i-shaped antibody engineering enables conformational tuning of biotherapeutic receptor agonists.

The ability to leverage antibodies to agonize disease relevant biological pathways has tremendous potential for clinical investigation. Yet while antibodies have been successful as antagonists, immune mediators, and targeting agents, they are not readily effective at recapitulating the biology of natural ligands. Among the important determinants of antibody agonist activity is the geometry of target receptor engagement. Here, we describe an engineering approach inspired by a naturally occurring Fab-Fab homotypic interaction that constrains IgG in a unique i-shaped conformation. i-shaped antibody (iAb) engineering enables potent intrinsic agonism of five tumor necrosis factor receptor superfamily (TNFRSF) targets. When applied to bispecific antibodies against the heterodimeric IL-2 receptor pair, constrained bispecific IgG formats recapitulate IL-2 agonist activity. iAb engineering provides a tool to tune agonist antibody function and this work provides a framework for the development of intrinsic antibody agonists with the potential for generalization across broad receptor classes.

Engineering interferons and interleukins for cancer immunotherapy.

Cytokines are a class of potent immunoregulatory proteins that are secreted in response to various stimuli and act locally to regulate many aspects of human physiology and disease. Cytokines play important roles in cancer initiation, progression, and elimination, and thus, there is a long clinical history associated with the use of recombinant cytokines to treat cancer. However, the use of cytokines as therapeutics has been limited by cytokine pleiotropy, complex biology, poor drug-like properties, and severe dose-limiting toxicities. Nevertheless, cytokines are crucial mediators of innate and adaptive antitumor immunity and have the potential to enhance immunotherapeutic approaches to treat cancer. Development of immune checkpoint inhibitors and combination immunotherapies has reinvigorated interest in cytokines as therapeutics, and a variety of engineering approaches are emerging to improve the safety and effectiveness of cytokine immunotherapy. In this review we highlight recent advances in cytokine biology and engineering for cancer immunotherapy.

A human orthogonal IL-2 and IL-2Rß system enhances CAR T cell expansion and antitumor activity in a murine model of leukemia.

Interleukin-2 (IL-2) is a central T cell cytokine that promotes T cell proliferation and effector function; however, toxicity due to its pluripotency limits its application to enhance CAR T cell immunotherapy. Previously, mouse IL-2 and its cognate receptor were engineered to create an orthogonal (ortho) cytokine-cytokine receptor pair capable of delivering an IL-2 signal without toxicity. Here, we engineered a human orthogonal IL-2 (ortho-hIL-2) and human orthogonal IL-2Rß (ortho-hIL-2Rß) pair, containing human-specific mutations. Ortho-hIL-2 is selective toward ortho-hIL-2Rß­expressing cells with no appreciable signaling on wild-type T cells. Ortho-hIL-2 induces IL-2 receptor signaling and supports proliferation of both an IL-2­dependent cell line and primary T cells transduced to express the ortho-hIL-2Rß. Using CD19-specific chimeric antigen receptor (CAR) T cells, we show that ortho-hIL-2 induces a dose-dependent increase in ortho-hIL-2Rß+ CAR T cell expansion in vivo by as much as 1000-fold at 2 weeks after adoptive transfer into immunodeficient mice bearing CD19+ Nalm6 leukemia xenografts. Ortho-hIL-2 can rescue the antileukemic effect of an otherwise suboptimal CAR T cell dose. In addition, ortho-hIL-2 administration initiated at the time of leukemic relapse after CAR T cell therapy can rescue an otherwise failed antileukemic response. These data highlight the potential of combining an orthogonal cytokine approach with T cell­based immunotherapies to augment the antitumor efficacy of engineered T cells.

Polymerase-guided base editing enables in vivo mutagenesis and rapid protein engineering.

