorporation of tyrosine analogs at specific sites in proteins was performed using an engineered orthogonal tRNA-tRNA synthetase pair from an archaebacterium. The unique azide and alkyne side chains in GLuc and the antibody fragment scFv facilitated conjugation by click chemistry. GLuc-scFv conjugates were shown to differentiate between cells expressing a surface target of the scFv and cells that did not carry this marker.

Escherichia coli-based production of a tumor idiotype antibody fragment – tetanus toxin fragment C fusion protein vaccine for B cell lymphoma

The unique immunoglobulin idiotype expressed on the surface of B lymphoma cells can be used as an effective antigen in tumor-specific vaccines when fused to immunostimulatory proteins and cytokines. A DNA vaccine encoding for an idiotype antibody single chain Fv (scFv) fragment fused to the Tetanus Toxin Fragment C (TTFrC) has been shown to induce protective anti-tumor responses. Protein-based strategies may be more desirable since they provide greater control over dosage, duration of exposure, and in vivo distribution of the vaccine. However, production of fusion protein vaccines containing complex disulfide bonded idiotype antibodies and antibody-derived fragments is challenging. We use an Escherichia coli-based cell-free protein synthesis platform as well as high-level expression of E. coli inclusion bodies followed by refolding for the rapid generation of an antibody fragment - TTFrC fusion protein vaccine. Vaccine proteins produced using both methods were shown to elicit anti-tumor humoral responses as well as protect from tumor challenge in an established B cell lymphoma mouse model. The development of technologies for the rapid production of effective patient-specific tumor idiotype-based fusion protein vaccines provides opportunities for clinical application.

Abstract 259: Ibrutinib enhances the anti-tumor efficacy of CTLA-4 blockade in lymphoma and colon cancer models

The anti-CTLA-4 antibody ipilimumab was the first immune checkpoint blockade antibody to show significant clinical efficacy. However, only a subset of the treated melanoma patients had long-term survival benefits (Schadendorf, D., et al. ECCO 2013). Combining ipilimumab with an agent that modulates immunity by a different mechanism may improve efficacy. Ibrutinib is a first-in-class, oral, covalent inhibitor of Bruton9s tyrosine kinase (Btk), an essential enzyme in the B-cell receptor signaling pathway. Ibrutinib also inhibits the interleukin-2-inducible Tec kinase (Itk) that is required for the activation/survival of Th2 T cells. This activity is consistent with preclinical studies that demonstrated ibrutinib could potentiate Th1 immunity (Dubovsky, JA., et al. Blood 2013). We investigated whether ibrutinib could enhance the anti-tumor effect of CTLA-4 blockade using the A20 B-cell lymphoma and the CT26 colon carcinoma mouse models. BALB/c mice (9-11/group) were implanted with tumor cells subcutaneously on the hind flanks. Animals bearing 80-100 mm3 (D x d2 x 0.4) tumors were dosed daily with oral ibrutinib (24 mg/kg) with or without the anti-mouse CTLA-4 antibody 9D9 (100 μg/mouse) given every 2 days by intraperitoneal injections. Animals in the lymphoma and colon cancer models were treated for 13 and 37 days, respectively. Tumor volume was measured and survival recorded for up to 2 months after tumor inoculation. Neither the A20 tumors, which express Btk, nor the CT26 tumors were inhibited by ibrutinib. In the A20 model, although 9D9 alone resulted in complete tumor regression in 7 of 9 treated animals, tumors in 2 of the 7 mice relapsed after treatment had ended. In contrast, 9D9 in combination with ibrutinib led to rapid, complete, and durable responses in 7 of 9 treated animals that lasted until the end of the study. In the CT26 model, treatment with 9D9 alone had minimal effects on tumor growth, but the addition of ibrutinib led to complete tumor regression in 6 of 10 treated animals. Thus, ibrutinib enhanced the anti-tumor efficacy of an anti-CTLA-4 antibody in B-cell lymphoma and colon carcinoma mouse models. The mechanism of tumor eradication by the combination therapy is under investigation and will be discussed at presentation. Citation Format: Patrick Ng, Daniel Lu, Betty Chang. Ibrutinib enhances the anti-tumor efficacy of CTLA-4 blockade in lymphoma and colon cancer models. [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 259. doi:10.1158/1538-7445.AM2015-259

