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

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.

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.

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.

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.

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.

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.

Molecular events contributing to cell death in malignant human hematopoietic cells elicited by an IgG3-avidin fusion protein targeting the transferrin receptor.

We have previously reported that an anti-human transferrin receptor IgG3-avidin fusion protein (anti-hTfR IgG3-Av) inhibits the proliferation of an erythroleukemia-cell line. We have now found that anti-hTfR IgG3-Av also inhibits the proliferation of additional human malignant B and plasma cells. Anti-hTfR IgG3-Av induces internalization and rapid degradation of the TfR. These events can be reproduced in cells treated with anti-hTfR IgG3 cross-linked with a secondary Ab, suggesting that they result from increased TfR cross-linking. Confocal microscopy of cells treated with anti-hTfR IgG3-Av shows that the TfR is directed to an intracellular compartment expressing the lysosomal marker LAMP-1. The degradation of TfR is partially blocked by cysteine protease inhibitors. Furthermore, cells treated with anti-hTfR IgG3-Av exhibit mitochondrial depolarization and activation of caspases 9, 8, and 3. The mitochondrial damage and cell death can be prevented by iron supplementation, but cannot be fully blocked by a pan-caspase inhibitor. These results suggest that anti-hTfR IgG3-Av induces lethal iron deprivation, but the resulting cell death does not solely depend on caspase activation. This report provides insights into the mechanism of cell death induced by anti-TfR Abs such as anti-hTfR IgG3-Av, a molecule that may be useful in the treatment of B-cell malignancies such as multiple myeloma.

Vaccination with novel combinations of anti-HER2/neu cytokines fusion proteins and soluble protein antigen elicits a protective immune response against HER2/neu expressing tumors.

We have previously demonstrated that anti-HER2/neu IgG3-(IL-2), (IL-12)-IgG3, or IgG3-(GM-CSF) antibody fusion proteins (mono-AbFPs) elicit anti-tumor activity against murine tumors expressing HER2/neu when used as adjuvants of extracellular domain of HER2/neu (ECD(HER2)) protein vaccination. We have now studied the effect of combinations of IL-2 and IL-12 or IL-12 and GM-CSF mono-AbFPs during vaccination with ECD(HER2). In addition, we developed two novel anti-HER2/neu IgG3-cytokine fusion proteins in which IL-2 and IL-12 or IL-12 and GM-CSF were fused to the same IgG3 molecule (bi-AbFPs). (IL-12)-IgG3-(IL-2) and (IL-12)-IgG3-(GM-CSF) were properly assembled and retained both cytokine activity and the ability to bind antigen. Vaccination of mice with ECD(HER2) and a combination of cytokines as either bi-AbFPs or two mono-AbFPs activated both Thl and Th2 immune responses and resulted in significant protection against challenge with a HER2/neu expressing tumor. Our results suggest that this approach will be effective in the prevention and/or treatment of HER2/neu expressing tumors.

A human biotin acceptor domain allows site-specific conjugation of an enzyme to an antibody-avidin fusion protein for targeted drug delivery.

We have previously constructed an antibody-avidin (Av) fusion protein, anti-transferrin receptor (TfR) IgG3-Av, which can deliver biotinylated molecules to cells expressing the TfR. We now describe the use of the fusion protein for antibody-directed enzyme prodrug therapy (ADEPT). The 67 amino acid carboxyl-terminal domain (P67) of human propionyl-CoA carboxylase alpha subunit can be metabolically biotinylated at a fixed lysine residue. We genetically fused P67 to the carboxyl terminus of the yeast enzyme FCU1, a derivative of cytosine deaminase that can convert the non-toxic prodrug 5-fluorocytosine to the cytotoxic agent 5-fluorouracil. When produced in Escherichia coli cells overexpressing a biotin protein ligase, the FCU1-P67 fusion protein was efficiently mono-biotinylated. In the presence of 5-fluorocytosine, the biotinylated fusion protein conjugated to anti-rat TfR IgG3-Av efficiently killed rat Y3-Ag1.2.3 myeloma cells in vitro, while the same protein conjugated to an irrelevant (anti-dansyl) antibody fused to Av showed no cytotoxic effect. Efficient tumor cell killing was also observed when E. coli purine nucleoside phosphorylase was similarly targeted to the tumor cells in the presence of the prodrug 2-fluoro-2'-deoxyadenosine. These results suggest that when combined with P67-based biotinylation, anti-TfR IgG3-Av could serve as a universal delivery vector for targeted chemotherapy of cancer.

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 C(H)3 region of a human IgG3 specific for the rat transferrin receptor. It forms strong, noncovalent interactions with biotinylated molecules such as glucose oxidase and beta-galactosidase, and delivers them into the rat myeloma cell line Y3-Ag1.2.3 through receptor-mediated endocytosis. Importantly, the beta-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.