Random mutagenesis is a technique used to generate diversity and engineer biological systems. In vivo random mutagenesis generates diversity directly in a host organism, enabling applications such as lineage tracing, continuous evolution, and protein engineering. Here we describe TRIDENT (TaRgeted In vivo Diversification ENabled by T7 RNAP), a platform for targeted, continual, and inducible diversification at genes of interest at mutation rates one-million fold higher than natural genomic error rates. TRIDENT targets mutagenic enzymes to precise genetic loci by fusion to T7 RNA polymerase, resulting in mutation windows following a mutation targeting T7 promoter. Mutational diversity is tuned by DNA repair factors localized to sites of deaminase-driven mutation, enabling sustained mutation of all four DNA nucleotides at rates greater than 10-4 mutations per bp. We show TRIDENT can be applied to routine in vivo mutagenesis applications by evolving a red-shifted fluorescent protein and drug-resistant mutants of an essential enzyme.

Targeting the myeloid checkpoint receptor SIRPα potentiates innate and adaptive immune responses to promote anti-tumor activity.

BACKGROUND: Signal regulatory protein α (SIRPα) is a myeloid-lineage inhibitory receptor that restricts innate immunity through engagement of its cell surface ligand CD47. Blockade of the CD47-SIRPα interaction synergizes with tumor-specific antibodies and T-cell checkpoint inhibitors by promoting myeloid-mediated antitumor functions leading to the induction of adaptive immunity. Inhibition of the CD47-SIRPα interaction has focused predominantly on targeting CD47, which is expressed ubiquitously and contributes to the accelerated blood clearance of anti-CD47 therapeutics. Targeting SIRPα, which is myeloid-restricted, may provide a differential pharmacokinetic, safety, and efficacy profile; however, SIRPα polymorphisms and lack of pan-allelic and species cross-reactive agents have limited the clinical translation of antibodies against SIRPα. Here, we report the development of humanized AB21 (hAB21), a pan-allelic anti-SIRPα antibody that binds human, cynomolgus monkey, and mouse SIRPα alleles with high affinity and blocks the interaction with CD47. METHODS: Human macrophages derived from donors with various SIRPα v1 and v2 allelic status were used to assess the ability of hAB21 to enhance phagocytosis. HAB21_IgG subclasses were evaluated for targeted depletion of peripheral blood mononuclear cells, phagocytosis and in vivo efficacy in xenograft models. Combination therapy with anti-PD1/anti-PD-L1 in several syngeneic models was performed. Immunophenotyping of tissues from MC38 tumor-bearing mice treated with AB21 and anti-PD-1 was evaluated. PK, PD and tolerability of hAB21 were evaluated in cynomolgus monkeys. RESULTS: SIRPα blockade with hAB21 promoted macrophage-mediated antibody-dependent phagocytosis of tumor cells in vitro and improved responses to rituximab in the Raji human tumor xenograft mouse model. Combined with PD-1/PD-L1 blockade, AB21 improved response rates by facilitating monocyte activation, dendritic cell activation, and T cell effector functions resulting in long term, durable antitumor immunity. In cynomolgus monkeys, hAB21 has a half-life of 5.3 days at 10 mg/kg and complete target occupancy with no hematological toxicity or adverse findings at doses up to 30 mg/kg. CONCLUSIONS: The in vitro and in vivo antitumor activity of hAB21 broadly recapitulates that of CD47 targeted therapies despite differences in ligand expression, binding partners, and function, validating the CD47-SIRPα axis as a fundamental myeloid checkpoint pathway and its blockade as promising therapeutic intervention for treatment of human malignancies.

Discovery of high affinity, pan-allelic, and pan-mammalian reactive antibodies against the myeloid checkpoint receptor SIRPα.