Abstract 251: Combining Ibrutinib with immune checkpoint blockade to induce therapeutic antitumor immune response in solid tumors

The ability of T-cell responses to effectively attack tumors is restricted by multiple negative checkpoint regulators. Monoclonal antibodies blocking negative regulators of T -cells, such as CTLA-4 and PD-L1 can amplify pre-existing immune responses and evoke new therapeutic responses. Ibrutinib (Imbruvica®), a covalent inhibitor of BTK has shown therapeutic efficacy in several blood cancers, by a mechanism thought to be a direct effect on a critical signaling pathway intrinsic to the malignant B cell. Since BTK is predominantly expressed in hematopoietic cells, solid tumors have not been an obvious target of this drug. An additional target of ibrutinib is the inhibition of ITK, a key enzyme in the survival of Th2 T cells. It has been reported that ibrutinib can alter the balance of Th1 and Th2 T cells leading to enhanced immune responses. Here we report that the combination of ibrutinib and a-PD-L1 antibody suppresses tumor growth, reduces lung metastasis and improves survival of mice bearing solid tumors such as 4T1-triple negative breast cancer and CT26-colon cancer. Moreover, the combination of ibrutinib and a-PD-L1 was able to overcome ibrutinib resistance in mouse models of lymphoma and myeloma. We present evidence that the synergistic therapeutic activity of Ibrutinib with anti PD1 blockade results in enhanced T cell anti-tumor immune responses. In preclinical models, by combining inhibitory checkpoint blockade while shifting the immune response towards CD4 Th1 with ibrutinib, immune tolerance to tumors can be overcome. These results provide rationale for several newly planned clinical trials combining ibrutinib with PD-1 blockade in solid tumor and exploration of such a combination in hematologic malignancies. Citation Format: Idit Sagiv-Barfi, Holbrook Kohrt, Debra Czerwinski, Patrick Ng, Betty Chang, Ronald Levy. Combining Ibrutinib with immune checkpoint blockade to induce therapeutic antitumor immune response in solid tumors. [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 251. doi:10.1158/1538-7445.AM2015-251

Abstract 648: Semi-synthetic peptibodies are a novel personalized therapeutic with activity against lymphoma in vitro and in vivo

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The complementarity determining region, or idiotype, of the surface immunoglobulin receptor is a true tumor-specific marker on B-cell lymphomas unique to each patient. Antibodies against idiotype can induce complete regression of lymphoma in patients, but since this requires the generation of a custom monoclonal antibody for each patient, it has not been practical. We have developed a method for targeting idiotype using a novel construct that we refer to as a “semi-synthetic peptibody”. It consists of a synthetic peptide ligand for idiotype that is covalently linked to the amino terminus of a recombinant IgG Fc domain. Peptide ligands for idiotype can be identified by high throughput screens of random peptide libraries and produced inexpensively by solid-phase synthesis. Linkage of these idiotype ligands to the Fc domain enhances their pharmacokinetics and augments their anti-tumor effect by activating innate immune effectors. Since each patient-specific synthetic peptide can be chemically linked to a common IgG Fc domain, this modular design yields a custom therapeutic that may be more practical to produce than a unique biologic monoclonal antibody. We demonstrate that semi-synthetic peptibodies bind specifically to the idiotype of tumor cells and induce apoptosis by crosslinking surface immunoglobulin. Additionally, they trigger antibody dependent cellular cytotoxicity, antibody mediated phagocytosis, and complement-mediated lysis of opsonized lymphoma cells in vitro. They possess a favorable pharmacokinetic profile and are sufficient to clear tumor in SCID mice challenged intravenously with a luciferase-labeled human lymphoma cell line. Tumor clearance in vitro and in vivo mediated by peptibody was superior to that achievable with an anti-idiotype monoclonal antibody. Thus, semi-synthetic anti-idiotype peptibodies demonstrate multimodal activity against lymphoma cells in vitro and clear human lymphoma in a disseminated xenograft model. This modular, semi-synthetic design may enable a personalized and targeted therapy that is feasible to produce for patients with B-cell lymphoma. Citation Format: James Torchia, Patrick Ng, Homer Chen, Kipp Weiskopf, Ronald Levy. Semi-synthetic peptibodies are a novel personalized therapeutic with activity against lymphoma in vitro and in vivo . [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 648. doi:10.1158/1538-7445.AM2014-648