Targeting the CD47-signal-regulatory protein α (SIRPα) pathway represents a novel therapeutic approach to enhance anti-cancer immunity by promoting both innate and adaptive immune responses. Unlike CD47, which is expressed ubiquitously, SIRPα expression is mainly restricted to myeloid cells and neurons. Therefore, compared to CD47-targeted therapies, targeting SIRPα may result in differential safety and efficacy profiles, potentially enabling lower effective doses and improved pharmacokinetics and pharmacodynamics. The development of effective SIRPα antagonists is restricted by polymorphisms within the CD47-binding domain of SIRPα, necessitating pan-allele reactive anti-SIRPα antibodies for therapeutic intervention in diverse patient populations. We immunized wild-type and human antibody transgenic chickens with a multi-allele and multi-species SIRPα regimen in order to discover pan-allelic and pan-mammalian reactive anti-SIRPα antibodies suitable for clinical translation. A total of 200 antibodies were isolated and screened for SIRPα reactivity from which approximately 70 antibodies with diverse SIRPα binding profiles, sequence families, and epitopes were selected for further characterization. A subset of anti-SIRPα antibodies bound to both human SIRPα v1 and v2 alleles with high affinity ranging from low nanomolar to picomolar, potently antagonized the CD47/SIRPα interaction, and potentiated macrophage-mediated antibody-dependent cellular phagocytosis in vitro. X-ray crystal structures of five anti-SIRPα antigen-binding fragments, each with unique epitopes, in complex with SIRPα (PDB codes 6NMV, 6NMU, 6NMT, 6NMS, and 6NMR) are reported. Furthermore, some of the anti-SIRPα antibodies cross-react with cynomolgus SIRPα and various mouse SIRPα alleles (BALB/c, NOD, BL/6), which can facilitate preclinical to clinical development. These properties provide an attractive rationale to advance the development of these anti-SIRPα antibodies as a novel therapy for advanced malignancies. Abbreviations: ADCC: antibody-dependent cellular cytotoxicity; ADCP: antibody-dependent cellular phagocytosis; CFSE: carboxyfluorescein succinimidyl ester; Fab: fragment antigen binding; Fc: fragment crystallizable; FcγR: Fcγ receptor; Ig: immunoglobulin; IND: investigational new drug; MDM⊘: monocyte-derived macrophage; NOD: non-obese diabetic; scFv: single chain fragment variable; SCID: severe combined immunodeficiency; SIRP: signal-regulatory protein.

Selective targeting of engineered T cells using orthogonal IL-2 cytokine-receptor complexes.

Interleukin-2 (IL-2) is a cytokine required for effector T cell expansion, survival, and function, especially for engineered T cells in adoptive cell immunotherapy, but its pleiotropy leads to simultaneous stimulation and suppression of immune responses as well as systemic toxicity, limiting its therapeutic use. We engineered IL-2 cytokine-receptor orthogonal (ortho) pairs that interact with one another, transmitting native IL-2 signals, but do not interact with their natural cytokine and receptor counterparts. Introduction of orthoIL-2Rß into T cells enabled the selective cellular targeting of orthoIL-2 to engineered CD4+ and CD8+ T cells in vitro and in vivo, with limited off-target effects and negligible toxicity. OrthoIL-2 pairs were efficacious in a preclinical mouse cancer model of adoptive cell therapy and may therefore represent a synthetic approach to achieving selective potentiation of engineered cells.

Localized CD47 blockade enhances immunotherapy for murine melanoma.

CD47 is an antiphagocytic ligand broadly expressed on normal and malignant tissues that delivers an inhibitory signal through the receptor signal regulatory protein alpha (SIRPα). Inhibitors of the CD47-SIRPα interaction improve antitumor antibody responses by enhancing antibody-dependent cellular phagocytosis (ADCP) in xenograft models. Endogenous expression of CD47 on a variety of cell types, including erythrocytes, creates a formidable antigen sink that may limit the efficacy of CD47-targeting therapies. We generated a nanobody, A4, that blocks the CD47-SIRPα interaction. A4 synergizes with anti-PD-L1, but not anti-CTLA4, therapy in the syngeneic B16F10 melanoma model. Neither increased dosing nor half-life extension by fusion of A4 to IgG2a Fc (A4Fc) overcame the issue of an antigen sink or, in the case of A4Fc, systemic toxicity. Generation of a B16F10 cell line that secretes the A4 nanobody showed that an enhanced response to several immune therapies requires near-complete blockade of CD47 in the tumor microenvironment. Thus, strategies to localize CD47 blockade to tumors may be particularly valuable for immune therapy.

Decoupling the Functional Pleiotropy of Stem Cell Factor by Tuning c-Kit Signaling.

Most secreted growth factors and cytokines are functionally pleiotropic because their receptors are expressed on diverse cell types. While important for normal mammalian physiology, pleiotropy limits the efficacy of cytokines and growth factors as therapeutics. Stem cell factor (SCF) is a growth factor that acts through the c-Kit receptor tyrosine kinase to elicit hematopoietic progenitor expansion but can be toxic when administered in vivo because it concurrently activates mast cells. We engineered a mechanism-based SCF partial agonist that impaired c-Kit dimerization, truncating downstream signaling amplitude. This SCF variant elicited biased activation of hematopoietic progenitors over mast cells in vitro and in vivo. Mouse models of SCF-mediated anaphylaxis, radioprotection, and hematopoietic expansion revealed that this SCF partial agonist retained therapeutic efficacy while exhibiting virtually no anaphylactic off-target effects. The approach of biasing cell activation by tuning signaling thresholds and outputs has applications to many dimeric receptor-ligand systems.

Durable antitumor responses to CD47 blockade require adaptive immune stimulation.

Therapeutic antitumor antibodies treat cancer by mobilizing both innate and adaptive immunity. CD47 is an antiphagocytic ligand exploited by tumor cells to blunt antibody effector functions by transmitting an inhibitory signal through its receptor signal regulatory protein alpha (SIRPα). Interference with the CD47-SIRPα interaction synergizes with tumor-specific monoclonal antibodies to eliminate human tumor xenografts by enhancing macrophage-mediated antibody-dependent cellular phagocytosis (ADCP), but synergy between CD47 blockade and ADCP has yet to be demonstrated in immunocompetent hosts. Here, we show that CD47 blockade alone or in combination with a tumor-specific antibody fails to generate antitumor immunity against syngeneic B16F10 tumors in mice. Durable tumor immunity required programmed death-ligand 1 (PD-L1) blockade in combination with an antitumor antibody, with incorporation of CD47 antagonism substantially improving response rates. Our results highlight an underappreciated contribution of the adaptive immune system to anti-CD47 adjuvant therapy and suggest that targeting both innate and adaptive immune checkpoints can potentiate the vaccinal effect of antitumor antibody therapy.

"Velcro" engineering of high affinity CD47 ectodomain as signal regulatory protein α (SIRPα) antagonists that enhance antibody-dependent cellular phagocytosis.

CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the "don't-eat-me" signal, to macrophages upon engaging its receptor signal regulatory protein α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined "Velcro" engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRPα compared with CD47. Herein, we have demonstrated that "Velcro" engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy.

The neonatal Fc receptor, FcRn, as a target for drug delivery and therapy.

Immunoglobulin G (IgG)-based drugs are arguably the most successful class of protein therapeutics due in part to their remarkably long blood circulation. This arises from IgG interaction with the neonatal Fc receptor, FcRn. FcRn is the central regulator of IgG and albumin homeostasis throughout life and is increasingly being recognized as an important player in autoimmune disease, mucosal immunity, and tumor immune surveillance. Various engineering approaches that hijack or disrupt the FcRn-mediated transport pathway have been devised to develop long-lasting and non-invasive protein therapeutics, protein subunit vaccines, and therapeutics for treatment of autoimmune and infectious disease. In this review, we highlight the diverse biological functions of FcRn, emerging therapeutic opportunities, as well as the associated challenges of targeting FcRn for drug delivery and disease therapy.

Fusion of a short peptide that binds immunoglobulin G to a recombinant protein substantially increases its plasma half-life in mice.

We explore a strategy to substantially increase the half-life of recombinant proteins by genetic fusion to FcIII, a 13-mer IgG-Fc domain binding peptide (IgGBP) originally identified by DeLano and co-workers at Genentech [DeLano WL, et al. (2000) Science 287:1279-1283]. IgGBP fusion increases the in vivo half-life of proteins by enabling the fusion protein to bind serum IgG, a concept originally introduced by DeLano and co-workers in a patent but that to the best of our knowledge has never been pursued in the scientific literature. To further investigate the in vitro and in vivo properties of IgGBP fusion proteins, we fused FcIII to the C-terminus of a model fluorescent protein, monomeric Katushka (mKate). mKate-IgGBP fusions are easily expressed in Escherichia coli and bind specifically to human IgG with an affinity of ∼ 40 nM and ∼ 20 nM at pH 7.4 and pH 6, respectively, but not to mouse or rat IgG isotypes. mKate-IgGBP binds the Fc-domain of hIgG1 at a site overlapping the human neonatal Fc receptor (hFcRn) and as a consequence inhibits the binding of hIgG1 to hFcRn in vitro. High affinity binding to human IgG also endows mKate-IgGBP with a long circulation half-life of ∼ 8 hr in mice, a 75-fold increase compared to unmodified mKate. Thus, IgGBP fusion significantly reduces protein clearance by piggybacking on serum IgG without substantially increasing protein molecular weight due to the small size of the IgGBP. These attractive features could result in protein therapies with reduced dose frequency and improved patient compliance.

Periplasmic production via the pET expression system of soluble, bioactive human growth hormone.

A pET based expression system for the production of recombinant human growth hormone (hGH) directed to the Escherichia coli periplasmic space was developed. The pET22b plasmid was used as a template for creating vectors that encode hGH fused to either a pelB or ompA secretion signal under control of the strong bacteriophage T7 promoter. The pelB- and ompA-hGH constructs expressed in BL21 (λDE3)-RIPL E. coli are secreted into the periplasm which facilitates isolation of soluble hGH by selective disruption of the outer membrane. A carboxy-terminal poly-histidine tag enabled purification by Ni(2+) affinity chromatography with an average yield of 1.4 mg/L culture of purified hGH, independent of secretion signal. Purified pelB- and ompA-hGH are monomeric based on size exclusion chromatography with an intact mass corresponding to mature hGH indicating proper cleavage of the signal peptide and folding in the periplasm. Both pelB- and ompA-hGH bind the hGH receptor with high affinity and potently stimulate Nb2 cell growth. These results demonstrate that the pET expression system is suitable for the rapid and simple isolation of bioactive, soluble hGH from E. coli.

Engineering neonatal Fc receptor-mediated recycling and transcytosis in recombinant proteins by short terminal peptide extensions.

The importance of therapeutic recombinant proteins in medicine has led to a variety of tactics to increase their circulation time or to enable routes of administration other than injection. One clinically successful tactic to improve both protein circulation and delivery is to fuse the Fc domain of IgG to therapeutic proteins so that the resulting fusion proteins interact with the human neonatal Fc receptor (FcRn). As an alternative to grafting the high molecular weight Fc domain to therapeutic proteins, we have modified their N and/or C termini with a short peptide sequence that interacts with FcRn. Our strategy was motivated by results [Mezo AR, et al. (2008) Proc Natl Acad Sci USA 105:2337-2342] that identified peptides that compete with human IgG for FcRn. The small size and simple structure of the FcRn-binding peptide (FcBP) allows for expression of FcBP fusion proteins in Escherichia coli and results in their pH-dependent binding to FcRn with an affinity comparable to that of IgG. The FcBP fusion proteins are internalized, recycled, and transcytosed across cell monolayers that express FcRn. This strategy has the potential to improve protein transport across epithelial barriers, which could lead to noninvasive administration and also enable longer half-lives of therapeutic proteins.